Atherosclerosis and cardiovascular complications are prevalent among patients undergoing chronic hemodialysis (HD). In this population, peripheral polymorphonuclear leukocytes (PMNLs) are primed, releasing pro-inflammatory mediators, such as elastase. Elastase is normally inhibited by a specific inhibitor, avoiding undesirable degradation of cellular and extracellular components. This study tested the hypothesis that in states of non-infectious inflammation, elastase is released by PMNLs and acts in an uncontrolled manner to inflict vascular damage. Blood was collected from HD patients and healthy controls (HC). PMNL intracellular and surface expression of elastase was determined by q-PCR, Western blotting and Flow cytometry. The elastase activity was evaluated using a fluorescent substrate. The levels of serum alpha1-anti trypsin (alpha1-AT), the natural elastase inhibitor, were determined by Western blot. Free active elastase was elevated in HD sera, while the levels of alpha1-AT, were decreased compared to HC. The levels of the intracellular elastase enzyme and its activity were lower in HD PMNLs despite similar expression levels of elastase mRNA. Elastase binding to PMNL cell surface was higher in HD compared to HC. The increased circulating levels of free active elastase released from primed HD PMNLs, together with the higher cell surface-bound enzyme and the lower levels of alpha1-AT result in the higher elastase activity in HD sera. This exacerbated elastase activity could lead to the endothelial dysfunction, as hypothesized. In addition, it suggests that free circulating elastase can serve as a new biomarker and therapeutic target to reduce inflammation and vascular complications in hemodialysis patients.
Geranium seemannii Peyr is a perennial plant endemic in central Mexico that has been widely used for its diuretic effect, but the responsible compound of this effect is unknown as well as the mechanism by which the diuretic effect is achieved. Geraniin is one of the compounds isolated from this kind of geranium. This study was designed to determinate whether geraniin possesses diuretic activity and to elucidate the mechanism of action. Geraniin was extracted and purified from Geranium seemannii Peyr. Male Wistar rats were divided into four groups: 1) Control, 2) 75 mg/kg of geraniin, 3) 20 mg/Kg of furosemide, and 4) 10 mg/Kg of hydrochlorothiazide. Each treatment was administered by gavage every 24 h for 7 days. The urinary excretion of electrolytes and the fractional excretion of sodium (FENa) were determined. To uncover the molecular target of geraniin, Xenopus laevis oocytes were microinjected with cRNAs encoding the Na-Cl cotransporter (NCC) and the Na-K-2Cl cotransporter NKCC2 to functionally express these cotransporters. Geraniin significantly increased diuresis, natriuresis and calciuresis to a similar extent as was observed in the furosemide-treated rats. Consistent with the furosemide-like effect, in Xenopus oocytes, geraniin significantly reduced the activity of NKCC2, with no effect on NCC activity. In contrast with furosemide, the effect of geraniin on NKCC2 was irreversible, apparently due to its inhibitory effect on the heat shock protein 90. Our observations suggest that geraniin could have a potential role in the treatment of hypertension or edematous states
Gestational potassium retention, most of which occurs during late pregnancy, is essential for fetal development. The purpose of this study was to examine mechanisms underlying changes in potassium handling by the kidney and colon in pregnancy. We found that potassium intake and renal excretion increased in late pregnancy while fecal potassium excretion remained unchanged and that pregnant rats exhibited net potassium retention. By qPCR we found markedly increased H+/K+-ATPase type2 (HKA2) mRNA expression in cortex and OM of late pregnant vs virgin. ROMK mRNA was unchanged in the cortex, but apical ROMK abundance (by immunofluorescence) was decreased in pregnant vs virgin in the distal convoluted tubule (DCT) and connecting tubule (CNT). BKa protein abundance in intercalated cells in cortex and outer medullary collecting ducts (by immunohistochemistry) fell in late pregnancy. In the distal colon we found increased HKA2 mRNA and protein abundance (western blot) and decreased BKα protein with no observed changes in mRNA. Therefore, the potassium retention of pregnancy is likely to be due to increased collecting duct potassium reabsorption (via increased HKA2) and decreased potassium secretion in (via decreased ROMK and BK), as well as increased colonic reabsorption via HKA2.
Our laboratory recently made the novel observation that 5-hydroxytryptamine 1F (5-HT1F) receptor activation induces mitochondrial biogenesis (MB), the production of new, functional mitochondria, in vitro and vivo. We sought to determine the mechanism linking the 5-HT1F receptor to MB in renal proximal tubule cells. Using LY344864, a selective 5-HT1F receptor agonist, we determined that the 5-HT1F receptor is coupled to Gαi/o and induces MB through Gβ dependent activation of Akt, endothelial nitric oxide (eNOS), cyclic guanosine-monophosphate (cGMP), protein kinase G (PKG) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α). We also report that the 5-HT1F receptor signals through a second, Gβ dependent pathway that is linked by Akt phosphorylation of Raf. In contrast to the activated Akt pathway, Raf phosphorylation reduced ERK1/2 and FOXO3a phosphorylation, suppressing an inhibitory MB pathway. These results demonstrate that the 5-HT1F receptor regulates MB through Gβ dependent dual mechanisms that activate a stimulatory MB pathway, Akt/eNOS/cGMP/PKG/PGC-1α, while simultaneously repressing an inhibitory MB pathway, Raf/MEK/ERK/FOXO3a. Novel mechanisms of MB provide the foundation for new chemicals that induce MB to treat acute and chronic organ injuries.
Podocyte dysfunction and loss is an early event and a hallmark of proteinuric kidney diseases. Podocyte's normal function is maintained via its unique cellular architecture that relies on an intracellular network of filaments, including filamentous actin (F-actin) and microtubules, that provides mechanical support. Damage to this filamentous network leads to changes in cellular morphology and results in podocyte injury, dysfunction, and death. Conversely, stabilization of this network protects podocytes and ameliorates proteinuria. This suggests that stabilization of podocyte architecture via its filamentous network could be a key therapeutic strategy for proteinuric kidney diseases. However, development of podocyte-directed therapeutics, especially those that target the cell's filamentous network, is still lacking, partly due to unavailability of appropriate cellular assays for use in a drug-discovery environment. Here, we describe a new high content screening based methodology that utilizes a multi-parametric approach to analyze phenotypic data. Implementation of this methodology on a podocyte-based screening assay with a library of 2121 compounds identified paullone-derivatives as a novel group of podocyte protective compounds. We find that three compounds - kenpaullone, 1-azakenpaullone, and alsterpaullone - dose-dependently protect podocytes from puromycin aminonucleoside (PAN) mediated injury in vitro, by reducing PAN-induced changes in both the filamentous actin and microtubules, with alsterpaullone providing maximal protection. Mechanistic studies further show that alsterpaullone suppressed PAN-induced activation of signaling downstream of GSK3β and p38 mitogen-activated protein kinase. In vivo, it reduced ADR-induced glomerular injury in a zebrafish model. Together, these results identify paullone-derivatives as novel podocyte protective agents for future therapeutic development.
Aquaporin-2 (AQP2) is a water channel protein expressed in principal cells (PCs) of the kidney collecting ducts (CDs) and plays a critical role in mediating water reabsorption and urine concentration. AQP2 undergoes both regulated trafficking mediated by vasopressin (VP) and constitutive recycling, which is independent of VP. For both pathways, actin cytoskeletal dynamics is a key determinant of AQP2 trafficking. We report here that manganese chloride (MnCl2) is a novel and potent regulator of AQP2 trafficking in cultured cells and in the kidney. MnCl2 treatment promoted internalization and intracellular accumulation of AQP2. The effect of MnCl2 on the intracellular accumulation of AQP2 was associated with activation of RhoA and actin polymerization without modification of AQP2 phosphorylation. Although the level of total and phosphorylated AQP2 did not change, MnCl2 treatment impeded VP-induced phosphorylation of AQP2 at its serine residues 256, 264, and 269 and dephosphorylation at serine 261. In addition, MnCl2 significantly promoted F-actin polymerization along with downregulation of RhoA activity, and prevented VP-induced membrane accumulation of AQP2. Finally, MnCl2 treatment in mice resulted in significant polyuria and reduced urinary concentration, likely due to intracellular relocation of AQP2 in the PCs of kidney CDs. More importantly, the reduced urinary concentration caused by MnCl2 treatment in animals was not corrected by VP. In summary, our study identified a novel effect of MnCl2 on AQP2 trafficking through modifying RhoA activity and actin polymerization, and uncovered its potent impact on water diuresis in vivo.
We previously demonstrated that renal tubular peptidylarginine deiminase-4 (PAD4) is induced after ischemia reperfusion (IR) injury and this induction of PAD4 exacerbates ischemic acute kidney injury (AKI) by promoting renal tubular inflammation and neutrophil infiltration. However, the mechanisms of renal tubular PAD4 induction after IR remain unknown. Here, we tested the hypothesis that ATP, a pro-inflammatory danger associated molecular pattern (DAMP) ligand released from necrotic cells after IR injury, induces renal tubular PAD4 and exacerbates ischemic AKI via P2 purinergic receptor activation. ATP as well as ATPS (a non-metabolizable ATP analog) induced PAD4 mRNA, protein and activity in human and mouse renal proximal tubule cells. Supporting the hypothesis that ATP induces renal tubular PAD4 via P2X7 receptor activation, A804598 (a selective P2X7 receptor antagonist) blocked the ATP-mediated induction of renal tubular PAD4 whereas BzATP (a selective P2X7 receptor agonist) mimicked the effects of ATP by inducing renal tubular PAD4 expression and activity. Moreover, ATP-mediated calcium influx in renal proximal tubule cells was blocked by A804598 and was mimicked by BzATP. P2X7 activation by BzATP also induced PAD4 expression and activity in mouse kidney in vivo. Finally, supporting a critical role for PAD4 in P2X7-mediated exacerbation of renal injury, BzATP exacerbated ischemic AKI in PAD4 wild type mice but not in PAD4 deficient mice. Taken together, our studies show that ATP induces renal tubular PAD4 via P2X7 receptor activation to exacerbate renal tubular inflammation and injury after IR.
Background: The intrarenal renin angiotensin system (RAS) is activated in polycystic kidney disease. We have recently shown in the Pkd1 mouse that Gen 2 antisense oligonucleotide (ASO), which suppresses angiotensinogen (Agt) synthesis, is efficacious in slowing kidney cyst formation compared to lisinopril. The aim of this current study was to determine 1) if unilateral nephrectomy accelerates cystogenesis in Pkd1 mice (as previously shown in cilia knockout mice), and 2) whether Agt ASO can slow the progression in this accelerated cystic mouse model. Methods: Adult Pkd1conditional floxed allele mice expressing cre were administered tamoxifen resulting in global knockout of Pkd1. Three weeks after tamoxifen injection, mice underwent left unilateral nephrectomy. Mice were then treated with Agt ASO (75 mg/kg/wk) or aliskiren (20 mg/kg/d) +Agt ASO or control for total of 8 weeks. Results: Unilateral nephrectomy accelerated kidney cyst formation compared to non-nephrectomized mice. Both Agt ASO and Aliskiren+Agt ASO treatment significantly reduced plasma and urinary Agt levels. Blood pressure was lowest in Aliskiren+Agt ASO among all treatment groups and control group had the highest BP. All mice developed significant kidney cysts at 8 wks after nephrectomy but Agt ASO and Aliskiren+Agt ASO group had fewer kidney cysts compared to control. Renal pAKT, pS6 levels and apoptosis were significantly suppressed in those receiving Agt ASO compared control. Conclusions: These results indicate that suppressing Agt using an ASO slowed the progression of accelerated cystic kidney disease induced by unilateral nephrectomy in Pkd1 mice by suppressing intrarenal RAS, mTOR pathway and cell proliferation.
The Epithelial Na+ Channel, ENaC, is the final arbiter of sodium excretion in the kidneys. As such, discretionary control of ENaC by hormones is critical to the fine-tuning of electrolyte and water excretion and consequently, blood pressure. Casein kinase 2 (CK2) phosphorylates ENaC. Phosphorylation by CK2 is necessary for normal ENaC activity. We tested the physiological importance of CK2 regulation of ENaC as the degree to which ENaC activity is dependent on CK2 phosphorylation in the living organism is unknown. This was addressed using patch clamp analysis of ENaC in split-open collecting ducts in complete with whole animal physiological studies of sodium excretion in mice. We also used ENaC harboring CK2 phosphorylation site mutations to elaborate mechanism. We found that ENaC activity in ex vivo preparations of murine collecting duct had a significant decrease in activity in response to selective antagonism of CK2. In whole animal experiments selective antagonism of CK2 caused a natriuresis similar to benzamil, but not additive to benzamil, suggesting an ENaC dependent mechanism. Regulation of ENaC by CK2 was abolished by mutation of the canonical CK2 phosphorylation sites in beta and gamma ENaC. Together, these results demonstrate that the appropriate regulation of ENaC by CK2 is necessary for the normal physiological role played by this key renal ion channel in the fine-tuning of sodium excretion.
The characteristic features of chronic peritoneal injury with peritoneal dialysis (PD) are submesothelial fibrosis and neoangiogenesis. Transforming growth factor (TGF)-β and vascular endothelial growth factor (VEGF)-A are the main mediators of fibrosis and neoangiogenesis, respectively; however, the effect of the interaction between them on the peritoneum is not well known. In this study, we investigated the relationship between TGF-β1 and VEGF-A in inducing peritoneal fibrosis using human tissues and dialysate, cultured cells, and animal models. The VEGF-A concentration correlated with the dialysate-to-plasma ratio of creatinine (D/P Cr) (P<0.001) and TGF-β1 (P<0.001) in human PD effluent. VEGF-A mRNA levels increased significantly in the peritoneal tissues of human ultrafiltration failure (UFF) patients, and correlated with number of vessels (P<0.01) and peritoneal thickness (P<0.001). TGF-β1 increased VEGF-A production in human mesothelial cell-lines and fibroblast cell-lines, and TGF-β1-induced VEGF-A was suppressed by TGF-β receptor I (TGFβR-I) inhibitor. Incremental peak values of VEGF-A mRNA stimulated by TGF-β1 in human cultured mesothelial cells derived from PD patients with a range of peritoneal membrane functions correlated with D/P Cr (P<0.05). To evaluate the regulatory mechanisms of VEGF-A and neoangiogenesis in vivo, we administered TGFβR-I inhibitor intraperitoneally in a rat chlorhexidine-induced peritoneal injury (CG) model. TGFβR-I inhibitor administration in the CG model decreased peritoneal thickness (P<0.001), the number of vessels (P<0.001), and VEGF-A levels (P<0.05). These results suggest that neoangiogenesis is associated with fibrosis through the TGF-β1-VEGF-A pathway in mesothelial cells and fibroblasts. These findings are important when considering the strategy for management of UFF in PD patients.
Serelaxin is a novel recombinant human relaxin-2 that has been investigated for the treatment of acute heart failure. However, its effects on renal function, especially on the renal microcirculation remain incompletely characterized. Our immunoexpression studies localized RXFP1 receptors on vascular smooth muscle cells and endothelial cells of afferent arterioles and on principal cells of collecting ducts. Clearance experiments were performed in male and female normotensive rats and Ang II infused male rats. Serelaxin increased mean arterial pressure slightly and significantly increased renal blood flow, urine flow and sodium excretion rate. Group analysis of all serelaxin infusion experiments showed significant increases in GFR. During infusion with subthreshold levels of Ang II, serelaxin did not alter mean arterial pressure, renal blood flow, GFR, urine flow or sodium excretion rate. Heart rates were elevated during serelaxin infusion alone (37±5%) and in Ang II infused rats (14±2%). In studies using the in vitro isolated juxtamedullary nephron preparation, superfusion with serelaxin alone (40 ng/ml) significantly dilated afferent arterioles (10.8±1.2 vs. 13.5±1.1 µm) and efferent arterioles (9.9±0.9 vs. 11.9±1.0 µm). During Ang II superfusion, serelaxin did not alter afferent or efferent arteriolar diameters. During NO synthase inhibition (L-NNA), afferent arterioles also did not show any vasodilation during serelaxin infusion. In conclusion, serelaxin increased overall renal blood flow, urine flow, GFR and sodium excretion and dilated the afferent and efferent arterioles in control conditions, but these effects were attenuated or prevented in the presence of exogenous Ang II and NO synthase inhibitor.
The donor glomerular filtration rate (GFR) measured before kidney donation is a strong determinant of recipient graft outcome. No tubular function markers have been identified that can similarly be used in donors to predict recipient outcomes. In the current study we investigated whether the predonation tubular maximum reabsorption capacity of phosphate (TmP-GFR), which may be considered as a functional tubular marker in healthy kidney donors, is associated with recipient GFR at one year after transplantation, a key determinant of long-term outcome. We calculated the predonation TmP-GFR from serum and 24h-urine phosphate and creatinine levels in 165 kidney donors, and recipient 125I-iothalamate GFR and eGFR (CKD-EPI) at 12 months after transplantation. Kidney donors were 51±10 years old, 47% were men, and mean GFR was 118±26 mL/min. The donor TmP-GFR was associated with recipient GFR 12 months after transplantation (GFR 6.0 mL/min lower per 1 mg/dL decrement of TmP-GFR), which persisted after multivariable adjustment for donor age, sex, predonation GFR and blood pressure and other potential confounders. Results were highly similar when eGFR at 12 months was taken as the outcome. Tubular damage markers KIM-1 and NGAL were low and not associated with recipient GFR. A lower donor TmP-GFR before donation, which may be considered to represent a functional measure of tubular phosphate reabsorption capacity, is independently associated with a lower recipient GFR one year after transplantation. These data are the first to link donor tubular phosphate reabsorption with recipient GFR post-transplantation.
Renal blood flow (RBF) provides important information regarding renal physiology and nephropathies. Arterial spin labeling-magnetic resonance imaging (ASL-MRI) is a non-invasive method of measuring blood flow without exogenous contrast media. However, low signal/noise ratio and respiratory motion artifacts are challenges for RBF measurements in small animals. Our objective was to evaluate the feasibility and reproducibility of RBF measures by ASL-MRI using respiratory-gating and navigator correction methods to reduce motion artifacts. ASL-MRI images were obtained from the kidneys of Sprague-Dawley (SD) rats on a 7 tesla Varian MRI system with a spin-echo imaging sequence. After 4 days, the study was repeated to evaluate its reproducibility. RBF was also measured in animals under unilateral nephrectomy and in renal artery stenosis (RST) to evaluate the sensitivity in high and low RBF models, respectively. RBF was also evaluated in Dahl salt-sensitive (SS) rats and spontaneous hypertensive rats (SHR). In SD rats the cortical RBFs (cRBF) were 305±59 and 271.8±39 ml/min/100 g tissue in the right and left kidneys, respectively. Re-test analysis revealed no differences (p=0.2). The test-retest reliability coefficient was 92±5%. The cRBFs before and after the nephrectomy were 296.8±30 and 428.2±45 ml/min/100 g (p=0.02), respectively. The kidneys with RST exhibited a cRBF decrease compared with sham animals (86±17.6 vs. 198±33.7 ml/min/100 g tissue; p<0.01). The cRBFs in SD, Dahl-ss and SHR rats were not different (p=0.35). We conclude that ASL-MRI performed with navigator correction and respiratory gating is a feasible and reliable non-invasive method for measuring RBF in rats.
Cellular, molecular, and ultrastructural nephron changes associated with ischemia reperfusion injury-induced acute kidney injury (IRI-AKI) are not completely understood. Here, a multidisciplinary study was used to identify nephron changes in a mouse model of IRI-AKI. Histological analyses indicated distended Bowman's glomerular spaces and proximal and distal tubules. Increased filtrate volume in nephrons was caused by reduced water reabsorption by severely damaged proximal tubule brush borders and blocked flow of filtrate into collecting tubules by mucoprotein casts in distal tubules. Immunohistochemistry revealed protein AKI biomarkers in proximal tubules and glomeruli but not in distal tubules. Nuclear magnetic resonance spectroscopy revealed several metabolites that increased such as valine alanine and lactate. Other metabolites such as trigonelline, succinate, 2-oxoisocaproate, and 1- methyl-nicotinamide decreased or were absent in urine following IRI due to altered kidney function or metabolism. Urinary glucose increased due to reduced reabsorption by damaged proximal tubule brush borders. Scanning electron microscopy revealed flattening of podocytes and pedicals surrounding glomerular capillaries and transmission electron microscopy (TEM) revealed effacement of podocyte pedicals, both consistent with increased hydrostatic pressure in nephrons following IRI-AKI. TEM revealed shortened proximal tubule microvilli in IRI kidneys with diminished lamina propia. TEM showed dramatic loss of mitochondria in distal tubule epithelia of IRI kidneys and emergence of multivesicular bodies of endosomes indicating ongoing cellular death. Collectively, the data define ultrastructural changes to nephrons and altered kidney metabolism associated with IRI-AKI.
p66Shc is one of the three adaptor proteins encoded by Shc1 gene, which are expressed in many organs including the kidney. Recent studies shed new light on several key questions concerning the signaling mechanisms mediated by p66Shc. The central goal of this review article is to summarize recent findings on p66Shc and the role it plays in kidney physiology and pathology. This manuscript provides a review of the various mechanisms whereby p66Shc has been shown to function within the kidney through a wide range of actions. The mitochondrial and cytoplasmic signaling of p66Shc, as it relates to production of reactive oxygen species (ROS) and renal pathologies, is further discussed.
Destabilized heme proteins release heme, and free heme is toxic. Heme is now recognized as an agonist for the TLR4 receptor. This study examined whether the TLR4 receptor mediates the nephrotoxicity of heme, specifically, the effects of heme on renal blood flow and inflammatory responses. We blocked TLR4 signaling by the specific antagonist TAK-242. Intravenous administration of heme to mice promptly reduced renal blood flow, an effect attenuated by TAK-242. In vitro, TAK-242 reduced heme-elicited activation of NF-B and its downstream gene MCP-1; in contrast, TAK-242 failed to reduce heme-induced activation of the anti-inflammatory transcription factor, Nrf2, and its downstream gene HO-1. TAK-242 did not reduce heme-induced renal MCP-1 upregulation in vivo. TAK-242 did not reduce dysfunction and histologic injury in the glycerol model of heme protein-induced acute kidney injury (AKI), findings corroborated by studies in TLR4+/+ and TLR4-/- mice. We conclude that: 1) acute heme-mediated renal vasoconstriction occurs through TLR4 signaling; 2) proinflammatory effects of heme in renal epithelial cells involve TLR4 signaling, whereas the anti-inflammatory effects of heme do not; 3) TLR4 signaling does not mediate the proinflammatory effects of heme in the kidney; 4) major mechanisms underlying glycerol-induced, heme protein-mediated AKI do not involve TLR4 signaling. These findings in the glycerol model are in stark contrast with findings in all other AKI models studied to-date and emphasize the importance of TLR4-independent pathways of heme protein-mediated injury in this model. Finally, these studies urge caution when using observations derived in vitro to predict what may occur in vivo.
Dyssynchrony of circadian rhythms are associated with various disorders, including cardiovascular and metabolic diseases. The cell autonomous molecular clock maintains circadian control, however, environmental factors that may cause circadian dyssynchrony either within or between organ systems are poorly understood. Our lab recently reported that the endothelin B (ETB) receptor functions to facilitate Na+ excretion in a time of day dependent manner. Therefore, the current study was designed to determine whether high salt (HS) intake leads to circadian dyssynchrony within the kidney, and whether the renal endothelin system contributes to control of the renal molecular clock. We observed that HS feeding led to region-specific alterations in circadian clock components within the kidney. For instance, HS caused a significant 5.5-hour phase delay in the peak expression of Bmal1 and suppressed Cry1 and Per2 expression in the renal inner medulla, but not the renal cortex, of control rats. The phase delay in Bmal1 expression appears to be mediated by endothelin (ET-1) because this phenomenon was not observed in the ETB deficient rat. In cultured inner medullary collecting duct cells, ET-1 suppressed Bmal1 mRNA expression. Furthermore, Bmal1 knockdown in these cells reduced epithelial Na+ channel expression. These data reveal that HS feeding leads to intra-renal circadian dyssynchrony mediated, in part, through activation of ETB receptors within the renal inner medulla.
Increased kidney oxygen consumption causing tissue hypoxia is suggested as a common pathway to chronic kidney disease. Mammalian target of rapamycin (mTOR) regulates cell proliferation and mitochondrial function. mTOR inhibitors, e.g. rapamycin, are used clinically to prevent graft rejection. mTOR has been identified as a key player in diabetes, which has stimulated the use of mTOR inhibitors to counter diabetic nephropathy. However, the effect of mTOR inhibition on kidney oxygen consumption is unknown. We therefore investigated the effects of mTOR inhibition on in vivo kidney function, oxygen homeostasis and glomerular permeability. Control and streptozotocin-induced diabetic rats were chronically treated with rapamycin and the functional consequences studied fourteen days thereafter. In both groups, mTOR inhibition induced mitochondrial uncoupling resulting in increased total kidney oxygen consumption and decreased intrarenal oxygen availability. Concomitantly, mTOR inhibition induced tubular injury, as estimated from urinary excretion of kidney injury molecule-1 (KIM-1), and reduced urinary protein excretion. The latter corresponded to reduced sieving coefficient for large molecules. In conclusion, mTOR inhibition induces mitochondrial dysfunction leading to decreased oxygen availability in normal and diabetic kidneys, which translates to increased KIM-1 in the urine. Reduced proteinuria after mTOR inhibition is an effect of reduced glomerular permeability for large molecules. Since hypoxia has been suggested as a common pathway to development of chronic kidney disease, mTOR inhibition to patients with pre-existing nephropathy should be used with caution since it may accelerate the progression of disease.
Hyperinsulinemia has been hypothesized to cause hypertension in obesity, type 2 diabetes and metabolic syndrome through a renal mechanism. However, it has been challenging to isolate renal mechanisms in chronic experimental models due, in part, to technical difficulties. In this study, we tested the hypothesis that a renal mechanism underlies insulin hypertension. We developed a novel technique to permit continuous insulin infusion through the renal artery in conscious rats for 7 days. Mean arterial pressure increased by approximately 10 mmHg in rats that were infused intravenously (IV) with insulin and glucose. Renal artery doses were 20% of the intravenous doses and did not raise systemic insulin levels or cause differences in blood glucose. The increase in blood pressure was not different from the IV group. MAP did not change in vehicle-infused rats and there were no differences in renal injury scoring due to the renal artery catheter. GFR, PRA and urinary sodium excretion did not differ between groups at baseline and did not change significantly with insulin infusion. Thus, by developing a novel approach for chronic, continuous renal artery insulin infusion, we provided new evidence that insulin causes hypertension in rats through actions initiated within the kidney.
The Notch pathway has been reported to control tissue damage in acute kidney diseases. To investigate potential beneficial nephroprotective effects of targeting Notch, we developed chemically functionalized -secretase inhibitors (GSIs) targeting -glutamyltranspeptidase (-GT) and/or -glutamylcyclotransfase (-GCT), two enzymes overexpressed in the injured kidney, and evaluated them in in vivo murine models of acute tubular and glomerular damage. Exposure of the animals to disease-inducing drugs together with the functionalized GSIs improved proteinuria and, to some extent, kidney dysfunction. The expression of genes involved in the Notch pathway, acute inflammatory stress responses and the renin-angiotensin system was enhanced in injured kidneys, which could be downregulated upon administration of functionalized GSIs. Immunohistochemistry staining and western blots demonstrated enhanced activation of Notch1 as detected by its cleaved active intracellular domain during acute kidney injury, and this was down-regulated by concomitant treatment with the functionalized GSIs. Thus, targeted -secretase-based prodrugs developed as substrates for -GT/-GCT have the potential to selectively control Notch activation in kidney diseases with subsequent regulation of the inflammatory stress response and the renin-angiotensin pathways.
Early life stress (ELS) in humans is associated with elevated pro-inflammatory markers. We hypothesized that ELS induces activation of the immune response in a rat model of ELS, maternal separation (MatSep), in adulthood. MatSep involves separating pups from the dam from postnatal day 2 to postnatal day 14 for 3hrs/day. Control rats are non-separated littermates. We determined circulating and renal immune cell numbers, renal immune cell activation markers, renal cytokine levels, and the renal inflammatory gene expression response to low dose lipopolysaccharide (LPS) in male MatSep and control rats. We observed that MatSep did not change the percentage of gated events for circulating CD3+, CD4+, CD8+ and CD4+/Foxp3+ cells or absolute numbers of mononuclear and T cells in the circulation and kidneys; however, MatSep led to an increase in activation of renal neutrophils as well as CD44+ cells. Renal toll-like receptor 4 (TLR4) and interleukin 1 beta (IL-1β) was significantly increased in MatSep rats, specifically in the outer and inner medulla and distal nephron, respectively. Evaluation of renal inflammatory genes, showed that in response to a low dose LPS challenge (intravenous, 2mg/kg) a total of 20 genes were significantly altered in kidneys from MatSep rats (17 genes were upregulated and 3 were downregulated), as opposed to no significant differences in gene expression in control vs. control + LPS groups. Taken together, these findings indicate that MatSep induces priming of the immune response in the kidney.
Acute kidney injury (AKI) arising from diverse etiologies is characterized by mitochondrial dysfunction. The peroxisome proliferator-activated receptor coactivator-1alpha (PGC1α), a master regulator of mitochondrial biogenesis, has been shown to be protective in AKI. Interestingly, reduction of PGC1α has also been implicated in the development of diabetic kidney disease and renal fibrosis. The beneficial renal effects of PGC1α make it a prime target for therapeutics aimed at ameliorating AKI, forms of chronic kidney disease (CKD), and their intersection. This review summarizes the current literature on the relationship between renal health and PGC1α and proposes areas of future interest.
Vascular topology and morphology is critical in the regulation of blood flow and the transport of small solutes including oxygen, carbon dioxide, nitric oxide and hydrogen sulfide. Renal vascular morphology is particularly challenging as many arterial walls are partially wrapped by the walls of veins. In the absence of a precise characterization of three-dimensional branching vascular geometry, accurate computational modeling of the intrarenal transport of small diffusible molecules is impossible. An enormous manual effort was required to achieve a relatively precise characterization of rat renal vascular geometry, highlighting the need for an automated method for analysis of branched vasculature morphology to allow characterization of the renal vascular geometry of other species, including humans. We present a semi-supervised method for three-dimensional morphometric analysis of renal vasculature images generated by computed tomography. We derive quantitative vascular attributes important to mass transport between arteries, veins and the renal tissue, and present methods for their computation for a three-dimensional vascular geometry. To validate the algorithm, we compare automated vascular estimates with subjective manual measurements for a portion of rabbit kidney. While increased image resolution can improve outcomes, our results demonstrate that the method can quantify the morphological characteristics of artery-vein pairs, comparing favorably with manual measurements. Similar to the rat, we show that rabbit artery-vein pairs become less intimate along the course of the renal vasculature, but the total wrapped mass transfer coefficient increases then decreases. This new method will facilitate new quantitative physiological models describing the transport of small molecules within the kidney.
Advanced glycation end products (AGEs) play a role in pathogenesis of diabetic nephropathy (DN). Myo-inositol Oxygenase (MIOX) has been implicated in tubulo-interstitial injury in the context of DN. We investigated the effect of AGEs on MIOX expression and delineated mechanisms that lead to tubulo-interstitial injury. Status of MIOX, RAGE and relevant cellular signaling pathways activated following AGE:RAGE interaction were examined in tubular cells and kidneys of AGE-BSA treated mice. Solid phase assay revealed an enhanced binding of RAGE with AGE-BSA, -laminin and -collagen IV. The cells treated with AGE-BSA had increased MIOX activity/expression and promoter activity. This was associated with activation of various signaling kinases of PI3K-AKT pathway, and increased expression of NF-B, TGF-β and fibronectin, which was negated with the treatment of MIOX/RAGE-siRNA. Concomitant with MIOX up-regulation there was an increased generation of reactive oxygen species (ROS), which could be abrogated with MIOX/RAGE-siRNA treatment. The kidneys of mice treated with AGE-BSA had significantly high urinary A/C ratio, up-regulation of MIOX, RAGE and NF-B along with influx of monocytes into the tubulo-interstitium, increased expression of MCP-1, IL-6 and fibronectin, and generation of ROS. Such perturbations were abrogated with the concomitant treatment of inhibitors MIOX or RAGE (D-glucarate and FPS-ZM1). These studies support a role of AGE:RAGE interaction in the activation of PI3K-AKT pathway and up-regulation of MIOX, with excessive generation of ROS, increased expression of NF-B, inflammatory cytokines, TGF-β and fibronectin. Collectively, these observations highlight the relevance of the biology of MIOX in the contribution towards tubulo-interstitial injury in DN.
Complement factor C5a has two known receptors, C5aR mediating pro-inflammatory effects and C5L2, a potential C5a scavenger. We previously identified C5a/C5aR signaling as a potent profibrotic pathway in the kidney. Here we tested for the first time the role of C5L2 in renal fibrosis. In unilateral ureteral obstruction (UUO)-induced kidney fibrosis, the expression of C5aR and C5L2 increased similarly and gradually as fibrosis progressed and was particularly prominent in injured dilated tubules. Genetic deficiency of either C5aR or C5L2 significantly reduced UUO-induced tubular injury. Expression of key pro-inflammatory mediators, however, significantly increased in C5L2- compared to C5aR-deficient mice, but this had no effect on the number of renal infiltrating macrophages or T-cells. Moreover, in C5L2-/--mice the cytokine and MMP-inhibitor TIMP-1 was specifically enhanced. Consequently, in C5L2-/--mice the degree of renal fibrosis was similar to WT, albeit with reduced mRNA expression of some fibrosis-related genes. In contrast, C5aR-/--mice had significantly reduced renal fibrosis compared to WT- and C5L2-/--mice in UUO. In vitro experiments with primary tubular cells demonstrated, that deficiency for either C5aR or C5L2 led to a significantly reduced expression of tubular injury- and fibrosis-markers. Vice versa, stimulation of WT tubular cells with C5a significantly induced the expression of these markers, whereas absence of either receptor abolished this induction. In conclusion, in experimental renal fibrosis C5L2 and C5aR both contribute to tubular injury, and, while C5aR acts profibrotic, C5L2 does not play a role in extracellular matrix accumulation, arguing against C5L2 functioning simply as a decoy receptor.
PPARA is nuclear hormone receptor that promotes fatty acid β-oxidation (FAO) and oxidative phosphorylation (OXPHOS). We and others have recently shown that PPARA and its target genes are downregulated, and FAO and OXPHOS are impaired in autosomal dominant polycystic kidney disease (ADPKD). However, whether PPARA and FAO/OXPHOS are causally linked to ADPKD progression is not entirely clear. We report that expression of PPARA and FAO/OXPHOS genes is downregulated and in-vivo β-oxidation rate of 3H-labelled triolein is reduced in Pkd1RC/RC mice, a slowly-progressing orthologous model of ADPKD that closely mimics the human ADPKD phenotype. To evaluate the effects of upregulating PPARA, we conducted a 5-month, randomized, pre-clinical trial by treating Pkd1RC/RC mice with fenofibrate, a clinically-available PPARA agonist. Fenofibrate treatment resulted in increased expression of PPARA and FAO/OXPHOS genes, upregulation of peroxisomal and mitochondrial biogenesis markers, and higher β-oxidation rates in Pkd1RC/RC kidneys. MRI-assessed total kidney volume and total cyst volume, kidney-weight-to-body-weight ratio, cyst index, and serum creatinine levels were significantly reduced in fenofibrate-treated compared to untreated littermate Pkd1RC/RC mice. Moreover, fenofibrate treatment was associated with reduced kidney cyst proliferation and infiltration by inflammatory cells including M2-like macrophages. Finally, fenofibrate treatment also reduced bile duct cyst number, cyst proliferation, and liver inflammation and fibrosis. In conclusion, our studies suggest that promoting PPARA activity to enhance mitochondrial metabolism may be a useful therapeutic strategy for ADPKD.
Fibroblast growth factor 23 (FGF23) production is upregulated by iron deficiency and hypoxia. However, the influence of acute blood loss, and the resulting increases in circulating erythropoietin, on FGF23 production is unknown. Using wild-type C57BL/6 mice, we show that acute loss of 10% total blood volume leads to an increase in plasma C-terminal FGF23 (cFGF23) levels within six hours, while plasma levels of intact FGF23, phosphate, calcium, parathyroid hormone, iron, and ferritin remain similar to control mice without acute blood loss. Volume resuscitation with PBS did not significantly alter these findings. The increase in plasma cFGF23 levels in bled animals was accompanied by increased plasma erythropoietin levels at 6 hours. Administration of erythropoietin led to an acute increase in plasma cFGF23 levels similar to that observed in acute blood loss. Fgf23 mRNA expression was increased 20-fold in bone marrow, but not in bone, of bled versus control mice, suggesting bone marrow as a key source of elevated plasma FGF23 levels following acute blood loss. To extend these findings to humans, we measured plasma cFGF23 levels in 131 critically ill patients admitted to the intensive care unit. In univariate and multivariate models, we found a positive association between number of red blood cell transfusions, an indirect indicator of acute blood loss, and plasma cFGF23 levels. We conclude that FGF23 production is rapidly increased after acute blood loss, and that erythropoietin may be the mediator of this increase. Thus, erythropoietin may represent a novel physiologic regulator of FGF23 production.
Hypertensive patients have an increased risk to develop kidney cancer. We have shown in vivo, that besides elevating blood pressure, angiotensin II causes DNA damage dose-dependently. Here, the role of blood pressure in the formation of DNA damage is studied. Mice lacking one of the two murine angiotensin II type 1 receptor (AT1R) subtypes, AT1aR, were equipped with osmotic minipumps, delivering angiotensin II during 28 days. Parameters of oxidative stress and DNA damage of kidneys and hearts of AT1aR-knockout mice were compared to wildtype (C57BL/6) mice receiving angiotensin II, and additionally, to wildtype (WT) mice treated with candesartan, an antagonist of both AT1R-subtypes. In WT mice, angiotensin II induced hypertension, reduced kidney function and led to a significant formation of reactive oxygen species (ROS). Furthermore, genomic damage was markedly increased in this group. All these responses to angiotensin II could be attenuated by concurrent administration of candesartan. In AT1aR-deficient mice treated with angiotensin II, systolic pressure was not increased, and renal function was not affected. However, angiotensin II still led to an increase of ROS in kidneys and hearts of these animals. Additionally, genomic damage in the form of double-strand breaks, was significantly induced in kidneys of AT1aR-deficient mice. Our results show that angiotensin II induced ROS production and DNA damage even without the presence of AT1aR and independently of blood pressure changes.
Acute nephron reduction such as after living kidney donation may increase the risk of hypertension. Uninephrectomy induces major hemodynamic changes in the remaining kidney resulting in rapid increase of single nephron GFR and fluid delivery in the distal nephron. Decreased sodium (Na) fractional reabsorption after the distal tubule has been reported after uninephrectomy in animals preserving volume homeostasis. In the present study, we thought to specifically explore the effect of unilateral nephrectomy on ENaC subunits expression in mice. We show that -ENaC subunit surface expression was specifically downregulated after uninephrectomy, while the expression of the aldosterone-sensitive α-ENaC and α1-Na,K-ATPase subunits as well as of NKCC2 and NCC were not significantly altered. As acute nephron reduction induces a rapid increase of single nephron GFR resulting in a higher tubular fluid flow, we speculated that local mechanical factors such as fluid-shear stress (FSS) were involved in Na reabsorption regulation after uninephrectomy. We further explore such hypothesis in vitro in a model of FSS applied on highly differentiated collecting duct principal cells. We found that FSS specifically downregulates β-ENaC and -ENaC subunits at the transcriptional level through an unidentified heat-insensitive paracrine basolateral factor. The primary cilium as a potential mechanosensor was not required. In contrast, PKA and cPLA2 were involved, but we could not demonstrate a role for cyclooxygenase or epoxygenase metabolites.
Urinary dysfunction is a common complaint following spinal cord injury (SCI), and is a leading issue for individuals with SCI that impacts their quality of life. One urinary complication that has received little attention is SCI-induced polyuria, even though SCI individuals will significantly restrict their fluid intake to decrease urine production leading to a sequelae of medical complications. Understanding the mechanisms instigating the development of polyuria will allow us to target interventions which may alleviate polyuria symptoms leading to significant improvements in the quality of life and urinary health of SCI individuals. In a rat SCI contusion model, an increase in the amount of urine excreted over a 24 hour period (p ≤ 0.001) was found at two weeks post-injury. The urine excreted was more dilute with decreased urinary creatinine and specific gravity (p ≤ 0.001). Several factors important in fluid balance regulation - vasopressin (AVP), natriuretic peptides, and corticosterone (CORT) also changed significantly post-injury. AVP levels decreased (p = 0.042) while atrial natriuretic peptide (ANP) and CORT increased (p = 0.005; p = 0.031, respectively) at two weeks post-injury. There was also a positive correlation between the increase in ANP and urine volume post-injury (p = 0.033). The changes in AVP, ANP and CORT are conducive to producing polyuria, and the timing of these changes coincides with the development of SCI-induced polyuria. This study identifies several therapeutic targets that could be used to ameliorate polyuria symptoms and improve quality of life in SCI individuals.
The Foxp3-expressing CD4+ regulatory T cells (Tregs), which is a subset of the helper T-cells (Th), constitute one of the major mechanisms of peripheral tolerance. Tregs prevent abnormal activation of the immune system throughout the lifespan, thus protecting from autoimmune and inflammatory diseases. Recent studies have elucidated the role of Tregs beyond autoimmunity. Tregs play important functions in controlling not only innate and adaptive immune cell activation, but also regulate non-immune cell function during insults and injury. Inflammation contributes to a multitude of acute and chronic diseases affecting the kidneys. This review examines the role of Tregs in pathogenesis of renal inflammatory diseases and explores the approaches for enhancing Tregs for prevention and therapy of renal inflammation.
Recently we and others have found that hyperfiltration-associated increase in biomechanical forces, namely tensile stress and fluid flow shear stress (FFSS) can directly and distinctly alter podocyte structure and function. The ultrafiltrate flow over the major processes and cell body generates FFSS to podocyte. Our previous work suggests that COX2-PGE2-EP2 axis plays an important role in mechanoperception of FFSS in podocyte (Srivastava et al. Am J Physiol Renal Physiol 307: F1323-F1333, 2014). To address mechanotransduction of the perceived mechanical stimulus through EP2 receptor, cultured podocytes were exposed to FFSS (2 dynes/cm2) for 2hrs. Total RNA from cells at the end of treatment, 2h post-FFSS and 24h post-FFSS was used for whole exon array analysis. The differentially regulated genes (p<0.01) were analyzed using bioinformatics tools Enrichr and Ingenuity Pathway Analysis to predict pathways/ molecules. Candidate pathways were validated using Western blot analysis, and then further confirmed to be resulting from a direct effect of PGE2 on podocytes. Results show that FFSS-induced mechanotransduction as well as exogenous PGE2 activate the Akt-GSK3β-β-catenin (Ser552) and ERK/MAPK but not the cAMP-PKA signal transduction cascades. These pathways are reportedly associated with FFSS-induced and EP2-mediated signaling in other epithelial cells as well. Current regimen for treating hyperfiltration-mediated injury largely depends on targeting the Renin-Angiotensin-Aldosterone System. Present study identifies specific transduction mechanisms and provides novel information on the direct effect of FFSS on podocytes. These results suggest that targeting EP2 receptor-mediated signaling pathways holds therapeutic significance for delaying progression chronic kidney disease secondary to hyperfiltration.
The cortical collecting duct (CCD) forms part of the aldosterone-sensitive distal nephron and plays an essential role in maintaining the NaCl balance and acid-base status. The CCD epithelium comprises principal cells as well as different types of intercalated cells. Until recently, transcellular Na+ transport was thought to be restricted to principal cells, while (acid-secreting) type A and (bicarbonate-secreting) type B intercalated cells were associated with the regulation of acid-base homeostasis. This review describes how this traditional view has been upended by several discoveries in the last decade. A series of studies has shown that type B intercalated cells can mediate electroneutral NaCl reabsorption by a mechanism involving Na+-dependent and Na+-independent Cl-/HCO3- exchange, and that is energetically driven by basolateral vacuolar H+-ATPase pumps. Other research indicates that type A intercalated cells can mediate NaCl secretion, through a bumetanide-sensitive pathway that is energized by apical H+,K+-ATPase type 2 pumps operating as Na+/K+ exchangers. We also review recent findings on the contribution of the paracellular route to NaCl transport in the CCD. Lastly, we describe cross-talk processes, by which one CCD cell type impacts Na+/Cl- transport in another cell type. The mechanisms that have been identified to date demonstrate clearly the interdependence of NaCl and acid-base transport systems in the CCD. They also highlight the remarkable versatility of this nephron segment.
Cystic epithelia acquire mesenchymal-like features in polycystic kidney disease (PKD). In this phenotypic alteration, it is well known that transforming growth factor (TGF)-β/Smad3 signaling is involved, however, there is emerging new data on Smad3 phosphoisoforms: Smad3 phosphorylated at linker regions (pSmad3L) and COOH terminal regions (pSmad3C) and both (pSmad3L/C). pSmad3L/C has a pathological role in colorectal cancer. Mesenchymal phenotype-specific cell responses in TGF-β/Smad3 pathway are implicated in carcinomas. In this study, we confirmed mesenchymal features and examined Smad3 phosphoisoforms in the cpk mouse, a model of autosomal recessive PKD. Kidney sections were stained with antibodies against mesenchymal markers and domain-specific phospho-Smad3. TGF-β, pSmad3L, pSmad3C, JNK, CDK4 and c-Myc were evaluated by western blotting. Co-phosphorylation of pSmad3L/C was assessed by immunoprecipitation. α-smooth muscle actin, which indicate mesenchymal features, was expressed higher in cpk mice. pSmad3L expression was increased in cpk mice and was predominantly localized in the nuclei of tubular epithelial cells in cysts; however, pSmad3C was equally expressed in both cpk and control mice. Levels of pSmad3L, JNK, CDK4 and c-Myc protein in nuclei were significantly higher in cpk mice than in controls. Immunoprecipitation showed that Smad3 was co-phosphorylated (pSmad3L/C) in cpk mice. Smad3 knockout/cpk double mutant mice revealed amelioration of cpk abnormalities. These findings suggest that up-regulating c-Myc through the JNK/CDK4-dependent pSmad3L pathway may be key to the pathophysiology in cpk mice. In conclusion, a qualitative rather than a quantitative abnormality of the TGF-β/Smad3 pathway is involved in PKD, and may be a target for disease-specific intervention.
Prostate smooth muscle contraction is critical for etiology and treatment of male lower urinary tract symptoms (LUTS), and is promoted by small monomeric GTPases (RhoA, Rac). GTPases may be activated by guanosine nucleotide exchange factors (GEFs). GEFs of the cytohesin family may indirectly activate Rac, or ADP ribosolyation factor (ARF) GTPases directly. Here we investigated the expression of cytohesin family GEFs, and effects of the cytohesin inhibitor secinH3 on smooth muscle contraction and GTPase activities in human prostate tissues. Of all four cytohesin isoforms, cytohesin-1 and -2 showed the highest expression in RT-PCR. Western blot and fluorescence staining suggested that cytohesin-2 may be the predominant isoform in prostate smooth muscle cells. Contractions induced by norepinephrine, the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analogue U46619, endothelins 1 and 3, as well as neurogenic contractions induced by electric field stimulation (EFS) were reduced by secinH3 (30 µM). Inhibition of EFS-induced contractions appeared to have similar efficacy than inhibition by the α1-adrenoceptor antagonist tamsulosin (300 nM). Combined application of secinH3 plus tamsulosin caused larger inhibition of EFS-induced contractions than tamsulosin alone. Pull down assays demonstrated inhibition of the small monomeric GTPase ARF6 by secinH3, but no inhibition of RhoA or Rac1. In conclusion, we suggest that a cytohesin/ARF6 pathway takes part in smooth muscle contraction. This may open attractive new possibilities in medical treatment of male LUTS.
This study in α-chloralose anesthetized cats discovered an excitatory peroneal nerve-to-bladder reflex. A urethral catheter was used to infuse the bladder with saline and record bladder pressure changes. Electrical stimulation was applied to the superficial peroneal nerve to trigger a reflex bladder activity. With the bladder distended at a volume about 90% of bladder capacity, superficial peroneal nerve stimulation (PNS) at 1-3 Hz and threshold (T) intensity for inducing muscle twitching on the posterior thigh induced large amplitude (40-150 cmH2O) bladder contractions. PNS (1-3 Hz, 1-2T) applied during cystometrograms (CMGs) when the bladder was slowly (1-3 ml/min) infused with saline, significantly (p<0.01) reduced bladder capacity to about 80% of the control capacity and significantly (p<0.05) enhanced reflex bladder contractions. To determine the impact of PNS on tibial nerve stimulation (TNS) induced changes in bladder function, PNS was delivered following TNS. TNS of 30-minute duration produced long-lasting post-stimulation inhibition and significantly (p<0.01) increased bladder capacity to 140.5±7.6% of the control capacity. During the post-TNS inhibition period, PNS (1-3 Hz, 1-4T) applied during CMGs completely restored bladder capacity to the control level and significantly (p<0.05) increased the duration of reflex bladder contractions to about 200% of control. The excitatory peroneal nerve-to-bladder reflex could also be activated by transcutaneous PNS using skin surface electrodes attached to the dorsal surface of the foot. These results raise the possibility of developing novel neuromodulation therapies to treat underactive bladder and non-obstructive urinary retention.
Adenosine, a regulator of cardiovascular development and renal function, constricts renal afferent arterioles by inducing intracellular Ca2+ ([Ca2+]i) elevation in smooth muscle cells (SMCs) via activation of its cognate A1 receptors (A1Rs). Mechanisms that underlie A1R-dependent [Ca2+]i elevation in renal vascular SMCs are not fully resolved. Whether A1R expression and function in preglomerular microvessels are dependent on postnatal kidney maturation is also unclear. In this study, we show that selective activation of A1Rs by 2-chloro-N6-cyclopentyladenosine (CCPA) does not stimulate store-operated Ca2+ entry in afferent arterioles isolated from neonatal pigs. However, CCPA-induced [Ca2+]i elevation is dependent on phospholipase C and transient receptor potential cation channel, subfamily C, member 3 (TRPC3). Basal [Ca2+]i concentration was unchanged in afferent arterioles isolated from newborn (0-day-old) pigs when compared with their 20-day-old counterparts. By contrast, CCPA treatment resulted in significantly larger [Ca2+]i in afferent arterioles from 20-day-old pigs. A1R protein expression levels in the kidneys and afferent arterioles were unaltered in 0-day- versus 20-day-old pigs. However, TRPC3 channel protein expression level was ~ 92 and 78% higher in 20-day-old pig kidneys and afferent arterioles, respectively. These data suggest that activation of A1Rs elicits receptor-operated Ca2+ entry in porcine afferent arterioles, the level of which is dependent on postnatal maturation of TRPC3 channels. We propose that TRPC3 channels may contribute to the physiology and pathophysiology of A1Rs.
Important progress has been made on the cytokine signalling in response to kidney injury in the past decade, especially cytokine signalling mediated by extracellular vesicles (EVs). For example, EVs released by injured renal tubular epithelial cells (TECs) can regulate inter-cellular communications and influence tissue recovery via both regulating the expression and transferring cytokines, growth factors, as well as other bioactive molecules at the site of injury. The effects of EVs on kidney tissue seem to vary depending on the sources of EVs; however, the literature data are often inconsistent. For example, EVs derived from mesenchymal stem cells (MSC-EVs) and endothelial progenitor cells (EPC-EVs) were shown to have both beneficial and harmful effects on injured renal tissue in rodents. Cautions are thus needed on the interpretation of these data as contradictory findings on EVs may not only be related to the origin of EVs, they can also be caused by the different methods used for EV isolation, and the physiological and pathological state of the tissues/cells under which they were obtained. Here, we review and discuss our current understanding related to the immunomodulatory function of EVs in renal tubular repair in the hope to encourage further investigation on mechanisms related to both the anti-inflammatory and the reparative roles of EVs, and to better define the therapeutic potential of EVs in renal diseases.
Proteinuria has been reported in cancer patients receiving agents that target the transmembrane receptor neuropilin-1 (Nrp1) suggesting potential adverse effects on glomerular function. Here we show that Nrp1 is highly expressed by mesangial cells and that genetic deletion of the Nrp1 gene from PDGFR+ mesangial cells results in proteinuric disease and glomerulosclerosis, leading to renal failure and death within 6 weeks of age in mice. The major defect is a failure of mesangial cell migration that is required to establish the mature glomerular tuft. In vitro data show the potent chemotactic effect of PDGFB is lost in Nrp1-deficient mesangial cells. Biochemical analyses reveal that Nrp1 is required for PDGFB-dependent phosphorylation of p130Cas, a large scaffold molecule that is involved in motility of other cell types. In stark contrast, matrix adhesion and activation of Erk and Akt, which mediate proliferation of mesangial cells in response to PDGFB, are unaffected by the absence of Nrp1. Taken together these results identify a critical cell-autonomous role for Nrp1 in the migratory behavior of mesangial cells and may help explain the renal effects that occur in patients receiving Nrp1 inhibitory drugs.
The voiding spot assay (VSA) on filter paper is an increasingly popular method for studying lower urinary tract physiology in mice. However, the ways VSA are performed differ significantly between laboratories, and many variables are introduced compared to the mouse's normal housing situation. Rodents are intelligent social animals, and it is increasingly understood that social and environmental stresses have significant effects on their physiology. Surprisingly, little is known about whether change of environment during VSA affects mouse voiding, and what the best methodologies are for retaining 'natural' micturition patterns. It is well known that stress-related neuropeptide corticotropin-releasing factor (CRF) is significantly elevated and induces dramatic voiding changes when rodents encounter stresses. Therefore, we hypothesized that changes in the environmental situation could potentially alter voiding during VSA. We have examined multiple factors to test whether they affect female mouse voiding patterns during VSA, including cage type, cage floor, water availability, water bottle location, single or group housing, and different handlers. Our results indicate that mice are surprisingly sensitive to changes of cage type and floor surface, water bottle location, and single/group housing, each of which induces significant changes in voiding patterns, indicative of a stress response. In contrast, changing handler or four hours of water deprivation did not affect voiding patterns. Our data indicate that VSA should be performed under conditions as close as possible to the mouse's normal housing. Optimizing VSA methodology will be useful in uncovering voiding alterations in both genetic and disease models of lower urinary dysfunctions.
Afferent arteriole (Af-Art) resistance is modulated by 2 intrinsic nephron feedbacks: 1) vasoconstrictor tubuloglomerular feedback (TGF), which is mediated by NKCC2 in the macula densa and blocked by furosemide; and 2) vasodilator connecting tubule glomerular feedback (CTGF), which is mediated by ENaC in the connecting tubule and blocked by benzamil. High salt intake reduces the Af-Art vasoconstrictor ability in Dahl salt-sensitive rats (Dahl SS). Previously, we measured CTGF indirectly using the differences between the TGF responses with and without CTGF inhibition. We recently developed a method to measure CTGF more directly by simultaneously inhibiting NKCC2 and the Na/H exchanger (NHE). We hypothesize that during the simultaneous inhibition of NKCC2 and NHE in vivo, CTGF causes Af-Art dilatation, as reflected by an increase in stop-flow pressure (PSF) in Dahl SS that is enhanced with a high salt intake. In the presence of furosemide alone, increasing nephron perfusion did not change the PSF in either Dahl salt-resistant rats (Dahl SR) or in Dahl SS. When furosemide and the NHE inhibitor dimethylamiloride were perfused simultaneously, the increased tubular flow caused Af-Art dilatation, as demonstrated by increased PSF. This increase was greater in Dahl SS (4.5±0.4 mmHg) than in Dahl SR (2.5±0.3 mmHg; P < 0.01). CTGF causes this vasodilation since benzamil completely blocked this effect. However, a high salt intake did not augment Af-Art dilatation. We conclude that during the simultaneous inhibition of NKCC2 and NHE in the nephron, CTGF induces Af-Art dilatation, and a high salt intake failed to enhance this effect.
We tested whether rat descending vasa recta (DVR) undergo regulatory adaptions after the kidney is exposed to ischemia. Left kidneys (LK) were subjected to 30 minute renal artery cross clamp. After 48 hours, the post-ischemic LK and contralateral right kidney (RK) were harvested for study. When compared to DVR isolated from either sham operated LK or the contralateral RK, post-ischemic LK DVR markedly increased their NO generation. The selective inducible NOS (iNOS) inhibitor, 1400W, blocked the NO response. Immunoblots from outer medullary homogenates showed a parallel 2.6 fold increase in iNOS expression (P=0.01). Microperfused post-ischemic LK DVR exposed to angiotensin II (AngII, 10 nM), constricted less than those from the contralateral RK, and constricted more when exposed to 1400W (10 microM). Resting membrane potentials of pericytes from post-ischemic LK DVR pericytes were hyperpolarized relative to contralateral RK pericytes (62.0±1.6 vs 51.8±2.2 mV, respectively P<0.05) or those from sham operated LK (54.9±2.1 mV, P<0.05). Blockade of NO generation with 1400W did not repolarize post-ischemic pericytes (62.5±1.4 vs 61.1±3.4 mV), however control pericytes were hyperpolarized by exposure to NO donation from SNAP (51.5±2.9 to 62.1±1.4 mV, P<0.05). We conclude that post ischemic adaptations intrinsic to the DVR wall occur after ischemia. A rise in 1400W sensitive NO generation and iNOS expression occurs that is associated with diminished contractile responses to AngII. Pericyte hyperpolarization occurs that is not explained by the rise in ambient NO generation within the DVR wall.
Augmented intratubular angiotensin (ANG) II is a key determinant of enhanced distal Na+ reabsorption via activation of epithelial Na+ channels (ENaC) and other transporters, which leads to the development of high blood pressure (BP). In ANGII-induced hypertension, there is increased expression of the prorenin receptor (PRR) in the collecting duct (CD), which has been implicated in the stimulation of the sodium transporters and resultant hypertension. The impact of PRR deletion along the nephron on BP regulation and Na+ handling remains controversial. In the present study, we investigate the role of PRR in the regulation of renal function and BP using a mouse model with specific deletion of PRR in the CD (CDPRR-KO). At basal conditions, CDPRR-KO mice had decreased renal function and lower systolic BP associated with higher fractional Na+ excretion and lower ANGII levels in urine. After 14 days of ANGII infusion (400ng/kg/min), the increases in systolic BP were mitigated in CDPRR-KO mice. CDPRR-KO mice had lower abundance of cleaved αENaC and ENaC, as well as lower ANGII and renin content in urine compared to WT. In isolated CD from CDPRR-KO mice, patch clamp studies demonstrated that ANGII-dependent stimulation of ENaC activity was reduced due to fewer active channels and lower open probability. These data indicate that CD PRR contributes to renal function and BP responses during chronic ANGII infusion by enhancing renin activity, increasing ANGII, and activating ENaC in the distal nephron segments.
Recent evidence suggests that a greater density of pericytes in renal cadaveric allografts is associated with better recovery following transplant. The physiological mechanism(s) through which pericyte density may be beneficial is not well understood. The goal of this study was to test the hypothesis that lower medullary pericyte density is associated with greater renal injury following ischemia reperfusion (IR) in a rat model, providing a basis for future studies to better understand pericytes in a pathological environment. To test our hypothesis, we determined the association between medullary pericyte density and renal injury in spontaneously hypertensive rats (SHR) following 45 minutes of warm bilateral IR. We found that there was a significant negative relationship between pericyte density and plasma creatinine (slope=-0.03, p=0.02) and blood urea nitrogen (slope=-0.5, p=0.01) in female but not male SHR. Pericyte density was negatively associated with medullary peritubular capillary (PT) congestion in both sexes following IR (male: slope=-0.04, p=0.009; female: slope=-0.03, p=0.0001). To further test this relationship, we utilized a previously reported method to reduce pericyte density in SHR. Medullary erythrocyte congestion in vasa recta (VR) and PT significantly increased following IR in both sexes when pericyte density was pharmacologically decreased (VR: p=0.03; PT: p=0.03). Our data support the hypothesis that pericyte density is negatively associated with the development of IR injury in SHR which may be mediated by erythrocyte congestion in the medullary vasculature.
Urinary tract infection (UTI) is a broad term referring to an infection of the kidneys, ureters, bladder and/or urethra. Due to its prevalence, frequent recurrence and rising resistance to antibiotics, UTI has become a challenge in clinical practice. Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic disorder of the kidney and is characterized by the growth of fluid-filled cysts in both kidneys. Progressive cystic enlargement, inflammation and interstitial fibrosis result in nephron loss with subsequent decline in kidney function. ADPKD patients frequently develop UTI; however, the cellular and molecular mechanisms responsible for the high UTI incidence in ADPKD patients remain virtually unaddressed. Emerging evidence suggests that α-intercalated cells (α-ICs) of the collecting ducts function in the innate immune defense against UTI. α-ICs inhibit bacterial growth by acidifying urine and secreting neutrophil gelatinase-associated lipocalin (NGAL) that chelates siderophore-containing iron. It is necessary to determine, therefore, if ADPKD patients with recurrent UTI have a reduced number and/or impaired function of α-ICs. Identification of the underlying cellular and molecular mechanisms may lead to the development of novel strategies to reduce UTI in ADPKD.
Although all-trans-retinoic acid (ATRA) provides protection against a variety of conditions in vivo, particularly ischemia, the molecular mechanisms underpinning these effects remain unclear. The present studies were designed to assess potential mechanisms by which ATRA affords cytoprotection against renal toxicants in LLC-PK1 cells. Pretreatment of LLC-PK1 cells with ATRA (25 μM) for 24 hr afforded cytoprotection against oncotic cell death induced by p-aminophenol (PAP), 2-(glutathion-S-yl)hydroquinone (MGHQ), and iodoacetamide but not against apoptotic cell death induced by cisplatin. Inhibition of protein synthesis with cycloheximide blunted ATRA protection, indicating essential cell survival pathways must be engaged prior to toxicant exposure in order to provide cytoprotection. Interestingly, ATRA did not prevent the PAP-induced generation of ROS, nor did it alter glutathione levels. Moreover, ATRA had no significant effect on Nrf2 protein expression, and the Nrf2 inducers sulforaphane and MG132 did not influence ATRA cytoprotection suggesting cytoprotective pathways beyond those that influence ROS levels contribute to ATRA protection. In contrast, ATRA rapidly (15 min) induced levels of the cellular stress kinases p-ERK and p-AKT at concentrations of ATRA (10 and 25 μM) required for cytoprotection. Consistent with a role for p-ERK in ATRA-mediated cytoprotection, inhibition of p-ERK with PD98059 reduced the ability of ATRA to afford protection against PAP toxicity. Collectively, these data suggest that p-ERK and its downstream targets, independent of ROS and antioxidant signaling, are important contributors to the cytoprotective effects of ATRA against oncotic cell death.
The renal medulla, considered critical for the regulation of salt and water balance and long-term blood pressure control, is enriched in anandamide and two of its major metabolizing enzymes, cyclooxygenase-2 (COX-2) and fatty acid amide hydrolase (FAAH). Infusion of anandamide (15, 30 and 60 nmol/min/kg) into the renal medulla of C57BL/6J mice stimulated diuresis and salt excretion in a COX-2- but not COX-1-dependent manner. To determine if endogenous endocannabinoids in the renal medulla can elicit similar effects, the effects of intramedullary isopropyl dodecyl fluorophosphate (IDFP), which inhibits the two major endocannabinoid hydrolases, were studied. IDFP treatment increased the urine formation rate and sodium excretion in a COX-2- but not COX-1-dependent manner. Neither anandamide or IDFP affected the glomerular filtration rate. Neither systemic (0.625 mg/kg/30 min, intravenous) or intramedullary (15 nmol/min/kg/30 min) IDFP pretreatment prior to intramedullary anandamide (15-30 nmol/min/kg) strictly blocked effects of anandamide, suggesting that hydrolysis of anandamide was not necessary for its diuretic effect. Intramedullary IDFP had no effect on renal blood flow but stimulated renal medullary blood flow. The effects of IDFP on urine flow rate and medullary blood flow were FAAH-dependent as demonstrated using FAAH knockout mice. Analysis of mouse urinary prostaglandin E2 (PGE2) concentrations by high performance liquid chromatography-electrospray ionization tandem mass spectrometry showed that IDFP treatment decreased urinary PGE2. These data are consistent with a role of FAAH and endogenous anandamide acting through a COX-2-dependent metabolite to regulate diuresis and salt excretion in the mouse kidney.
Multiple vaginal parities have been reported to be an important risk factor for stress urinary incontinence (SUI). Simulated birth trauma with single vaginal distention (VD) has been used to induce the SUI condition in animals; however, the effect of multiple simulated birth traumas on the urethral continence function has not been well characterized. Therefore, we examined the effects of multiple VDs on urethral functions in vivo and the changes in gene expressions of several molecules in the urethra using female SD rats, which were divided into 3 groups; sham, VD-1 (single VD), and VD-3 groups (3 times of VDs every 2 weeks). Two weeks after the final VD, leak point pressure (LPP) and urethral responses during sneezing were evaluated. Also, changes in mRNA levels of urethral molecules were quantified with RT-PCR. The VD-1 group did not show any change in LPP with only a tendency of decrease in amplitudes of the urethral responses during sneezing (A-URS); however, the VD-3 group showed a significant decrease in LPP and urethral responses such as baseline urethral pressure and A-URS accompanied with SUI episodes during sneezing. Nicotinic receptor subtypes and TGF-β1 were significantly increased in both VD-1 and VD-3 groups while TNFR-1, IL-6, collagens and MMP-9 were significantly increased only in the VD-3 group. These data indicate that rats with multiple simulated birth traumas exhibit the profound impairment of urethral continence function and that these functional changes are associated with those in cytokines, extracellular matrix molecules and nicotinic receptor subtypes in the urethra.
Posterior urethral valves are the most common cause of partial bladder outlet obstruction (PBOO) in the pediatric population. Pathological changes in the bladder developed during PBOO are responsible for long-lasting voiding dysfunction in this population despite early surgical interventions. Increasing evidence showed PBOO induces an upregulation of Hypoxia inducible factors (HIFs) and their transcriptional target genes, and they play a role in pathophysiological changes in the obstructed bladders. We hypothesized that blocking HIF pathways can prevent PBOO-induced bladder dysfunction. PBOO was surgically created by ligation of the bladder neck in male C57BL/6J mice for 2 weeks. PBOO mice received intraperitoneal injection of either saline or 17-DMAG (Alvespimycin, 3mg/kg) every 48 h starting from day 1 post-surgery. Sham operated animals received injection of saline at the same schedule as PBOO mice and served as controls. The bladders were harvested after 2 weeks, and basal activity and evoked contractility of the detrusor smooth muscle (DSM) were evaluated in vitro. Bladder function was assessed in vivo by void spot assay and cystometry in conscious, unrestrained mice. Results indicated the 17-DMAG treatment preserved DSM contractility, and partially prevented the development of detrusor over activity in obstructed bladders. In addition, PBOO caused a significant increase in the frequency of micturition which was significantly reduced by 17-DMAG treatment. The 17-DMAG treatment improved urodynamic parameters, including increases in the bladder pressure at micturition and non-void contractions observed in PBOO mice. These results demonstrate that treatment with 17-DMAG, a HIF inhibitor significantly alleviated PBOO-induced bladder pathology in vivo.
The intrarenal autocrine-paracrine dopamine (DA) system mediates a significant fraction of the natriuresis in response to a salt load. DA inhibits a number of Na+ transporters to effect sodium excretion, including the proximal tubule Na+/H+ exchanger-3 (NHE3). DA represent a single hormone that regulates NHE3 at multiple levels including translation, degradation, endocytosis, and protein phosphorylation. Since cell surface NHE3 protein is determined by the balance between exocytotic insertion and endocytotic retrieval, we examined whether DA acutely affects the rate of NHE3 exocytosis in a cell culture model. DA inhibited NHE3 exocytosis at a dose-dependent manner with a half maximal around 10-6 M. The DA effect on NHE3 exocytosis was blocked by inhibition of protein kinase A (PKA) and by Brefeldin A, which inhibits endoplasmic reticulum-to-Golgi transport. NHE3 directly interacts with the coatomer protein COP based on yeast-two-hybrid and co-immunoprecipitation. Since NHE3 has been shown to be recycled back to the cell membrane after endocytosis, we measured NHE3 recycling using a biochemical re-insertion assay and showed that re-insertion of NHE3 back to the membrane is also inhibited by DA. In conclusion, amongst the many mechanisms by which DA reduce apical membrane NHE3 and induces proximal tubule natriuresis, one addition mechanism is inhibition of exocytotic insertion and re-insertion of NHE3 into the apical cell surface.
Intravesical Prostaglandin E2 (PGE2) was previously used to induce overactive bladder (OAB) symptoms as it reduces bladder capacity in rats and causes a "strong urgency sensation" in healthy women. However, the mechanism by which this occurs is unclear. To clarify how PGE2 reduces bladder capacity, 100 µM PGE2 was administered intravesically during open, single-fill cystometry with simultaneous measurement of sphincter EMG in the urethane-anesthetized female Wistar rat. PGE2 was also applied to the urethra or bladder selectively by use of a ligature at the bladder neck prior to (urethra) or during (bladder) closed-outlet, single-fill cystometry. Additional tests of urethral perfusion with PGE2 were made. PGE2 decreased bladder capacity, increased voiding efficiency, and increased sphincter EMG during open cystometry compared to saline controls. The number of non-voiding contractions did not change with PGE2, however, bladder compliance decreased. During closed-outlet cystometry, PGE2 applied only to the bladder or the urethra did not decrease bladder capacity. Urethral infusion of PGE2 decreased urethral perfusion pressure. Taken together, these results suggest that intravesical PGE2 may decrease bladder capacity by targeting afferents in the proximal urethra. This may occur through urethral relaxation and decreased bladder compliance, both of which may increase activation of proximal urethra afferents from distension of the proximal urethra. This hypothesis stands in contrast to many hypotheses of urgency that focus on bladder dysfunction as the primary cause of OAB symptoms. Targeting the urethra, particularly urethral smooth muscle, may be a promising avenue for the design of drugs and devices to treat OAB.
Myogenic response, a phenomenon in which resistance size arteries and arterioles swiftly constrict or dilate in response to an acute elevation or reduction, respectively in intravascular pressure is a key component of renal autoregulation mechanisms. Although it is well-established that the renal system is functionally immature in neonates, mechanisms that regulate neonatal renal blood flow (RBF) remain poorly understood. In this study, we investigated the hypothesis that members of the transient receptor potential vanilloid (TRPV) channels are molecular components of renal myogenic constriction in newborns. We show that unlike TRPV1-3, TRPV4 channels are predominantly expressed in neonatal pig preglomerular vascular smooth muscle cells (SMCs). Intracellular Ca2+ ([Ca2+]i) elevation induced by osmotic cell swelling was attenuated by TRPV4, L-type Ca2+, and stretch-activated Ca2+ channel blockers, but not phospholipase A2 inhibitor. Blockade of TRPV4 channels reversed steady-state myogenic tone and inhibited pressure-induced membrane depolarization, [Ca2+]i elevation, and constriction in distal interlobular arteries. A step increase in arterial pressure induced efficient autoregulation of renal cortical perfusion and total RBF in anesthetized and mechanically ventilated neonatal pigs. Moreover, intrarenal arterial infusion of TRPV4 channel blockers HC 067047 and RN 1734 attenuated renal autoregulation in the pigs. These data suggest that renal myogenic autoregulation is functional in neonates. Our findings also indicate that TRPV4 channels are mechanosensors in neonatal pig preglomerular vascular SMCs, and contribute to renal myogenic autoregulation.
Adult-onset autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either the PKD1 or PKD2 gene, leading to malfunction of their gene products, polycystin 1 or 2. Histone deacetylase 6 (HDAC6) expression and activity are increased in PKD1-mutant renal epithelial cells. Here we studied the effect of ACY-1215, a specific HDAC6 inhibitor, on cyst growth in ADPKD. Treatment with ACY-1215 slowed cyst growth in a mouse model of ADPKD that forms massive cysts within 3 weeks after knockout of polycystin 1 function. It also prevented cyst formation in MDCK.2 cells, an in vitro model of cystogenesis and in an ADPKD cell line derived from the proximal tubules from a pkd1-/-.mouse (PN cells). In PN cells ACY-1215 also reduced the size of already established cysts. We found that ACY-1215 lowered cAMP levels and protein expression of adenylyl cyclase 6. Our results suggest that HDAC6 could potentially serve as a therapeutic target in ADPKD.
In autosomal dominant polycystic kidney disease (ADPKD) activation of the renin-angiotensin aldosterone system (RAAS) may contribute to hypertension and disease progression. Although previous studies focused on circulating RAAS-components, preliminary evidence suggests APDKD may increase urinary RAAS-components. Therefore, our aim was to analyze circulating and urinary RAAS-components in ADPKD. We cross-sectionally compared 60 patients with ADPKD to 57 patients with non-ADPKD chronic kidney disease (CKD). The two groups were matched by gender, estimated glomerular filtration rate (eGFR), blood pressure, and RAAS-inhibitor use. Despite similar plasma levels of angiotensinogen and renin, urinary angiotensinogen and renin excretion were 5- to 6-fold higher in ADPKD (P<0.001). These differences persisted when adjusting for group differences, and were present regardless of RAAS-inhibitor use. In multivariable analyses, ADPKD, albuminuria, and the respective plasma concentrations were independent predictors for urinary angiotensinogen and renin excretion. In ADPKD, both plasma and urinary renin correlated negatively with eGFR. Total kidney volume correlated with plasma renin and albuminuria, but not with urinary renin or angiotensinogen excretions. Albuminuria correlated positively with urinary angiotensinogen and renin excretions in ADPKD and CKD. In three ADPKD patients who underwent nephrectomy, the concentrations of albumin and angiotensinogen were highest in plasma followed by cyst fluid and urine; urinary renin concentrations were higher than cyst fluid. In conclusion, this study shows that, despite similar circulating RAAS-component levels, higher urinary excretions of angiotensinogen and renin are a unique feature of ADPKD. Future studies should address the underlying mechanism and whether this may contribute to hypertension or disease progression in ADPKD.
We developed a mathematical model of calcium (Ca) and phosphate (PO4) homeostasis in the rat to elucidate the hormonal mechanisms that underlie the regulation of Ca and PO4 balance. The model represents the exchanges of Ca and PO4 between the intestine, plasma, kidneys, bone, and the intracellular compartment, and the formation of Ca-PO4-fetuin A complexes. It accounts for the regulation of these fluxes by parathyroid hormone (PTH), vitamin D3, fibroblast growth factor 23 (FGF23), and Ca2+-sensing receptors. Our results suggest that the Ca and PO4 homeostatic systems are robust enough to handle small perturbations in the production rate of either PTH or vitamin D3. The model predicts that large perturbations in PTH or vitamin D3 synthesis have a greater impact on the plasma concentration of Ca2+ ([Ca2+]p) than on that of PO4 ([PO4]p); due to negative feedback loops, [PO4]p does not consistently increase when the production rate of PTH or vitamin D3 is decreased. Our results also suggest that following a large PO4 infusion, the rapidly exchangeable pool in bone acts as a fast, transient storage PO4 compartment (on the order of minutes), whereas the intracellular pool is able to store greater amounts of PO4 over several hours. Moreover, a large PO4 infusion rapidly lowers [Ca2+]p owing to the formation of CaPO4 complexes. A large Ca infusion, however, has a small impact on [PO4]p since a significant fraction of Ca binds to albumin. This mathematical model is the first to include all major regulatory factors of calcium and phosphate homeostasis.
Consumption of cannabis and various related products (cannabinoids) for both medicinal and recreational use is gaining popularity. Furthermore, regulatory changes are fostering a cultural shift towards increasing liberalization of cannabis use, thereby increasing the likelihood of even larger numbers of individuals being exposed in the future. The two different types of receptors (CB1 and CB2) that are activated by the pharmacologically active ingredients of cannabis are found in numerous tissues, including the kidneys. Experimental studies suggest that stimulation of these receptors using pharmacologic agents or their naturally occurring ligands could have both deleterious and beneficial effects on the kidneys, depending on receptor distribution, type of renal insult, or the timing of the activation during acute or chronic states of kidney injury. To date, the mechanisms by which the CB1 or CB2 receptors are involved in the pathology of these renal conditions remains to be fully described. Furthermore, a better understanding of the impact of exo- and endo-cannabinoids on the renal endocannabinoid system may lead to the development of new drugs to treat kidney disease and its complications. Given the increasing public health relevance of cannabis exposure, it is clear that more research is necessary to clarify the various physiologic and pathophysiologic effects of cannabis and related analogs on the kidney. This will help limit the deleterious effects of these substances while promoting their potential beneficial impact on renal function in various types of kidney diseases.
Intercalated cells of the collecting duct (CD) are critical for acid-base homeostasis and innate immune defense of the kidney. Little is known about the impact of acidosis on innate immune defense in the distal nephron. Urinary tract infections are mainly due to Escherichia coli and are an important risk factor for development of chronic kidney disease. While the effect of urinary pH on growth of E. coli is well established, in this study we demonstrate that acidosis increases urine antimicrobial activity due, at least in part, to induction of cathelicidin expression within the CD. Acidosis was induced in rabbits by adding NH4Cl to the drinking water and reducing food intake over 3 days or by casein supplementation. Microdissected CDs were examined for cathelicidin mRNA expression and antimicrobial activity and cathelicidin protein levels in rabbit urine were measured. Cathelicidin expression in CD cells was detected in kidney sections. CDs from acidotic rabbits expressed three times more cathelicidin mRNA than those isolated from normal rabbits. Urine from acidotic rabbits had significantly more antimicrobial activity (versus E. coli) than normal urine and most of this increased activity was blocked by cathelicidin antibody. The antibody had little effect on antimicrobial activity of normal urine. Urine from acidotic rabbits had at least twice the amount of cathelicidin protein as did normal urine. We conclude that metabolic acidosis not only stimulates CD acid secretion but also induces expression of cathelicidin and thereby enhancing innate immune defense against urinary tract infections via induction of antimicrobial peptide expression.
Osr1 is a transcriptional repressor that plays critical roles in maintaining the mesenchymal stem cell population within the developing kidney. Here, we report that newborn pups with a heterozygous null mutation in Osr1 exhibit a 21% incidence of vesico-ureteric reflux and have hydronephrosis and urinary tract duplications. Newborn pups have a short intravesical ureter resulting in a less competent uretero-vesical junction which arises from a delay in urinary tract development. We describe a new domain of Osr1 expression in the ureteral mesenchyme and within the developing bladder in the mouse. OSR1 was sequenced in one hundred and eighty-six children with primary vesico-ureteric reflux and seventeen have single nucleotide polymorphisms. Fifteen children have a common synonymous variant, rs12329305, one child has a rare non-synonymous variant, rs3440471, and one child has a rare 5'UTR variant, rs45535040. The impact of these SNPs is not clear, therefore, the role of OSR1 in human disease remains to be elucidated. Osr1 is a candidate gene implicated in the pathogenesis of vesico-ureteric reflux and CAKUT in mice.
Hypertension is a complex, multifaceted disorder, affecting approximately 1 on 3 adults in the United States. Although hypertension occurs in both men and women, there are distinct sex differences in the way in which they develop hypertension, with women having a lower incidence of hypertension until the sixth decade of life. Despite observed sex differences in hypertension, little is known about the molecular mechanisms underlying the development of hypertension in females, primarily due to their underrepresentation in both clinical and experimental animal studies. The first goal of this review is to provide a concise overview of the participation of women in clinical trials, including a discussion of the importance of including females in basic science research, as recently mandated by the NIH. The remaining portion of the review is dedicated to identifying clinical and experimental animal studies that concentrate on gender and sex differences in hypertensive kidney disease, ending with a proposed role for T cells in mediating sex differences in blood pressure.
Tubulointerstitial fibrosis (TIF) is a prominent factor in the progression of chronic kidney disease (CKD) regardless of etiology. ErbB4 expression levels were inversely correlated to renal fibrosis in human fibrotic kidneys. In both unilateral ureteral obstruction (UUO) and ischemia reperfusion injury followed by uninephrectomy (IRI/UNx) mouse models, expression levels of ErbB4 were elevated in the early stage of renal injury. Using mice with global ErbB4 deletion except for transgenic rescue in cardiac tissue (ErbB4-/-ht+), we determined that UUO induced similar injury in proximal tubules compared to wild-type mice but more severe injury in distal nephrons. TIF was apparent earlier and was more pronounced following UUO in ErbB4-/-ht+ mice. With ErbB4 deletion, UUO injury inhibited AKT phosphorylation and increased the percentage of cells in G2/M arrest. There was also increased nuclear immunostaining of YAP and increased expression of p-Smad3, snail1 and vimentin. These results indicate that ErbB4 deletion accelerates the development and progression of renal fibrosis in obstructive nephropathy. Similar results were found in a mouse IRI/UNx model. In conclusion, increased expression of ErbB4 in the early stages of renal injury may reflect a compensatory effect to lessen tubulointerstitial injury.
Cisplatin is a potent chemotherapeutic used for the treatment of many types of cancer. However, its dose-limiting side effect is nephrotoxicity leading to acute kidney injury (AKI) (29, 30). Patients who develop AKI have an increased risk of mortality and are more likely to develop chronic kidney disease (CKD) (5, 14, 31). Unfortunately, there are no therapeutic interventions for the treatment of AKI. It has been suggested that the lack of therapies is due in part to the fact that the established mouse model used to study cisplatin-induced AKI does not recapitulate the cisplatin dosing regimen patients receive (41). In recent years, work has been done to develop more clinically-relevant models of cisplatin-induced kidney injury, with much work focusing on incorporation of multiple, low doses of cisplatin administered over a period of weeks. These models can be used to recapitulate the development of CKD after AKI, and by doing so, increase the likelihood of identifying novel therapeutic targets for the treatment of cisplatin-induced kidney injury.
In the renal proximal tubule the secretion and reabsorption of glomerularly filtrated compounds is realized by a functional network of uptake and efflux transporters. The activity and localization of several transporters expressed at the apical tubular membrane is regulated by the membrane associated protein PDZ domain containing 1 (PDZK1). We aimed to characterize the transcriptional regulation of this modulator of renal transport. Coexpression analyses of PDZK1 and putative regulators were performed using human kidney samples. Protein and mRNA expression of PDZK1 in renal proximal tubule epithelial cells after adenoviral transfer and siRNA knockdown of transcription factor hepatocyte nuclear factor 1 alpha (HNF1α) was assessed by quantitative real-time PCR and Western blot analysis. Transactivation of the PDZK1 promoter was quantified in cell-based reporter gene assays. Subsequently, the binding of HNF1α to the PDZK1 promoter was verified by in silico analyses and chromatin immunoprecipitation assay. HNF1α positively regulated the promoter activity of PDZK1. Adenoviral overexpression of HNF1α in renal proximal tubule epithelial cells (RPTEC) increased PDZK1 mRNA and protein expression, whereas siRNA knockdown of HNF1α resulted in decreased expression of PDZK1. Our results show that HNF1α, which has previously been described as a modulator of several transporters of the renal transportosome, is also a key determinant of PDZK1 transcription.
The renal collecting duct (CD) contains two major cell types, intercalated (ICs) and principal cells (PCs). A previous report showed that deletion of β-1 integrin in the entire renal CD causes defective CD morphogenesis resulting in kidney dysfunction. However, subsequent deletion of β-1 integrin specifically in ICs, and PCs respectively did not cause any morphological defects in the CDs. The discrepancy between these studies prompts us to reinvestigate the role of β-1 integrin in CD cells, specifically in the PCs. We conditionally deleted β-1 integrin in mouse CD PCs using a specific AQP2 promoter Cre-LoxP system. The resulting mutant mice, β-1f/fAQP2-Cre+ had lower body weight, failed to thrive and died around 8 to 12 weeks. Their CD tubules were dilated and some of them contained cellular debris. Increased apoptosis and proliferation of PCs were observed in the dilated CDs. Trichrome staining and electron microscopy revealed the presence of peritubular and interstitial fibrosis that is associated with increased production of extracellular matrix proteins including collagen type IV and fibronectin, as detected by immunoblotting. Further analysis revealed a significantly increased expression of TGF-β induced protein, fibronectin, and TGF-β receptor-1 mRNAs, and concomitantly increased phosphorylation of SMAD-2 that indicates the activation of the TGF-β signaling pathway. Therefore, our data reveals that normal expression of β-1 integrin in PCs is a critical determinant of CD structural and functional integrity, and further supports the previously reported critical role of β-1 integrin in the development and/or maintenance of the CD structure and function.
During the early phase of angiotensin (ANG) II-dependent hypertension tubular prostaglandin E2 (PGE2) is increased. Renin synthesis and secretion in the collecting duct (CD) is upregulated by ANGII contributing to further intratubular ANGII formation. However, what happens first and whether the triggering mechanism is independent of tubular ANGII, remain unknown. PGE2 stimulates renin synthesis in juxtaglomerular (JG) cells via E-prostanoid (EP) receptors through cAMP/CREB pathway. EP receptors are also expressed in the CD. Here, we tested the hypothesis that renin is upregulated by PGE2 in CD cells. M-1 CD cell line expressed EP1, EP3 and EP4 but not EP2. Dose response experiments in the presence of AT1 receptor blockade with candesartan demonstrated that 10-6 M PGE2 maximally increases renin mRNA (~4 fold) and prorenin/renin protein levels (~2 fold). This response was prevented by micromolar doses of SC-19220 (EP1 antagonist), attenuated by the EP4 antagonist, L-161,982, and exacerbated by the highly selective EP3 antagonist, L-798106 (~10 fold increase). To further evaluate the signaling pathway involved we used the PKC inhibitor calphostin C and transfections with PKCα dominant negative (DN). Both strategies blunted the PGE2-induced increases in cAMP levels, CREB phosphorylation and augmentation of renin. Knockdown of EP1 receptor and CREB also prevented renin upregulation. These results indicate that PGE2 increases CD renin expression through EP1 receptor via PKC/cAMP/CREB pathway. Therefore, we conclude that during early stages of ANGII-dependent hypertension, there is augmentation of PGE2 that stimulates renin in the CD, resulting in increased tubular ANGII formation and further stimulation of renin.
Systemic inflammation in end-stage renal disease (ESRD) is an established risk factor for mortality and a catalyst for other complications which are related to a premature aging phenotype, including muscle wasting, vascular calcification and other forms of premature vascular disease, depression, osteoporosis and frailty. Uremic inflammation is also mechanistically related to mechanisms involved in the aging process, such as telomere shortening, mitochondrial dysfunction, and altered nutrient sensing, which can have direct effect on cellular and tissue function. In addition to uremia-specific causes such as abnormalities in the phosphate- Klotho axis, there are remarkable similarities between the pathophysiology of uremic inflammation and so-called "inflammaging" in the general population. Potentially relevant, but still somewhat unexplored in this respect are abnormal or misplaced protein structures as well as abnormalities in tissue homeostasis, which evoke danger signals through damage associated molecular patters (DAMPS) as well as the senescence associated secretory phenotype (SASP). Systemic inflammation, in combination with the loss of kidney function, can impair the resilience of the body to external and internal stressors by reduced functional and structural tissue reserve, and by impairing normal organ crosstalk, thus providing an explanation for the greatly increased risk of homeostatic breakdown in this population. In this review, the relation between uremic inflammation and a premature aging phenotype, as well as potential causes and consequences are discussed.
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common monogenic hereditary disorders in humans characterized by fluid-filled cysts, primarily in the kidneys. Cux1, a cell cycle regulatory gene highly expressed during kidney development, is elevated in the cyst-lining cells of Pkd1 mutant mice, and in human ADPKD cells. However, forced expression of Cux1 is insufficient to induce cystic disease in transgenic mice, or to induce rapid cyst formation after cilia disruption in the kidneys of adult mice. Here we report a double mutant mouse model that has a conditional deletion of the Pkd1 gene in the renal collecting ducts together with a targeted mutation in the Cux1 gene (Pkd1CD;Cux1tm2Ejn). While kidneys isolated from newborn Pkd1CD mice exhibit cortical and medullary cysts, kidneys isolated from newborn Pkd1CD;Cux1tm2Ejn -/- mice did not show any cysts. Because, Cux1tm2Ejn -/- are perinatal lethal, we evaluated Pkd1CD mice that were heterozygote for the Cux1 mutation. Similar to the newborn Pkd1CD;Cux1tm2Ejn -/- mice, newborn Pkd1CD;Cux1tm2Ejn +/- mice did not show any cysts. Comparison of Pkd1CD and Pkd1CD;Cux1tm2Ejn +/- mice at later stages of development showed a reduction in the severity of PKD in the Pkd1CD;Cux1tm2Ejn +/- mice. Moreover, we observed an increase in expression of the cyclin kinase inhibitor p27, a target of Cux1 repression, in the rescued collecting ducts. Taken together, our results suggest that Cux1 expression in PKD is not directly involved in cystogenesis, but promotes cell proliferation required for expansion of existing cysts, primarily by repression of p27.
A low-protein diet (LPD) protects against the progression of renal injury in patients with chronic kidney disease (CKD). However, LPD may accelerate muscle wasting in these patients. Both exercise and branched chain amino acids (BCAA) are known to increase muscle protein synthesis by activating the mammalian target of rapamycin (mTOR) pathway. The aim of this study is to investigate whether endurance exercise and BCAA play a role for increasing muscle protein synthesis in LPD-fed CKD (5/6 nephrectomized) rats. Both CKD and sham rats were pair-fed on LPD or LPD fortified with a BCAA diet (BD), and approximately half animals in each group was subjected to treadmill exercise (15 m/min, 1 hour/day, 5 days/week). After 7 weeks, renal function was measured, and soleus muscles were collected to evaluate muscle-protein synthesis. Renal function did not differ between LPD- and BD-fed CKD rats, and the treadmill exercise did not accelerate renal damage in either group. The treadmill exercise slightly increased the phosphorylation of p70s6 kinase, a marker of mTOR activity, in the soleus muscle of LPD-fed CKD rats compared with the sham group. Furthermore, BCAA supplementation of the LPD-fed, exercise-trained CKD rats restored the phosphorylation of p70s6 kinase to the same level observed in the sham group; however, the corresponding induced increase in muscle-protein synthesis and muscle mass was marginal. These results indicate that the combination of treadmill exercise and BCAA stimulates cell signaling to promote muscle protein synthesis; however, the implications of this effect for muscle growth remain to be clarified.
Accumulating data indicate that renal uric acid (UA) handling is altered in diabetes and by hypoglycemic agents. In addition, hyperinsulinemia is associated with hyperuricemia and hypouricosuria. However, the underlying mechanisms remain unclear. In this study, we aimed to investigate how diabetes and hypoglycemic agents alter the levels of renal UA transporters. In insulin-depleted diabetic rats with streptozotocin treatment, both UA excretion and fractional excretion of UA (FEUA) were increased, suggesting that tubular handling of UA is altered in this model. In the membrane fraction of the kidney, the expression of urate transporter 1 (URAT1) was significantly decreased, whereas that of ATP-binding cassette sub-family G member 2 (ABCG2) was increased, consistent with the increased renal UA clearance. Administration of insulin to the diabetic rats, but not of the SGLT2 inhibitor ipragliflozin, decreased UA excretion and alleviated UA transporter level changes. To confirm the contribution of insulin in the regulation of urate transporters, normal rats received insulin and separately ipragliflozin. Insulin significantly increased URAT1 and decreased ABCG2 levels, resulting in increased UA reabsorption. In contrast, SGLT2 inhibitor did not alter URAT1 or ABCG2 levels, although blood glucose levels were similarly reduced. Furthermore, we found that insulin significantly increased endogenous URAT1 levels in the membrane fraction of NRK-52E cells, the kidney epithelial cell line, demonstrating the direct effects of insulin on renal UA transport mechanisms. These results suggest a previously unrecognized mechanism for the anti-uricosuric effects of insulin, and provide novel insights into the renal UA handling in the diabetic state.
Sex and age influence susceptibility to acute kidney injury (AKI), with young females exhibiting lowest incidence. In these studies, we investigated mechanisms which may underlie the sex/age based dissimilarities. Cisplatin (Cp)-induced AKI resulted in morphological evidence of injury in all groups. A minimal rise in plasma creatinine (PCr) was seen in Young Females, while in Aged Females, PCr rose precipitously. Relative to Young Males, Aged Males showed significantly, but temporally, comparably elevated PCr. Notably, Aged Females showed significantly greater mortality, while Young Females exhibited none. Tissue KIM-1 and plasma NGAL were significantly lower in Young Females than all others. IGFBP7 levels were modestly increased in both Young groups. IGFBP7 levels in Aged Females were significantly elevated at baseline relative to Aged Males, and increased linearly through day 3, when these levels were comparable in both Aged groups. Plasma cytokine levels similarly showed a pattern of protective effects preferentially in Young Females. Expression of the drug transporter MATE2 did not explain the sex/age distinctions. Heme oxygenase-1 (HO-1) levels (~28kD species) showed elevation at day 1 in all groups with highest levels seen in Young Males. Exclusively in Young Females, these levels returned to baseline on day 3, suggestive of a more efficient recovery. In aggregate, we demonstrate, for the first time, a distinctive pattern of response to AKI in Young Females relative to males which appears to be significantly altered in aging. These distinctions may offer novel targets to exploit therapeutically in both females and males in the treatment of AKI.
Female spontaneously hypertensive rats (SHR) have more renal regulatory T cells (Tregs) than males, and greater levels of Tregs in female SHR is dependent on blood pressure (BP). However, the molecular mechanism responsible for greater Tregs in female SHR is unknown. Transforming growth factor (TGF)-β is a pleiotropic cytokine critical in the differentiation of naïve T cells into Tregs, and female SHR have higher TGF-β excretion than male SHR. The goals of the current study were to test the hypotheses that 1) female SHR have greater renal TGF-β expression than male SHR which is dependent on BP and 2) neutralizing TGF-β will decrease renal Tregs in female SHR. Renal cortices were isolated from 5 and 13 week old male and female SHR and TGF-β levels were measured via Western blot and ELISA. Adult female SHR have more free, active TGF-β1 than 5 week old female SHR (46% more) or male SHR (44% more than 5 week old males and 56% more than 13 week old male SHR). We confirmed greater TGF-β1 in adult female SHR was due to increases in BP and not sexual maturation by measuring TGF-β1 levels following treatment with BP lowering drugs or ovariectomy. Separate female SHR were treated with an antibody to TGF-β1,2,3; BP was measured and T cells were assessed in whole blood and the kidney. Neutralizing TGF-β had no effect on BP, although circulating Tregs decreased by 32% while Th17 cells increased by 64%. Renal Tregs were not altered by antibody treatment, although Th17 cells were decreased by 61%. In conclusion, although TGF-β promotes circulating Tregs in female SHR, it does not account for the sex difference in renal Tregs in SHR.
Exosomes are nano-sized vesicles produced and secreted by cells to mediate intercellular communication. The production and function of exosomes in kidney tissues and cells remain largely unclear. Hypoxia is a common patho-physiological condition in kidneys. This study was designed to characterize exosome production during hypoxia of rat renal proximal tubular cells (RPTC), investigate the regulation by hypoxia-inducible factor-1 (HIF-1), and determine the effect of the exosomes on ATP-depletion induced tubular cell injury. Hypoxia did not change the average sizes of exosomes secreted by RPTC cells, but it significantly increased exosome production in a time-dependent manner. HIF-1 induction with dimethyloxalyl glycine (DMOG) also promoted exosome secretion, whereas pharmacologic and genetic suppression of HIF-1 abrogated the increase of exosome secretion under hypoxia. The exosomes from hypoxic RPTC cells had inhibitory effects on apoptosis of RPTC cells following ATP-depletion. The protective effects were lost in the exosomes from HIF-1α knockdown cells. It is concluded that hypoxia stimulates exosome production and secretion in renal tubular cells. The exosomes from hypoxic cells are protective against renal tubular cell injury. HIF-1 mediates exosome production during hypoxia and contributes to the cytoprotective effect of the exosomes.
The early events that signal renal dysfunction in presymptomatic heart failure are unclear. We tested the hypothesis that functional and mechanistic changes occur in the kidney that precede the development of symptomatic heart failure. We employed a transgenic mouse model with cardiomyocyte-specific overexpression of mutant alpha-B-crystallin that develops slowly progressive cardiomyopathy. Presymptomatic transgenic mice displayed an increase in serum creatinine (1.17 ± 0.34 versus wild type 0.65 ± 0.16 mg/dL, p<0.05), and in urinary neutrophil gelatinase-associated lipocalin (NGAL, 278.92 ± 176.24 versus wild type 49.11 ± 22.79 ng/mL, p<0.05), but no renal fibrosis. Presymptomatic transgenic mouse kidneys exhibited a two-fold upregulation of the Ren1 gene, marked over-expression of renin protein in the tubules, and a worsened response to ischemia-reperfusion injury based on serum creatinine (2.77 ± 0.66 in transgenic mice versus 2.01 ± 0.58 mg/dL in wild type, p<0.05), urine NGAL (9198.79 ± 3799.52 in transgenic mice versus 3252.94 ± 2420.36 ng/mL in wild type, p<0.05), tubule dilation score (3.4 ± 0.5 in transgenic mice versus 2.6 ± 0.5 in wild type, p<0.05), tubule cast score (3.2 ± 0.4 in transgenic mice versus 2.5 ± 0.5 in wild type, p<0.05), and Tunel-positive nuclei (10.1 ± 2.1 in the transgenic group versus 5.7 ± 1.6 per 100 cells counted in wild type, p<0.01). Our findings indicate functional renal impairment, urinary biomarker elevations, and induction of renin gene and protein expression in the kidney that occur in early presymptomatic heart failure, which increase the susceptibility to subsequent acute kidney injury.
Via developmental programming, prenatal perturbations, such as exposure to glucocorticoids and maternal malnutrition alter kidney development and contribute to the development of hypertension. To examine the possibility that alterations in tubuloglomerular feedback (TGF) contribute to the development of hypertension in offspring following maternal dexamethasone treatment (Dex) in early gestation, studies were conducted in fetal sheep and lambs. Pregnant ewes were infused with dexamethasone (0.48 mg/h) at 26-28 days gestation. No differences were observed in mean arterial pressure, glomerular filtration rate or electrolyte excretion rates between the Dex and untreated fetuses or lambs. Gestational exposure to Dex markedly enhanced TGF sensitivity, as the turning point in Dex treated fetuses was significantly lower (12.9±0.9 nl/min; P<0.05) compared to untreated fetuses (17.0±1.0 nl/min). This resetting of TGF sensitivity persisted after birth (P<0.01). TGF reactivity did not differ between the groups in fetuses or lambs. In response to nitric oxide inhibition, TGF sensitivity increased (the turning point decreased) and reactivity increased in untreated fetuses and lambs, but these effects were blunted in the Dex treated fetuses and lambs. Our data suggest that an altered TGF response may be an underlying renal mechanism contributing to the development of hypertension in the Dex model of fetal programming. The lower tonic level of NO production in these dexamethasone exposed offspring may contribute to the development of hypertension as adults.
Mitochondrial dysfunction is increasingly recognized as an important factor in glomerular diseases. Previous study showed that mitochondrial fission contributed mitochondrial dysfunction. However, the mechanism of mitochondrial fission on mitochondrial dysfunction in aldosterone-induced podocyte injury remains ambiguous. This study aimed to investigate the pathogenic effect of mitochondrial fission both in vivo and in vitro. In an animal model of aldosterone-induced nephropathy, inhibition of the mitochondrial fission protein Drp1 (dynamin-related protein 1) suppressed aldosterone-induced podocyte injury. In cultured podocytes, aldosterone dose-dependently induced Drp1 expression. Knockdown of Drp1 inhibited aldosterone-induced mitochondrial fission, mitochondrial dysfunction and podocyte apoptosis. Furthermore, aldosterone dose-dependently induced p53 expression. Knockdown of p53 inhibited aldosterone-induced Drp1 expression, mitochondrial dysfunction and podocyte apoptosis. These findings implicated that aldosterone-induced mitochondrial dysfunction and podocyte injury mediated by p53/Drp1-dependent mitochondrial fission, which may provide opportunities for therapeutic intervention for podocyte injury.
We examined bladder and urethral sphincter activity in mice with or without spinal cord injury (SCI) after C-fiber afferent desensitization induced by capsaicin pretreatment and changes in electrophysiological properties of mouse bladder afferent neurons 4 weeks after SCI. Female C57BL/6N mice were divided into 4 groups; (1) spinal intact (SI)-control, (2) SI-capsaicin pretreatment (Cap), (3) SCI-control and (4) SCI-Cap groups. Continuous cystometry and external urethral sphincter (EUS)-electromyogram (EMG) were conducted under an awake condition. In Cap groups, capsaicin (25, 50 or 100 mg/kg) was injected subcutaneously 4 days before experiments. In SI-Cap group, 100 mg/kg capsaicin pretreatment significantly increased bladder capacity and decreased the silent period duration of EUS-EMG compared to SI-control group. In SCI-Cap group, 50 and 100 mg/kg capsaicin pretreatment respectively decreased the number of non-voiding contractions (NVCs), and the duration of reduced EUS activity during voiding compared to SCI-control group. In SCI mice, hexamethonium, a ganglionic blocker, almost completely blocked NVCs, suggesting that they are of neurogenic origin. Patch clamp recordings in capsaicin-sensitive bladder afferent neurons from SCI mice showed hyperexcitability evidenced by decreased spike thresholds and increased firing rate compare to SI mice. These results indicate that capsaicin-sensitive C-fiber afferent pathways, which become hyperexcitable after SCI, can modulate bladder and urethral sphincter activity in awake SI and SCI mice. Detrusor overactivity as shown by NVCs in SCI mice is significantly, but partially, dependent on capsaicin-sensitive C-fiber afferents; whereas the EUS relaxation during voiding is enhanced by capsaicin-sensitive C-fiber bladder afferents in SI and SCI mice.
Primary cilia have been called "the forgotten organelle" for over 20 years. As cilia now have their own Journal and several books devoted to their study, perhaps it's time to reconsider the moniker "forgotten organelle". In fact, during the drafting of this review, 12 relevant publications have been issued- we therefore apologize in advance for any relevant work we inadvertently omitted. What purpose is yet another ciliary review? The primary goal of this review is to specifically examine the evidence for and against the hypothesized flow-sensing function of primary cilia expressed by differentiated epithelia within a kidney tubule, bringing together differing disciplines and their respective conceptual and experimental approaches. We will show that understanding the biophysics/biomechanics of primary cilia provides essential information for understanding any potential role of ciliary function in disease. We will summarize experimental and mathematical models used to characterize renal fluid flow and incident force on primary cilia, to characterize the mechanical response of cilia to an externally applied force and discuss possible ciliary-mediated cell signaling pathways triggered by flow. Throughout, we stress the importance of separating the effects of fluid shear and stretch from the action of hydrodynamic drag.
Obesity is a global epidemic associated with an increased risk for lower urinary tract dysfunction. Inefficient voiding and urinary retention may arise in late-stage obesity when the expulsive force of the detrusor smooth muscle cannot overcome outlet resistance. Detrusor underactivity (DUA) and impaired contractility may contribute to the pathogenesis of non-obstructive urinary retention. We used cystometry and electrical stimulation of peripheral nerves (pudendal and pelvic n.) to characterize and improve bladder function in urethane-anesthetized obese-prone (OP) and obese-resistant (OR) rats following diet-induced obesity (DIO). OP rats exhibited urinary retention and impaired detrusor contractility following DIO, reflected as increased volume threshold, decreased peak micturition pressure, and decreased voiding efficiency (VE) compared to OR rats. Electrical stimulation of the sensory branch of the pudendal nerve did not increase VE, whereas patterned bursting stimulation of the motor branch of the pudendal nerve increased VE two-fold in OP rats. OP rats required increased amplitude of electrical stimulation of the pelvic nerve to elicit bladder contractions and maximum evoked bladder contraction amplitudes were decreased relative to OR rats. Collectively, these studies characterize a novel animal model of DUA that can be used to determine pathophysiology and suggest that neuromodulation is a potential management option for DUA.
Our previous study demonstrated that the abundance of extracellular matrix proteins was suppressed by store-operated Ca2+ entry in mesangial cells (MCs). The present study was conducted to investigate the underlying mechanism focused on the transforming growth factor beta 1 (TGFβ1) - Smad3 pathway, a critical pathway for ECM expansion in diabetic kidneys. We hypothesized that SOCE suppressed ECM protein expression by inhibiting this pathway in MCs. In cultured human MCs, we observed that TGFβ1 (5 ng/ml for 15 hours) significantly increased Smad3 phosphorylation as evaluated by immunoblot. However, this response was markedly inhibited by thapsigargin (1 µM), a classical activator of store-operated Ca2+ channel. Consistently, both immunocytochemistry and immunoblot showed that TGFβ1 significantly increased nuclear translocation of Smad3 which was prevented by pre-treatment with thapsigargin. Importantly, the thapsigargin effect was reversed by Lanthanum (La3+) (5 µM) and GSK-7975A (10 µM), both of which are selective blockers of store-operated Ca2+ channel. Furthermore, knockdown of Orai1, the pore-forming subunit of store-operated Ca2+ channel, significantly augmented TGFβ1-induced Smad3 phosphorylation. Overexpression of Orai1 augmented the inhibitory effect of thapsigargin on TGFβ1-induced phosphorylation of Smad3. In agreement with the data from cultured MCs, in vivo knockdown of Orai1 specific to MCs using a targeted nanoparticle siRNA delivery system resulted in marked increase in abundance of phosphorylated Smad3 and in nuclear translocation of Smad3 in glomerulus of mice. Taken together, our results indicate that store-operated Ca2+ entry in MCs negatively regulates the TGFβ1-Smad3 signaling pathway.
Overactive bladder (OAB) syndrome is a highly prevalent condition that may lead to medical complications and decreased quality of life. Emerging therapies focusing on selective electrical stimulation of peripheral nerves associated with lower urinary tract (LUT) function may provide improved efficacy and reduced side effects compared to sacral neuromodulation for the treatment of OAB symptoms. Prior studies investigating the effects of pelvic nerve (PelN) stimulation on LUT function were focused on promoting bladder contractions, and it is unclear if selective stimulation of the PelN would be beneficial for the treatment of OAB. Therefore, our motivation was to test the hypothesis that PelN stimulation would increase bladder capacity in the prostaglandin E2 (PGE2) rat model of OAB. The effects of intravesical PGE2 vs. vehicle and PelN stimulation after intravesical PGE2 on cystometric parameters were quantified in 17 urethane-anesthetized female Sprague-Dawley rats. Intravesical infusion of PGE2 resulted in decreased bladder capacity and increased voiding efficiency without a change in bladder contraction area under the curve, maximum contraction pressure, or contraction duration. Bladder capacity was also significantly decreased compared to vehicle confirming that the change in bladder capacity was mediated by PGE2. PelN stimulation reversed the PGE2-induced change in bladder capacity and increased the EUS EMG activity at a specific stimulation condition (1.0T amplitude at 10 Hz). These results confirm that the urodynamic changes reported in conscious rats are also observed under urethane anesthesia and that PelN stimulation is a novel and promising approach for the treatment of the symptoms of OAB.
The failure of the polycystins (PCs) to function in primary cilia is thought to be responsible for Autosomal Dominant Polycystic Kidney Disease (ADPKD). Primary cilia integrate multiple cellular signaling pathways, including calcium, cAMP, Wnt, and Hedgehog, which control cell proliferation and differentiation. It has been proposed that mutated PCs result in reduced intracellular calcium, which in turn upregulates cAMP, protein kinase A (PKA) signaling, and subsequently other proliferative signaling pathways. However, the role of PKA in ADPKD has not been directly ascertained in vivo, although the expression of the main regulatory subunit of PKA in cilia and other compartments (PKA-RIα, encoded by PRKAR1A) is increased in a mouse model orthologous to ADPKD. Therefore, we generated a kidney specific knockout of Prkar1a to examine the consequences of constitutive upregulation of PKA on wild-type and Pkd1 hypomorphic (Pkd1RC) backgrounds. Kidney specific loss of Prkar1a induced renal cystic disease and markedly aggravated cystogenesis in the Pkd1RC models. In both settings, it was accompanied by upregulation of Src, Ras, MAPK/ERK, mTOR, CREB, STAT3, Pax2 and Wnt signaling. On the other hand, Gli3 repressor activity was enhanced, possibly contributing to hydronephrosis and impaired glomerulogenesis in some animals. To assess the relevance of these observations in humans we looked for and found evidence for kidney and liver cystic phenotypes in the Carney complex, a tumoral syndrome caused by mutations in PRKAR1A. These observations expand our understanding of the pathogenesis of ADPKD and demonstrate the importance of PRKAR1A highlighting PKA as a therapeutic target in ADPKD.
Src family kinases (SFKs) belong to non-receptor protein tyrosine kinases (PTKs) and have been implicated in the regulation of numerous cellular processes, including cell proliferation, differentiation, migration and invasion, angiogenesis. The role and mechanisms of SFKs in tumorgenesis have been extensively investigated and some of SFK inhibitors are currently under clinical trials for tumor treatment. Recent studies have also demonstrated the importance of SFKs in regulating the development of various fibrosis related chronic diseases (e.g., idiopathic pulmonary fibrosis, liver fibrosis, renal fibrosis, and systemic sclerosis). In this article, we summarize the roles of SFKs in various chronic kidney diseases, including glomerulonephritis, diabetic nephropathy, HIV-associated nephropathy, ADPKD and obesity associated kidney disease and discuss the mechanisms involved.
Lithium is the mainstay treatment for patients with bipolar disorder, but generally causes nephrogenic diabetes insipidus (NDI), a disorder in which the renal urine concentrating ability has become vasopressin-insensitive. Li-NDI is caused by lithium uptake by collecting duct principal cells and downregulation of AQP2 water channels, which are essential for water uptake from pro-urine. Recently, we found that the prophylactic administration of acetazolamide to mice effectively attenuated Li-NDI. To evaluate whether acetazolamide might benefit lithium-treated patients, we administered acetazolamide to mice with established Li-NDI and six patients with a lithium-induced urinary concentrating defect. In mice, acetazolamide partially reversed lithium-induced polyuria and increased urine osmolality, which, however, did not coincide with increased AQP2 abundances. In patients, acetazolamide led to the withdrawal of two patients from the study due to side effects. In the four remaining patients acetazolamide did not lead to clinically relevant changes in maximal urine osmolality. Urine output was also not affected, although none of these patients demonstrated overt lithium-induced polyuria. In three out of four patients, acetazolamide treatment increased serum creatinine levels, indicating a decreased GFR. Strikingly, these three patients also showed a decrease in systemic blood pressure. Altogether, our data reveal that acetazolamide does not improve the urinary concentrating defect caused by lithium, but lowers the GFR, likely explaining the reduced urine output in our mice and in a recently reported patient with lithium-induced polyuria. The reduced GFR in patients prone to CKD development, however, warrants against application of acetazolamide in Li-NDI patients without long-term (pre)clinical studies.
The OK cell line derived from kidney of a female opossum Didelphis virginiana has proven to be a useful model in which to investigate the unique regulation of ion transport and membrane trafficking mechanisms in the proximal tubule (PT). Sequence data and comparison of the transcriptome of this cell line to eutherian mammal PTs would further broaden the utility of this culture model. However, genomic sequence for Didelphis virginiana is not available and although a draft genome sequence for the opossum Monodelphis domestica (sequenced in 2012 by the Broad Institute) exists, transcripts sequenced from both species show significant divergence. The Monodelphis domestica sequence is not highly annotated and the majority of transcripts are predicted rather than experimentally validated. Using deep RNA sequencing of the Didelphis virginiana OK cell line we characterized its transcriptome via de novo transcriptome assembly and alignment to the Monodelphis domestica genome. The quality of the de novo assembled transcriptome was assessed by the extent of homology to sequences in nucleotide and protein databases. Gene expression levels in the OK cell line, from both the de novo transcriptome and genes aligned to the Monodelphis domestica genome, were compared to publicly available rat kidney nephron segment expression data. Our studies demonstrate the expression in OK cells of numerous PT specific ion transporters and other key proteins relevant for rodent and human PT function. Additionally, the sequence and expression data reported here provide an important resource for genetic manipulation and other studies on PT cell function using these cells.
Both the incidence and prevalence of chronic kidney disease are increasing in the elderly population. Although aging is known to induce kidney injury, the underlying molecular mechanisms remain unclear. Sirtuin 1 (Sirt1), a longevity gene, is known to protect kidney cell injury from various cellular stresses. In previous studies, we showed that the podocyte-specific loss of Sirt1 aggravates diabetic kidney injury. However, the role of Sirt1 in aging-induced podocyte injury is not known. Therefore, in this study we sought to determine the effects of podocyte-specific reduction of Sirt1 in age-induced kidney injury. We employed the inducible podocyte-specific Sirt1 knockdown mice that express shRNA against Sirt1 (Pod-Sirt1RNAi) and control mice expressed shRNA for luciferase (Pod-LuciRNAi). We found that reduction of podocyte Sirt1 led to aggravated aging-induced glomerulosclerosis and and albuminuria. In addition, urinary level of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative stress, was markedly increased in aged Pod-Sirt1RNAi mice compared to aged Pod-LuciRNAi mice. Although podocyte-specific markers decreased in aged mice compared to the young controls, the decrease was further exacerbated in aged Pod-Sirt1RNAi compared to Pod-LuciRNAi mice. Interestingly, expression of cellular senescence markers was significantly higher in the glomeruli of Pod-Sirt1RNAi mice than Pod-LuciRNAi mice, suggesting that cellular senescence may contribute to podocyte loss in aging kidneys. Finally, we confirmed that Pod-Sirt1RNAi glomeruli were associated with reduced activation of transcription factors, PGC1α/PPAR, FOXO3, FOXO4, and p65 NF-B, through SIRT1-mediated deacetylation. Together, our data suggest that SIRT1 may be a potential therapeutic target to treat patients with aging-related kidney disease.
OBJECTIVE Several studies reported gender differences in aldosterone. It is unknown whether these differences are associated with differences in volume regulation. Therefore, we studied both aldosterone and extracellular volume in men and women on different sodium intakes. METHODS In healthy normotensive men (n=18) and premenopausal women (n=18) we investigated plasma aldosterone, blood pressure, and extracellular volume (125I-iothalamate), during both a low (target intake 50 mmol Na+/day) and high sodium intake (target intake 200 mmol Na+/day) in a cross-over set-up. Furthermore, we studied the adrenal response to angiotensin II infusion (0.3, 1.0 and 3.0 ng/kg/min for 1 h) on both sodium intakes. RESULTS Men had a significantly higher plasma aldosterone, extracellular volume and systolic blood pressure than women during a high sodium intake (p<0.05). During a low sodium intake, extracellular volume and blood pressure were higher in men as well (p<0.05), whereas the difference in plasma aldosterone was no longer significant (P=0.252). The adrenal response to exogenous angiotensin II was significantly lower in men than in women on both sodium intakes. CONCLUSIONS Constitutive gender differences in the regulation of aldosterone, characterized by a higher aldosterone and a lower adrenal response to exogenous angiotensin II infusion in men, are associated with a higher extracellular volume and blood pressure in men. These findings suggest that gender differences in the regulation of aldosterone contribute to differences in volume regulation between men and women.
Bladder outlet obstruction (BOO) triggers inflammation in the bladder through the NLRP3 inflammasome. BOO also activates fibrosis, which is largely responsible for the decompensation of the bladder in the chronic state. Because fibrosis can be driven by inflammation, we have explored a role for NLRP3 (and IL-1β produced by NLRP3) in the activation and progression of BOO-induced fibrosis. Female rats were divided into 5 groups: 1) control, 2) sham, 3) BOO + Vehicle, 4) BOO + the NLRP3 inhibitor glyburide or 5) BOO + the IL-1β receptor antagonist anakinra. Fibrosis was assessed by Masson's Trichrome Stain, collagen secretion via Sirius Red, and protein localization by immunofluorescence. BOO increased collagen production in the bladder which was blocked by glyburide and anakinra, clearly implicating the NLRP3/IL-1β pathway in fibrosis. The collagen was primarily found in the lamina propria and the smooth muscle, while IL-1 receptor 1 and prolyl 4-hydroylase (an enzyme involved in the intracellular modification of collagen) both localized to the urothelium and the smooth muscle. Lysyl oxidase, the enzyme involved in the final extracellular assembly of mature collagen fibrils, was found to some extent in the lamina propria where its expression was greatly enhanced during BOO. In vitro studies demonstrated isolated urothelial cells from BOO rats secreted substantially more collagen than controls, and collagen expression in control cultures could be directly stimulated by IL-1β. In summary, NLRP3-derived IL-1β triggers fibrosis during BOO, most likely through an autocrine loop in which IL-1β acts on urothelia to drive collagen production.
The cotransporter SGLT2 is responsible for 90% of the renal glucose reabsorption and we recently shown that MAP17 appears to work as a required beta subunit. We report in the present paper a detailed functional characterisation of human SGLT2 in co-expression with human MAP17 in Xenopus laevis oocytes. Addition of external glucose generates a large inward current in the presence of Na confirming an electrogenic transport mechanism. At a membrane potential of -50 mV, SGLT2 affinity constants for glucose and Na are 3.4 ±0.4 mM and 18 ± 6 mM, respectively. The change in the reversal potential of the cotransport current as a function of external glucose concentration clearly confirms a 1Na:1glucose transport stochiometry. SGLT2 is selective for glucose and alpha-methylglucose but it also transports, to a lower extent, galactose and 3-O-methyl-glucose. SGLT2 can be inhibited in a competitive manner by phlorizin (Pz, Ki=31 ±4 nM ) and by dapagliflozin (Ki= 0.75 ±0.3 nM). Similarly to SGLT1, SGLT2 can be activated by Na, Li and protons. Presteady-state currents for SGLT2 do exist but they are small in amplitude and relatively fast (a time constant of ~2 ms). The leak current defined as the Pz-sensitive current in the absence of substrate was extremely small in the case of SGLT2. In summary, in comparison to SGLT1, SGLT2 has a lower affinity for glucose, a transport stoichiometry of 1:1, very small pre-steady-state and leak currents, a 10 fold higher affinity for phlorizin and an affinity for dapagliflozin in the sub nM range.
We studied gender differences in NCC activity and expression in wild-type (WT) and AT1a receptor knockout (KO) mice. In renal clearance experiments, urine volume (UV), GFR, absolute Na and K (ENa; EK) and fractional Na and K (FENa; FEK) excretion were measured and compared at peak changes after bolus iv injection of hydrochlorothiazide (HCTZ; 30mg/kg). In WT, females responded more strongly than males to HCTZ, with larger fractional increases of UV (7.8- vs. 3.4-fold), ENa (11.7- vs. 5.7-fold), FENa (7.9-vs. 4.9-fold) and EK (2.8- vs. 1.4-fold). In contrast, there were no gender differences in the responses to the diuretic in KO mice; HCTZ produced greater effects on male KO than on WT, but similar effects on females. In WT, total (tNCC) and phosphorylated (pNCC) NCC protein expression were 1.8- fold and 4.6- fold higher in females compared to the males (P<0.05), consistent with the larger response to HCTZ. In KO mice, tNCC and pNCC increased significantly in males, to levels not different from those in females. There were no gender differences in the expression of the Na/H-Exchanger (NHE3) expression in WT; NHE3 protein decreased to similar extents in male and female KO animals, suggesting AT1a-mediated NHE3 expression in proximal tubules. The resulting increase in delivery of NaCl to the distal nephron may underlie increased NCC expression and activity in mice lacking AT1aR.
Contrast-induced nephropathy (CIN) is an important complication following diagnostic radiographic imaging and interventional therapy. It results from administration of intravascular iodinated contrast media (CM) and is currently the third most common cause of hospital-acquired acute kidney injury. CIN is associated with increased morbidity, prolonged hospitalization and higher mortality. Although the importance of CIN is widely appreciated, and its occurrence can be mitigated by the use of pre- and post-hydration protocols and low osmolar instead of high osmolar iodine-containing CM, specific prophylactic therapy is lacking. Remote ischemic preconditioning (RIPC), induced through short cycles of ischemia/reperfusion applied to the limb, is an intriguing new strategy which has been shown to reduce myocardial infarction size in patients undergoing emergency percutaneous coronary intervention. Furthermore, multiple proof of principle clinical studies have suggested benefit in several other ischemia/reperfusion syndromes, including stroke. Perhaps somewhat surprisingly, RIPC also is emerging as a promising strategy for CIN prevention. In this review, we discuss current clinical and experimental developments regarding the biology of CIN, concentrating on the pathophysiology of CIN, and cellular and molecular mechanisms by which limb ischemic preconditioning may confer renal protection in clinical and experimental models of CIN.
Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) plays a critical role in the regulation of blood pressure and fluid volume homeostasis. Mice lacking functional Npr1 (coding for GC-A/NPRA) exhibit hypertension and congestive heart failure. However, the underlying mechanisms remain largely less clear. The objective of the present study was to determine the physiological efficacy and impact of all-trans retinoic acid (ATRA) and sodium butyrate (NaBu) in ameliorating the renal fibrosis, inflammation, and hypertension in Npr1 gene-disrupted haplotype (1-copy; +/-) mice (50% expression levels of NPRA). Both ATRA and NaBu either alone or in combination, decreased the elevated levels of renal proinflammatory and profibrotic cytokines and lowered blood pressure in Npr1+/- mice compared with untreated controls. The treatment with ATRA-NaBu facilitated the dissociation of histone deacetylase (HDAC) 1 and 2 from signal transducer and activator of transcription 1 (STAT1) and enhanced its acetylation in the kidneys of Npr1+/- mice. The acetylated STAT1 formed complex with nuclear factor-B (NF-B) p65, thereby inhibiting its DNA-binding activity and downstream proinflammatory and profibrotic signaling cascades. The present results demonstrate that the treatment of the haplotype Npr1+/- mice with ATRA-NaBu, significantly lowered blood pressure and reduced the renal inflammation and fibrosis involving the interactive roles of HDAC, NF-B (p65), and STAT1. The current findings will help in developing the molecular therapeutic targets and new treatment strategies for hypertension and renal dysfunction in humans.
Diabetic nephropathy (DN), a microvascular complication of diabetes, has emerged as an important health problem worldwide. There is strong evidence to suggest that oxidative stress, inflammation, and fibrosis play a pivotal role in the progression of DN. Apigenin has been shown to possess antioxidant, anti-inflammatory, anti-apoptotic, anti-fibrotic, as well as anti-diabetic properties. Hence, we evaluated whether apigenin halts the development and progression of DN in streptozotocin (STZ)-induced diabetic rats. Male albino Wistar rats were divided into control, diabetic-control, apigenin treatment groups (5-20 mg/kg p.o. respectively), apigenin per se (20 mg/kg p.o.) and ramipril treatment group (2 mg/kg p.o.). A single injection of STZ (55 mg/kg i.p) was administered to all the groups, except control and per se groups, to induce type 1 diabetes mellitus. Rats with fasting blood glucose > 250mg/dl were included in the study and randomized to different groups. Thereafter, the protocol was continued for 8 months in all the groups. Apigenin (20 mg/kg) treatment attenuated renal dysfunction, oxidative stress and fibrosis (decreased TGF-β1, fibronectin and type IV collagen) in the diabetic rats. It also significantly prevented MAPK activation which inhibited inflammation (reduced TNF-α, IL-6 and NF-B expression) and apoptosis (increased expression of Bcl-2 and decreased Bax and Caspase-3). Furthermore, histopathological examination demonstrated reduced inflammation, collagen deposition and glomerulosclerosis in the renal tissue. In addition, all these changes were comparable to those produced by ramipril. Hence, apigenin ameliorated renal damage due to DN by suppressing oxidative stress, fibrosis and by inhibiting MAPK pathway.
Kidneys are highly vascularized and contain many distinct vascular beds. However, the origins and roles of developing renal endothelial cells in the formation of the kidney are unclear. We have shown that the Foxd1-positive renal stroma gives rise to endothelial marker expressing progenitors that are incorporated within a subset of peritubular capillaries; however, the significance of these cells is unclear. The purpose of this study was to determine whether deletion of Flk1 in the Foxd1 stroma was important for renal development. To that end, we conditionally deleted Flk1 (critical for endothelial cell development) in the renal stroma by breeding floxed Flk1 mice (Flk1fl/fl) with Foxd1cre mice to generate Foxd1cre; Flk1fl/fl (Flk1ST-/-) mice. We then performed FACsorting, histological, morphometric and metabolic analyses of Flk1ST-/- versus control mice. We confirmed decreased expression of endothelial markers in the renal stroma of Flk1ST-/- kidneys via Flow sorting and immunostaining, and upon interrogation of embryonic and postnatal Flk1ST-/- mice we found they had dilated peritubular capillaries. Three-dimensional (3D) reconstructions showed reduced ureteric branching, and fewer nephrons in developing Flk1ST-/- kidneys versus controls. Juvenile Flk1ST-/- kidneys displayed renal papillary hypoplasia and a paucity of collecting ducts. 24-hour urine collections revealed that postnatal Flk1ST-/- mice had urinary concentrating defects. Thus, while lineage-tracing revealed that the renal cortical stroma gave rise to a small subset of endothelial progenitors, these Flk1 expressing stromal cells are critical for patterning the peritubular capillaries. Also, loss of Flk1 in the renal stroma leads to non-autonomous patterning defects in ureteric lineages.
The dynamics of propagating myogenic contractions in the wall of the resting ex vivo urinary bladder of the rabbit were characterised by spatiotemporal maps and related to cyclic variation in intravesical pressure (pves). Patches of propagating contractions (PPCs) enlarged and involuted in near synchrony with peaks in intravesical pressure (pves) (3.85 mean ± 0.3cpm). and were preceded by regions of stretch. The maximum area of the bladder undergoing contraction (55.28±2.65 %) and the sizes of individual PPCs (42.61 ± 1.65 mm2) coincided with the peak in pves. PPCs, originated and propagated within temporary patch domains (TPDs) and comprised groups of near synchronous cyclic propagating individual contractions (PICs). The TPDs were located principally along the vertical axis of the anterior surface of the bladder. The sites of origin of PICs within PPCs were inconsistent, consecutive contractions often propagating in opposite directions along linear maps of strain rate. Similar patterns of movement occurred in areas of the anterior bladder wall that had been stripped of mucosa. Intravesical pressure (pves) varied cyclically with area of contraction and with the indices of aggregation of PPCs, indicating that they grew by peripheral enlargement and collision without annihilation. The synchronisation of PICs within PPCs was sometimes lost, uncoordinated PICs then occurring irregularly (between 4 and 20 cpm) having little effect on Pves The authors postulate that the formation and involution of PPCs within a TPD resulted from cyclic variation in excitation, that increased the incidence and distance over which component PICs propagated.
Regulation of lipogenesis by pathophysiological factors in the liver and skeletal muscle is well understood; however, regulation in the kidney is still unclear. To elucidate nutritional regulation of lipogenic factors in the kidney, we measured the renal expression of lipogenic transcriptional factors and enzymes during fasting and refeeding in chow-fed and high-fat-fed mice. We also examined the regulatory effect of the liver X receptor (LXR) on the expression of lipogenic factors. The renal gene expression of sterol regulatory element-binding protein (SREBP)-1c and fatty acid synthase (FAS) was reduced by fasting for 48 h and restored by refeeding, whereas the mRNA levels of forkhead box O (FOXO)1/3 were increased by fasting and restored by refeeding. Accordingly, protein levels of SREBP-1, FAS, and phosphorylated FOXO1/3 were reduced by fasting and restored by refeeding. The patterns of lipogenic factors expression in the kidney were similar to those in the liver and skeletal muscle. However, this phasic regulation of renal lipogenic gene expression was blunted in diet-induced obese mice. LXR agonist TO901317 increased the lipogenic gene expression and the protein levels of SREBP-1 precursor and FAS but not nuclear SREBP-1. Moreover, increases in insulin-induced genes mRNA and nuclear carbohydrate-responsive element binding protein (ChREBP) levels were observed in the TO901317-treated mice. These results suggest that the kidney shows flexible suppression and restoration of lipogenic factors following fasting and refeeding in lean mice, but this is blunted in obese mice. LXR is involved in the renal expression of lipogenic enzymes, and ChREBP may mediate the response.
Background Detrusor overactivity (DO) is the abnormal response of the urinary bladder to physiological stretch during the filling phase of the micturition cycle. The mechanisms of bladder smooth muscle compliance upon the wall stretch are poorly understood. We previously reported that the function of normal detrusor is regulated by TREK-1, a member of the mechano-gated subfamily of two-pore domain potassium (K2P) channels. In the present study, we aimed to identify the changes in expression and function of TREK-1 channels under pathological conditions associated with DO, evaluate the potential relationship between TREK-1 channels and cytoskeletal proteins in the human bladder, and test the possibility of modulation of TREK-1 channel expression by small RNAs. Results Expression of TREK-1 channels in DO specimens was 2.7-fold decreased in comparison to control bladders, and was associated with a significant reduction of the recorded TREK-1 currents. Isolated DO muscle strips failed to relax when exposed to a TREK-1 channel opener. Immunocytochemical labeling revealed close association of TREK-1 channels with cell cytoskeletal proteins and caveolins, with caveolae microdomains being severely disrupted in DO specimens. Small activating RNA tested in vitro provided evidence that expression of TREK-1 protein could be partially up-regulated. Conclusions Our data confirmed a significant down-regulation of TREK-1 expression in human DO specimens, and provided evidence of close association between the channel, cell cytoskeleton and caveolins. Up-regulation of TREK-1 expression by saRNA could be a future step for the development of in vivo pharmacological and genetic approaches to treat DO in humans.
Significant alterations in maternal calcium (Ca2+) and magnesium (Mg2+) balance occur during lactation. Ca2+ is the primary divalent cation mobilized into breast milk by demineralization of the skeleton and alterations in intestinal and renal Ca2+ transport. Mg2+ is also concentrated in breast milk, but the underlying mechanisms are not well understood. To determine the molecular alterations in Ca2+ and Mg2+ transport in the intestine and kidney during lactation, 3 groups of female mice consisting of either non-pregnant controls, lactating mice, or mice undergoing involution were examined. The fractional excretion of Ca2+, but not Mg2+, rose significantly during lactation. Renal 1-alpha hydroxylase and 24-OHase mRNA levels increased markedly as did plasma 1,25 dihydroxyvitamin D levels. This was accompanied by significant increases in intestinal expression of Trpv6 and S100g in lactating mice. However, no alterations in the expression of cation permeable claudins (-2, -12 or -15) were found in the intestine. In kidney, increased expression of Trpv5 and Calb1 was observed during lactation, while no changes in claudins involved in Ca2+ and Mg2+ transport (-2, -14, -16 or -19) were found. Consistent with the mRNA expression, both Calbindin-D28K and TRPV5 protein expression increased. Colonic Trpm6 expression increased during lactation, while renal Trpm6 remained unaltered. In conclusion, proteins involved in transcellular Ca2+ and Mg2+ transport pathways increase during lactation, while expression of paracellular transport proteins remained unchanged. Increased fractional Ca2+ excretion can be explained by vitamin D-dependent intestinal hyperabsorption and bone demineralization, despite enhanced transcellular Ca2+ uptake by the kidney.
Macrophage migration inhibitory factor (MIF) is a cytokine with pleiotropic actions that is produced by several organs and cell types. Depending on the target cell and the inflammatory context, MIF can engage its two component receptor complex CD74 and CD44, and the chemokine receptors CXCR2/4. MIF is constitutively expressed in renal proximal tubular cells, stored in intracellular preformed pools and is released at a low rate. Recently a second MIF-like protein (i.e. MIF-2/D-DT) has been characterized in mammals. Our study was aimed at examining the role of MIF-2/D-DT, which mediates tissue protection in the heart, in tubular cell regeneration from IR injury. We found that Mif-/-, Mif-2-/- and Cd74-/- mice had significantly worse tubular injury compared to WT control mice and that treatment with MIF-2/D-DT significantly improved recovery of injured epithelial cells. RNAseq analysis of kidney tissue from the IR injury model revealed MIF-2/D-DT treatment to stimulates SLPI and cyclin D1 expression. MIF-2/D-DT additionally activates of eIF2α and ATF4, two transcription factors involved in the integrated stress response (ISR), which is a cellular stress response activated by hypoxia, nutrient deprivation and oxygen radicals. MIF-2/D-DT also inhibited apoptosis and induced autophagy in hypoxia treated MPT cells. These results indicate that MIF-2/D-DT is an important factor in tubular cell regeneration and may be of therapeutic utility as a regenerative agent in the clinical setting of ischemic AKI.
Kidney transplantation (KTX) is a life-saving procedure for patients with end-stage renal disease. Expression levels of many genes in kidney vary between males and females which may play an essential role in the sex differences in graft function. However, whether these differences are affected after cross-sex-KTX is unknown. In the present study, we assessed post-operative changes in genotype, function and inflammatory responses of the grafts in same-sex and cross-sex-KTX. Single kidney transplants were performed between same and different sex C57BL/6 mice paired into 4 combination groups: female donor/female recipient (F/F); male donor/male recipient (M/M); female donor/male recipient (F/M); and male donor/female recipient (M/F). The remnant native kidney was removed 4 days post-transplant. Expression levels of genes related to the contractility of the afferent arteriole and tubular sodium reabsorption were assessed. Same-sex-KTX did not significantly alter the magnitude or sex difference pattern of gene expression in male or female grafts. Cross-sex-KTX showed an attenuated sex difference in gene expressions. The measurements of Endothelin 1, Endothelin ETA receptor, NKCC2 and ENaC subunits exhibited decreases in M/F compared with M/M, and increases in F/M compared with F/F. There were no significant differences in hemodynamics or sodium excretion in response to acute volume expansion for any sex combinations. Cross-sex-KTX stimulated more robust inflammatory responses than same-sex-KTX. IL-6 and KC mRNA levels elevated 5-20 fold in cross-sex-KTX compared with same-sex-KT. In conclusion, cross-sex-KTX alters gene expression levels and induces inflammatory responses, which might play an important role in long-term graft function.
Ferroptosis is an iron-dependent form of regulated, non-apoptotic cell death, which contributes to damage in models of acute kidney injury (AKI). Heme oxygenase-1 (HO-1) is a cytoprotective enzyme induced in response to cellular stress, and is protective against AKI due to its anti-apoptotic and anti-inflammatory properties. However, the role of HO-1 in regulating ferroptosis is unclear. The purpose of this study was to elucidate the role of HO-1 in regulating ferroptotic cell death in renal proximal tubule cells (PTCs). Immortalized PTCs obtained from HO-1+/+ and HO-1-/- mice were treated with erastin or RSL3, ferroptosis inducers, in the presence or absence of anti-oxidants, an iron source, or an iron chelator. Cells were assessed for changes in morphology and metabolic activity as an indicator of cell viability. Treatment of HO-1+/+ PTCs with erastin resulted in a time- and dose-dependent increase in HO-1 gene expression and protein levels compared to vehicle-treated controls. HO-1-/- cells showed increased dose-dependent erastin- or RSL3-induced cell death in comparison to HO-1+/+ PTCs. Iron supplementation with ferric ammonium citrate in erastin-treated cells decreased cell viability further in HO-1-/- PTCs compared with HO-1+/+ cells. Co-treatment with ferrostatin-1 (ferroptosis inhibitor), deferoxamine (iron chelator), or N-acetyl-L-cysteine (glutathione replenisher) significantly increased cell viability and attenuated erastin-induced ferroptosis in both HO-1+/+ and HO-1-/- PTCs. These results demonstrate an important anti-ferroptotic role of HO-1 in renal epithelial cells.
Inner medullary collecting duct (IMCD)-derived endothelin-1 (ET-1) is stimulated by volume expansion, in part through augmented luminal flow, whereupon it can elicit natriuresis and diuresis. Since flow can alter nitric oxide (NO) and reactive oxygen species (ROS), both of which can affect collecting duct salt transport, we asked whether NO and/or ROS mediate flow-stimulated IMCD ET-1. Mouse IMCD3 cells were exposed to flow and ET-1/GAPDH mRNA assessed. A shear stress of 10 dyne/cm2 for 1 hr increased ET-1 mRNA by 4-fold compared to no flow (ET-1 flow response). Global NO synthase (NOS) inhibition (L-NAME) reduced the ET-1 flow response, however pharmacologic inhibition of NOS1 or NOS2, NOS3 siRNA, arginase inhibition, removal of media L-Arg, or inhibition of NO-dependent signaling pathways (PKG, guanylyl cyclase or NFkB) did not affect the ET-1 flow response. Tempol reduced the ET-1 flow response; no further inhibition occurred with L-NAME. Superoxide dismutase, but not catalase, reduced the ET-1 flow response. Inhibition of NAPDH oxidase (NOX) (apocynin), pharmacologic inhibition of NOX1/4, or NOX4 siRNA reduced the ET-1 flow response. Finally, flow increased IMCD3 ROS production and this was inhibited by apocynin, NOX1/4 inhibition and, to a small extent, by L-NAME. Taken together, these data suggest that NOX4-derived ROS in general, and possibly superoxide in particular, are involved in flow-stimulated IMCD ET-1 production. To our knowledge, this is the first report of flow-stimulated ROS production by the CD, as well as the first report of such flow-stimulated CD ROS exerting a biologic effect.
Exercise has been overlooked as a potential therapy in chronic kidney disease (CKD), mainly due to a lack of understanding on its safety aspects. Notably, there is no data on renal function after exercise in CKD considering its stages. We investigated the acute effects of a 30-minute moderate-intensity aerobic exercise bout on glomerular filtration rate (GFR) and albuminuria in twenty-two non-dialysis CKD patients divided into: CKD stages 1 and 2 (CKD1-2) and; CKD stages 3 and 4 (CKD3-4). Eleven body mass index-, age-, and sex-matched healthy individuals served as control (CON). Blood and urine samples were collected before, immediately after, and up to 90 minutes post exercise for creatinine and albumin assessments. GFR was determined by creatinine clearance (GFRCr-Cl). All CKD patients had significantly lower peak oxygen uptake than CON. CKD1-2 and CKD3-4 had increasingly higher serum creatinine than CON (9.6±2.6 mg/L; 25.6±1.01 mg/L; 7.5±1.4 mg/L, respectively); however, no within-group changes in serum or urinary creatinine were observed across time. GFRCr-Cl was decreased in CKD1-2 and CKD3-4 when compared with CON (91±17 ml/min-1/1.73m2-1; 34±15 ml/min-1/1.73m2-1; 122±20 ml/min-1/1.73m2-1, respectively). Most importantly, exercise did not affect GFRCr-Cl in none of the groups across time. Albuminuria was significantly higher in CKD3-4 (297±284 µg/min-1) than in CON (5.4±1.4 µg/min-1), but no within-group changes were observed after exercise. In conclusion, a single 30-minute moderate-intensity aerobic exercise bout does not impair renal function in non-dialysis CKD patients, regardless of disease stage, supporting the notion that exercise training can be safe in this disease.
Sepsis outcomes are heavily dependent on the development of septic organ injury, but no interventions exist to interrupt or reverse this process. MicroRNA-223 (miR-223) is known to be involved in both inflammatory gene regulation and host-pathogen interactions key to the pathogenesis of sepsis. The goal of this study was to determine the role of miR-223 as a mediator of septic kidney injury. Using miR-223 knockout mice and multiple models of experimental sepsis, we found that miR-223 differentially influences acute kidney injury (AKI) based on the model used. In the absence of miR-223, mice demonstrated exaggerated AKI in sterile models of sepsis (LPS injection) and attenuated AKI in a live-infection model of sepsis (cecal ligation and puncture). We demonstrated that miR-223 expression is induced in kidney homogenate after cecal ligation and puncture, but not after LPS or fecal slurry injection. We investigated additional potential mechanistic explanations including differences in peritoneal bacterial clearance and host stool virulence. Our findings highlight the complex role of miR-223 in the pathogenesis of septic kidney injury, as well as the importance of differences in experimental sepsis models and their consequent translational applicability.
Novel therapeutic interventions for preventing or attenuating kidney injury following ischemia/reperfusion injury (IRI) remain a focus of significant interest. Currently, there are no definitive therapeutic or preventive approaches available for ischemic acute kidney injury (AKI). Our objective is to determine (a) whether renal arginase activity or expression is increased in renal IRI and (b) whether arginase plays a role in development of renal IRI. The impact of arginase activity and expression on renal damage was evaluated in male C57BL/6J (WT) and arginase-2 deficient (Arg2-/-) mice subjected to bilateral renal ischemia for 28min followed by reperfusion for 24h. ARG2 expression and arginase activity significantly increased following renal IRI, paralleling the increase in kidney injury. Pharmacological blockade or genetic deficiency of Arg2 conferred kidney protection in renal IRI. Arg2-/- mice had significantly attenuated kidney injury and lower plasma creatinine and blood urea nitrogen levels after renal IRI. Blocking arginases using S-(2-boronoethyl)-L-cysteine (BEC) 18h before ischemia mimicked arginase deficiency by reducing kidney injury, histopathological changes and kidney injury marker-1 expression, renal apoptosis, kidney inflammatory cell recruitment and inflammatory cytokines, kidney oxidative stress, and increased kidney NO production and eNOS phosphorylation, kidney PPAR co-activator (PGC)-1α expression, mitochondrial ATP, and preserved kidney mitochondrial ultrastructure compared to vehicle-treated IRI mice. Importantly, BEC-treated eNOS-knockout mice failed to reduce BUN and creatinine following renal IRI. These findings indicate that arginase-2 plays a major role in renal IRI, via an eNOS-dependent mechanism and that blocking ARG2 activity or expression could be a novel therapeutic approach for prevention of AKI.
The basal, intermediate, and superficial cell layers of the urothelium undergo rapid and complete recovery following acute injury; however, the effects of chronic injury on urothelial regeneration have not been well defined. To address this discrepancy, we employed a mouse model to explore urothelial changes in response to spinal cord injury (SCI), a condition characterized by life-long bladder dysfunction. One-day post SCI there was a focal loss of umbrella cells, which are large cells that populate the superficial cell layer and normally express uroplakins (UPKs) and KRT20, but not KRT5, KRT14, or TP63. In response to SCI, regions of urothelium devoid of umbrella cells were replaced with small superficial cells that lacked KRT20 expression and appeared to be derived in part from the underlying intermediate cell layer, including cells positive for KRT5 and TP63. We also observed KRT14-positive basal cells that extended thin cytoplasmic extensions, which terminated in the bladder lumen. Both KRT14-positive and KRT14-negative urothelial cells proliferated one-day post SCI, and by seven-days, cells in the underlying lamina propria, detrusor, and adventitia were also dividing. At 28-days post SCI, the urothelium appeared morphologically patent, and the number of proliferative cells decreased to baseline levels; however, patches of small superficial cells were detected that co-expressed UPKs, KRT5, KRT14, and TP63, but failed to express KRT20. Thus, unlike the rapid and complete restoration of the urothelium that occurs in response to acute injuries, regions of incompletely differentiated urothelium were observed even 28-days-post SCI.
TThe goal of this study was to address the role of ATP-citrate lyase (ACL), an enzyme that converts citrate to acetyl-CoA, in high glucose (HG)-induced histone acetylation and pro-fibrotic gene expression. Our recent ChIP-Seq studies have demonstrated that HG induces genome-wide histone hyperacetylation in mesangial cells (MCs). Here we showed that exposure of MCs to HG markedly increased histone acetylation at the H3K9/14 and H3K18 marks and induced the expression of potent pro-fibrotic factors TGF-β1, TGF-β3 and CTGF. The induction of these pro-fibrotic factors was further enhanced by histone deacetylase inhibitor but suppressed by histone acetyl-transferase inhibitor, confirming the importance of histone acetylation in this regulation. Interestingly, HG not only up-regulated ACL expression but also promoted ACL nuclear translocation, evidenced by increased ACL concentration and activity in the nuclear extracts. Consistent with this observation, transfection of MCs with a plasmid carrying GFP-ACL fusion protein led to GFP nuclear accumulation when cultured in HG condition. Silencing ACL with siRNAs alleviated HG-induced histone hyperacetylation as well as up-regulation of TGF-β1, TGF-β3, CTGF and extracellular matrix (ECM) proteins fibronectin and collagen type IV, whereas ACL overexpression further enhanced HG induction of histone acetylation as well as these pro-fibrotic factors and ECM proteins. Collectively, these observations demonstrate that HG promotes ACL expression and translocation into the nucleus, where ACL converts citrate to acetyl-CoA to provide the substrate for histone acetylation, leading to up-regulation of fibrogenic genes. Therefore, ACL plays a critical role in epigenetic regulation of diabetic renal fibrosis.
Recently, new methods for assessing renal function in conscious mice (transcutaneous assessment) and for counting and sizing all glomeruli in whole kidneys (MRI) were described. In the present study, these methods were used to assess renal structure and function in aging mice, and in mice born with a congenital low nephron endowment. Age-related nephron loss was analysed in adult C57BL/6 mice (10-50 weeks of age) and congenital nephron deficit was assessed in glial cell line-derived neurotrophic factor heterozygous (GDNF HET) null mutant mice. Renal function was measured through the transcutaneous quantitation of FITC-sinistrin half-life (t1/2) in conscious mice. MRI was used to image, count and size cationic-ferritin labelled glomeruli in whole kidneys ex vivo. Design-based stereology was used to validate the MRI measurements of glomerular number and mean volume. In adult C57BL/6 mice, older age was associated with fewer and larger glomeruli, and a rightwards shift in the glomerular size distribution. These changes coincided with a decrease in renal function. GNDF HET mice had a congenital nephron deficit that was associated with glomerular hypertrophy and exacerbated by aging. These findings suggest that glomerular hypertrophy and hyperfiltration are compensatory processes that can occur in conjunction with both age-related nephron loss and congenital nephron deficiency. The combination of measurement of renal function in conscious animals and quantitation of glomerular number, volume and volume distribution provides a powerful new tool for investigating aspects of renal aging and functional changes.
Sodium nitrite (NaNO2) is converted to nitric oxide (NO) in vivo and has vasodilatory and natriuretic effects. Our aim was to examine the effects of NaNO2 on hemodynamics, sodium excretion and GFR. In a single-blinded, placebo-controlled, cross-over study, we infused placebo (0.9% NaCl) or 0.58, 1.74, or 3.48 μmol NaNO2/kg/hour for two hours in twelve healthy subjects, after four days standard diet. Subjects were supine and water-loaded. We measured brachial and central blood pressure (BP), plasma concentrations of renin, angiotensin II, aldosterone, arginine vasopressin (P-AVP), and plasma nitrite (P-NO2-), GFR by Cr-EDTA clearance, fractional excretion of sodium (FENa) free water clearance (CH2O), and urinary excretion rate of guanosine 3',5'-cyclic monophosphate (U-cGMP). The highest dose reduced brachial systolic BP (5.6 mmHg, p=0.003), central systolic BP (5.6 mmHg, p=0.035) and CH2O (maximum change from 3.79 to 1.27 ml/min, p=0.031), and increased P-NO2- (from 0.065 to 0.766 μmol/l, p<0.001), while reducing U-cGMP (from 444 to 247 pmol/min, p=0.004). GFR, FENa, P-AVP and the components in the renin-angiotensin-aldosterone system did not change significantly. In conclusion, intravenous NaNO2 induced a dose-dependent reduction of brachial and central BP. The hemodynamic effect was not mediated by the renin-angiotensin-aldosterone system. NaNO2 infusion resulted in a vasopressin-independent decrease in CH2O and urine output, but no change in urinary sodium excretion or GFR. The lack of increase in cGMP accompanying the increase in NO2-, suggests a direct effect of nitrite or nitrate on the renal tubules and vascular bed with little or no systemic conversion to NO.
Long-term peritoneal dialysis (PD) therapy results in functional and structural alteration of the peritoneal membrane, including epithelial-to-mesenchymal transition (EMT). Interleukin 6 (IL-6) is a local pleiotropic cytokine, hypothesized to play an important role in EMT. This study was designed to investigate the role of IL-6 in EMT and peritoneal membrane dysfunction in long-term PD patients by assessing the level of IL-6 in dialysate and exploring the relationship between IL-6, the related signaling pathway JAK2/STAT3, and EMT, using in vitro cellular and molecular techniques. Plasma and dialysate levels of IL-6 were significantly higher in PD ultrafiltration failure patients compared to those in patients without ultrafiltration failure and were negatively correlated with measures of PD adequacy. In vitro, IL-6 treatment changed human peritoneal mesothelial cell phenotype from a typical cobblestone-like to a fibroblast-like appearance and increased cell viability. IL-6 treatment increased α-SMA and VEGF expression but decreased E-cadherin expression. IL-6 treatment activated the JAK/STAT signaling pathway. However, the JAK2/STAT3 inhibitor WP1066 prevented IL-6-induced activation of the JAK2/STAT3 pathway and EMT. We conclude that IL-6 promotes the EMT process, possibly by activating the JAK2/STAT3 signaling pathway. IL-6 may serve as a novel therapeutic target for preventing EMT, and preservation of the peritoneal membrane may arise from these studies.
The acute effects of Ang-(1-7) on the reabsorptive bicarbonate flow (JHCO3-) were evaluated using stationary microperfusion in vivo in the proximal tubules of spontaneously hypertensive rats (SHR) and their normotensive controls, Wistar-Kyoto (WKY) rats, using a microelectrode sensitive to H+. In WKY rats, the control JHCO3- was 2.40 ± 0.10 nmol.cm-2.s-1 (28); Losartan (10-7 M) or A779 (10-6 M, a specific Mas antagonist), alone or in combination with Losartan, decreased the JHCO3-. Ang-(1-7) had biphasic effects on JHCO3-: at 10-9 M, it inhibited and at 10-6, it stimulated the flow. S3226 (10-6 M, a specific NHE3 antagonist) decreased JHCO3- and changed the stimulatory effect of Ang-(1-7) to an inhibitory one but did not alter the inhibitory action of Ang-(1-7). In SHR, the control JHCO3- was 2.04 ± 0.13 nmol.cm-2.s-1 (14), and A779 and/or Losartan reduced the flow. Ang-(1-7) at 10-9 M increased JHCO3-, and Ang-(1-7) at 10-6 M reduced it. The effects of A779, Losartan and S3226 on the JHCO3- were similar to those found in WKY rats, which indicated that in SHR, the Ang-(1-7) action on the NHE3 was via Mas and AT1. The cytosolic calcium ([Ca2+]i), in the WKY or SHR rats was 100 nM and was increased by Ang-(1-7) at 10-9 or 10-6 M. In hypertensive animals, a high plasma level of Ang-(1-7) inhibited NHE3 in the proximal tubule, which mitigated the hypertension caused by the high plasma level of Ang II.
Enhanced expression of cyclooxygenase 2 (COX2) in podocytes contributes to glomerular injury in diabetic kidney disease, but some basal level of podocyte COX2 expression might be required to promote podocyte attachment and/or survival. To investigate the role of podocyte COX2 expression in diabetic kidney disease, we deleted COX2 specifically in podocytes in a mouse model of type 1 diabetes mellitus (Akita mice). Podocyte specific knockout (KO) of COX2 did not affect renal morphology or albuminuria in non-diabetic mice. Albuminuria was significantly increased in wild type (WT) and KO Akita mice compared to non-diabetic controls, and the increase in albuminuria was significantly greater in KO Akita mice compared to WT Akita mice at both 16- and 20-weeks of age. At the 20-week time point, mesangial expansion was also increased in WT and KO Akita mice compared to non-diabetic animals, and these histologic abnormalities were not improved by KO of COX2. Tubular injury was seen only in diabetic mice, but there were no significant differences between groups. Thus, KO of COX2 enhanced albuminuria and did not improve the histopathologic features of diabetic kidney disease. These data suggest that: 1. KO of COX2 in podocytes does not ameliorate diabetic kidney disease in Akita mice, and 2. Some basal level of podocyte COX2 expression in podocytes is necessary to attenuate the adverse effects of diabetes on glomerular filtration barrier function.
BACKGROUND: Tryptophan is metabolized along the kynurenine pathway, initially to kynurenine, and subsequently to cytotoxic 3-hydroxykynurenine. There is increasing interest in this pathway, because of its pro-inflammatory nature, and drugs interfering in it receive increasing attention. We aimed to investigate whether serum and urinary parameters of the tryptophan-kynurenine pathway, and particularly cytotoxic 3-hydroxykynurenine, are associated with systemic inflammation and long-term outcome in renal transplant recipients (RTR). METHODS: Data were collected in outpatient RTR with a functioning graft for >1 year. Tryptophan, kynurenine and 3-hydroxykynurenine in serum and urine were measured using LC-MS/MS. RESULTS: A total of 561 RTR (age 51±12 years; 56% male) were included at median 6.0 [2.6-11.6] years post-transplantation. Baseline median serum tryptophan was 40.0 [34.5-46.0] µmol/l; serum kynurenine was 1.8 [1.4-2.2] µmol/l; serum 3-hydroxykynurenine was 42.2 [31.0-61.7] nmol/l. Serum kynurenine and 3-hydroxykynurenine were strongly associated with parameters of systemic inflammation. During follow-up for 7.0 [6.2-7.5] years, 51 RTR (9%) developed graft failure and 120 RTR (21%) died. Both serum kynurenine and 3-hydroxykynurenine were independently associated with graft failure (HR 1.72 [1.23-2.41], P=0.002 and HR 2.03 [1.42-2.90], P<0.001). Serum 3-hydroxykynurenine was also independently associated with mortality (HR 1.37 [1.08-1.73], P=0.01), while serum kynurenine was not. Urinary tryptophan-kynurenine pathway parameters were not associated with outcome. CONCLUSIONS: Of tryptophan metabolites, serum 3-hydroxykynurenine is cross-sectionally most strongly and consistently associated with systemic inflammation and prospectively with adverse long-term outcome after kidney transplantation. Serum 3-hydroxykynurenine may be an interesting biomarker and target for the evaluation of drugs interfering in the tryptophan-kynurenine pathway.
Pathways implicated in diabetic kidney disease (DKD) are largely derived from animal models. To examine if renin-angiotensin system (RAS) changes in humans are concordant with rodent models, we measured concentration of angiotensinogen (AOG), cathepsin D(CTSD), angiotensin converting enzyme(ACE), ACE2 and enzymatic activities of ACE, ACE2 and aminopeptidase-A in FVB mice 13-20 weeks after streptozotocin (n=9) or vehicle (n=15) and people with longstanding type 1 diabetes, with (n=37) or without (n=81) DKD. In streptozotocin-treated mice, urine AOG and CTSD were 10.4-and 3.0-fold higher than controls, respectively (p-values <0.001). Enzymatic activities of ACE, ACE2 and APA were 6.2-, 3.2- and 18.8-fold higher, respectively, in diabetic animals (p-values <0.001). AngiotensinII was 2.4-fold higher in diabetic animals (p-value 0.017). Compared to people without DKD, those with DKD had higher urine AOG (170 vs. 15μg/g) and CTSD (147 vs. 31μg/g). In people with DKD, urine ACE concentration was 1.8-fold higher (1.4vs. 0.8μg/g without DKD), while its enzymatic activity was 0.6-fold lower (1.0 vs. 1.6 x 10 RFU/g without DKD). Lower ACE activity, not ACE protein concentration, was associated with ACE inhibitor (ACEI) treatment. After adjustment for clinical covariates, AOG, CTSD, ACE concentration and activity remained associated with DKD. In conclusion, in mice with streptozotocin-induced diabetes and humans with DKD, urine concentrations and enzymatic activities of several RAS components are concordantly increased, consistent with enhanced RAS activity and greater angiotensinII formation. ACEI use was associated with a specific reduction in urine ACE activity, not ACE protein concentration, suggesting it may be a marker of exposure to this therapy.
We recently reported that natriuresis produced by renal medullary salt loading is dependent on endothelin (ET)-1 and purinergic (P2) receptors in male rats. Since sex differences in ET-1 and P2 signaling have been reported, we decided to test whether ovarian sex hormones regulate renal medullary ET-1 and P2-dependent natriuresis. The effect of medullary NaCl loading on Na+ excretion was determined in intact and ovariectomized (OVX) female Sprague Dawley rats with and without ET-1 or P2 receptor antagonism. Isosmotic saline (284 mOsmol/kg H2O) was infused into the renal medullary interstitium of anesthetized rats during a baseline urine collection period, followed by isosmotic or hyperosmotic saline (1800 mOsmol/kg H2O) infusion. Medullary NaCl loading significantly enhanced Na+ excretion in intact and OVX female rats. ETA+B or P2 receptor blockade did not attenuate the natriuretic effect of medullary NaCl loading in intact females, whereas ETA+B or P2 receptor blockade attenuated the natriuretic response to NaCl loading in OVX rats. Activation of medullary P2Y2 & P2Y4 receptors by UTP infusion had no significant effect in intact females, but enhanced Na+ excretion in OVX rats. Combined ETA+B receptor blockade significantly inhibited the natriuretic response to UTP observed in OVX rats. These data demonstrate that medullary NaCl loading induces ET-1 and P2-independent natriuresis in intact females. In OVX, activation of medullary P2 receptors promotes ET-dependent natriuresis suggesting that ovarian hormones may regulate the interplay between the renal ET-1 and P2 signaling systems to facilitate Na+ excretion.
Angiotensin II (ANG II) has many biological effects in renal physiology, particularly in Ca2+ handling in the regulation of fluid and solute reabsorption. It involves the systemic endocrine renin-angiotensin system (RAS), but tissue and intracrine ANG II are also known. We have shown that ANG II induces heterodimerization of its AT1 and AT2 receptors (AT1R and AT2R) to stimulate sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity. Thus, we investigated whether ANG II-AT1R/AT2R complex is formed and internalized, and also looked at the intracellular localization of this complex to determine how its effect might be exerted on renal intracrine RAS. Living cell imaging of LLC-PK1 cells, quantification of extracellular ANG II, and use of the receptor antagonists, losartan and PD123319, showed that ANG II is internalized with AT1R/AT2R heterodimers as a complex in a microtubule-dependent and clathrin-independent manner, since colchicine - but not Pitstop2 - blocked this process. This result was confirmed by an increase of β-arrestin phosphorylation after ANG II treatment, clathrin-mediated endocytosis being dependent on dephosphorylation of β-arrestin. Internalized ANG II co-localized with an endoplasmic reticulum (ER) marker and increased levels of AT1R, AT2R and PKCα in ER-enriched membrane fractions. This novel evidence suggests the internalization of an ANG II-AT1/AT2 complex to target ER, where it might trigger intracellular Ca2+ responses.
The urinary tract is usually culture negative despite its close proximity to microbial flora. The precise mechanisms by which the kidneys and urinary tract defends against infection is not well understood. The initial kidney cells to encounter ascending pathogens are the collecting tubule cells which consist of principal cells (PCs) that express aquaporin 2 (AQP2) and intercalated cells (ICs) that express vacuolar H+-ATPase (V-ATPase, B1 subunit). We have previously shown that ICs are involved with the human renal innate immune defense. Here we generated two reporter mice, VATPase B1-cre+tdT+ mice to fluorescently label ICs and AQP2-cre+tdT+ mice to fluorescently label PCs and then performed flow sorting to enrich PCs and ICs for analysis. Isolated ICs and PCs along with proximal tubular cells were used to measure anti-microbial peptide (AMP) mRNA expression. ICs and PCs were significantly enriched for AMPs. Isolated ICs responded to uropathogenic Escherichia coli (UPEC)challenge in vitro and had higher RNase4 gene expression than control while both ICs and PCs responded to UPEC challenge in vivo by upregulating Defb1 mRNA expression. To our knowledge this is the first report of isolating murine collecting tubule cells and performing targeted analysis for multiple classes of AMPs.
Role and mechanism of renal PDI in blood pressure (BP) regulation has not been tested before. Here, we are testing this possibility in Sprague-Dawley (SD) rats. Rats were treated with PDI inhibitor bacitracin (100mg/kg, i.p./day for 14 days) followed by determining BP and renal AT1 receptor (AT1R) function in anesthetized rats. Renal AT1R function was determined as the ability of candesartan (an AT1R blocker) to increase diuresis and natriuresis. Second set of vehicle and bacitracin treated rats were used to determine biochemical parameters. Systolic BP increased in bacitracin-treated compared to vehicle-treated rats. Compared to vehicle, bacitracin-treated rats showed increased diuresis and natriuresis in response to candesartan, suggesting higher AT1R function in these rats. These were associated with higher renin activities in the plasma and renal tissues. Furthermore, urinary 8-isoprostane and kidney injury molecule-1 levels were higher whereas urinary antioxidant capacity was lower in bacitracin-treated rats. Renal protein carbonyl and nitrotyrosine levels also were higher in bacitracin compared to vehicle treated rats, suggesting oxidative stress burden in bacitracin-treated rats. Moreover, PDI activity decreased while its protein levels increased in renal tissues of bacitracin-treated rats. Also, nuclear levels of Nrf2 transcription factor, which regulates redox homeostasis, were decreased in bacitracin-treated rats. Further, tissue levels of Keap1, an Nrf2 inhibitory molecule, and tyrosine 216 phosphorylated GSK3β protein (GSK3β-pY216), an Nrf2 nuclear export protein, were increased in bacitracin treated rats. These results suggest that renal PDI by regulating keap1/Nrf2 pathway acts as an antioxidant, maintaining redox balance, renal AT1R function and BP in rats.
Renal tubular injury is the hallmark of cisplatin-induced nephrotoxicity. Caspase-11, a member of the caspase family, plays an important role in inflammation and cell death. However, its role in cisplatin-induced renal tubular injury remain unclear. We observed that caspase-11 expression was significantly elevated and the expression of caspase-11 mainly located in renal tubule in cisplatin-treated mice. Inhibition of caspase-11 by siRNA or wedelolactone (Wed) attenuated cisplatin-induced renal dysfunction and tubular injury. In primary renal tubular epithelial cells, cisplatin significantly promoted the expression and activation of caspase-11. Inhibition of caspase-11 by siRNA reduced cisplatin-induced cell apoptosis. Overexpression of caspase-11 promoted cell apoptosis by activating caspase-3-related apoptotic pathway. Furthermore, Co-immunoprecipitation results showed there was a direct interaction between caspase-11 and caspase-3. The fluorescence confocal microscopy results showed that caspase-11 and caspase-3 were co-localized in the cytoplasm of primary renal tubular epithelial cells. These results demonstrate that caspase-11 plays an important role in cisplatin-induced renal tubular injury. Caspase-11 promotes renal epithelial cell apoptosis by activating caspase-3-dependent apoptotic pathway. Caspase-11 may be a potential target for therapeutics against cisplatin-induced nephrotoxicity.
Diabetes is the leading cause of end stage renal disease, resulting in a significant health care burden and loss of economic productivity by affected individuals. As current therapies for progression of diabetic nephropathy (DN) are only moderately successful, identification of underlying mechanisms of disease is essential in order to develop more effective therapies. We showed previously that inhibition of arginase using S-(2-boronoethyl)-L-cysteine (BEC) or genetic deficiency of the arginase-2 isozyme was protective against key features of nephropathy in diabetic mouse models. However, those studies did not determine whether all markers of DN were dependent only on arginase-2 expression. The objective of this study was to identify features of DN that are associated specifically with expression of arginase-1 or -2. Elevated urinary albumin excretion rate and plasma urea levels, increases in renal fibronectin mRNA levels, and decreased renal medullary blood flow were associated almost completely and specifically with arginase-2 expression, indicating that arginase-2 selectively mediates major aspects of diabetic renal injury. However, increases in renal macrophage infiltration and renal TNF-α mRNA levels occurred independently of arginase-2 expression but were almost entirely abolished by treatment with BEC, indicating a distinct role for arginase-1. We therefore generated mice with a macrophage-specific deletion of arginase-1 (CD11bCre/Arg1fl/fl). CD11bCre/Arg1fl/fl mice had significantly reduced macrophage infiltration, but had no effect on albuminurea compared to Arg1fl/fl mice after 12 weeks of streptozotocin-induced diabetes. These results indicate that selective inhibition of arginase-2 would be effective in preventing or ameliorating major features of diabetic renal injury.
The direct detrusor relaxant effect of β3-adrenoceptor agonists as a primary mechanism to improve overactive bladder symptoms has been questioned. Among other targets, activation of β3-adrenoceptors down-modulate nerve-evoked acetylcholine (ACh) release, but there is insufficient evidence for the presence of these receptors on bladder cholinergic nerve terminals. Our hypothesis is that adenosine formed from the catabolism of cyclic AMP in the detrusor may act as a retrograde messenger via prejunctional A1 receptors to explain inhibition of cholinergic activity by β3-adrenoceptors. Isoprenaline (1 µM) decreased [3H]ACh release from stimulated (10 Hz, 200 pulses) human (-47±5%) and rat (-38±1%) detrusor strips. Mirabegron (0.1 µM, -53±8%) and CL316,243 (1 µM, -37±7%) mimicked isoprenaline (1 µM) inhibition and their effects were prevented by blocking β3-adrenoceptors with L748,337 (30 nM) and SR59230A (100 nM), respectively in human and rat detrusor. Mirabegron and isoprenaline increased extracellular adenosine in the detrusor. Blockage of A1 receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 100 nM) or the equilibrative nucleoside transporters (ENT) with dipyridamole (0.5 µM) prevented mirabegron and isoprenaline inhibitory effects. Dipyridamole prevented isoprenaline-induced adenosine outflow from the rat detrusor and this effect was mimicked by the ENT1 inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI, 30 µM). Cystometry recordings in anaesthetized rats demonstrated that SR59230A, DPCPX, dipyridamole and NBTI reversed the decrease in the voiding frequency caused by isoprenaline (0.1-1000 nM). Data suggest that inhibition of cholinergic neurotransmission by β3-adrenoceptors results from adenosine release via equilibrative nucleoside transporters and prejunctional A1 receptors stimulation in human and rat urinary bladder.
This study was carried out to analyze the developmental changes of bladder response to cholinergic stimulation in detail, highlighting calcium sensitization (CS) and its related pathways. Rats were divided into three groups in accordance with reported time of developmental milestones (newborn: day 1-4, youngster: day 5-14 and grown-up: day 15-21). Following cholinergic stimulation (carbachol 5 uM) the contractile response to detrusor were analyzed with respect to three phases (initial phasic, tonic and superimposed phasic contractions). Contractile responses were analyzed by dynamic and kinetic aspects. The responses were further compared in varying external calcium concentrations and in the presence of inhibitors of protein kinase C (PKC) and Rho kinase (ROCK), which are involved in CS. The responses of newborns were contrasted to the others by the short and brisk initial phasic contractions, prominent tonic contractions and delayed participation of irregular superimposed phasic contractions. With development, phasic contractions became prominent and tonic contractions diminished. These developmental changes of phasic contractions were reproduced when exposed to increasing calcium concentrations. Application of specific inhibitors and molecular phasic analysis revealed that PKC was functional in tonic contractions of the newborn, whereas ROCK took over its role with development. Within a few days of birth, rats' bladders experienced drastic changes of contractile mechanisms. These included dominance of phasic contractions from tonic contractions due to increased calcium dependence and the maturational shift of calcium sensitivity mechanism from PKC to ROCK.
The current paradigm regarding sodium handling in animals and humans postulates that total body sodium is regulated predominately via regulation of extracellular volume. Active sodium storage independent of volume retention is thought to be negligible. However, studies in animals, hypertensive patients and healthy humans suggest non-osmotic storage of sodium in skin. We hypothesized that tissue sodium concentrations ([Na]T) found in humans vary and reflect regulation due to variable glycosaminoglycan content due to variable expression of XYLT-1. 27 patients on dialysis and 21 living kidney transplant donors free of clinically detectable edema were studied. During surgery, abdominal skin, muscle and arteries were biopsied. [Na]T was determined by inductively coupled plasma - optical emission spectrometry, semiquantitative glycosaminoglycan content with Alcian stain, XYLT-1 expression by real-time PCR. [Na]T of arteries were ranging between 0.86 and 9.83 g/kg wet weight and were significantly higher in arteries (4.52 ± 1.82 g/kg) than in muscle (2.03 ± 1.41 g/kg; p<0.001) or skin (3.24 ± 2.26 g/kg wet weight; p=0.038). For individual patients [Na]T correlated for skin and arterial tissue (r=0.440, p=0.012). [Na]T also correlated significantly with blinded semiquantitative analysis of glycosaminoglycans staining (r=0.588, p=0.004). In arteries XYLT-1 expression was also correlated with [Na]T (r=0.392, p=0.003). Our data confirm highly variable [Na]T in human skin and muscle and extend this observation to [Na]T in human arteries. These data support the hypothesis of water-independent sodium storage via regulated glycosaminoglycan synthesis in human tissues, including arteries.
One of the many unresolved questions regarding the permeability of the glomerular filtration barrier is the reason behind the marked difference in permeability between albumin and polysaccharide probe molecules such as Ficoll and dextran of the same molecular size. Although the differences in permeability have been mainly attributed to charge effects, we have previously shown that this would require a highly charged filtration barrier, having a charge density that is ~10 times more than that on the albumin molecule. In this article, the classic two-pore model was extended by introducing size distributions on the solute molecules, making them conformationally flexible. Experimental sieving data for Ficoll from the rat glomerulus and from precision-made silicon nanopore membranes were analyzed using the model. For the rat glomerulus a small pore radius of 36.2 Å and a geometric standard deviation (gSD) for the Ficoll size-distribution of 1.16 was obtained. For the nanopore membranes, a gSD of 1.24 and a small-pore radius of 43 Å was found. Interestingly, a variation of only ~16% in the size of the polysaccharide molecule is sufficient to explain the difference in permeability between albumin and Ficoll. Also, in line with previous data, the effects of applying a size-distribution on the solute molecule are only evident when the molecular size is close to the pore size. Surely there is at least some variation in the pore radii and, likely, the gSD obtained in the current study is an overestimation of the "true" variation in the size of the Ficoll molecule.
Among solid organs the kidney's vascular network stands out because each nephron has 2 distinct capillary structures in series, and because tubuloglomerular feedback (TGF), one of the mechanisms responsible for blood flow autoregulation, is specific to renal tubules. TGF and the myogenic mechanism, acting jointly, autoregulate single nephron blood flow. Each generates a self-sustained periodic oscillation and an oscillating electrical signal that propagates upstream along arterioles. Similar electrical signals from other nephrons interact, allowing nephron synchronization. Experimental measurements show synchronization over fields of a few nephrons; simulations based on a simplified network structure that could obscure complex interactions predict more widespread synchronization. To permit more realistic simulations we made a cast of blood vessels in a rat kidney, performed micro-computed tomography at 2.5 μm resolution, and recorded 3-dimensional coordinates of arteries, afferent arterioles, and glomeruli. Non-terminal branches of arcuate arteries form tree-like structures requiring from 2 to 6 bifurcations to reach terminal branches at the tree tops. Terminal arterial structures were either paired branches at tops of arterial trees, from which 52.6 % of all afferent arterioles originated, or unpaired arteries not at tree tops yielding the other 22.9 % . The other 24.5 % originated directly from non-terminal arteries. Afferent arterioles near the cortico-medullary boundary were longer than those farther away, suggesting juxtamedullary nephrons have longer afferent arterioles. The distance separating origins of pairs of afferent arterioles varied randomly. The results suggest an irregular network tree structure with vascular nodes where arteriolar activity and local blood pressure interact.
Basement membranes (BMs) are a specialized form of extracellular matrix (ECM) which underlie nearly all cell layers and provide structural support for tissues and interact with cell surface receptors to determine cell behavior. Both macromolecular composition and stiffness of BM influence cell-BM interactions. Collagen IV is major constituent of BM that forms an extensively cross-linked oligomeric network. Its deficiency leads to BM mechanical instability as observed with glomerular basement membrane (GBM) in Alport Syndrome. These data have led to the hypothesis that collagen IV and its cross-links determine BM stiffness. A sulfilimine bond (S=N) between a methionine sulfur and lysine nitrogen cross-links collagen IV and is formed by the matrix enzyme peroxidasin. Using peroxidasin knock-out mice with reduced collagen IV sulfilimine cross-links, we find that renal tubular BM stiffness is reduced in these mice. Thus, this work provides the first direct experimental evidence that collagen IV sulfilimine cross-links contribute to BM mechanical properties and provides a foundation for future work on the relationship of BM mechanics to cell function in renal disease.
Uncoupling protein 2 (UCP2) plays critical roles in energy metabolism and cell survival. Previous investigations showed that UCP2 regulated the production of extracellular matrix and renal fibrosis. However, little is known about UCP2 in acute kidney injury. Here, we used UCP2 knockout mice to investigate the role of UCP2 in AKI model generated by renal ischemia/reperfusion (I/R) injury. The UCP2 global knockout mice were born and growth normal without kidney histological abnormality or renal dysfunctions. As compared with littermates, deletion of UCP2 exacerbated I/R-induced AKI while increase of UCP2 by conjugated linoleic acid (CLA) attenuated I/R injury. Tubular cell apoptosis and autophagy were induced by I/R. After injury, more tubular cell apoptosis and less autophagy were identified in the kidneys of knockout mice compared with their littermates and less apoptosis and more autophagy were observed in mice fed with CLA. In vitro, rotenone, an inhibitor of electron transport chain complex I, was applied to induce energy depletion in cultured tubular epithelial cells. As expected, rotenone/recovery (R/R) treatment induced tubular cell apoptosis and autophagy. UCP2 plasmid transfection reduced cell apoptosis and facilitated autophagy after R/R treatment, while UCP2 siRNA transfection sensitized cell apoptosis but reduced autophagy induced by R/R treatment. Interference of autophagy by its inhibitor 3-Methyladenine treatment or autophagy initiation protein Beclin-1 siRNA transfection resulted in tubular cell apoptosis. Thus, UCP2 attenuates I/R-induced AKI, probably by reducing cell apoptosis through protection of autophagy.
Unilaterally nephrectomized rats (UNx) have higher glomerular capillary pressure (PGC) that can cause significant glomerular injury in the remnant kidney. PGC is controlled by the ratio of afferent (Af-Art) and efferent arteriole resistance. Af-Art resistance in turn is regulated by two intrinsic feedback mechanisms: 1) Tubuloglomerular feedback (TGF) that causes Af-Art constriction in response to increased NaCl in the macula densa and 2) connecting tubule glomerular feedback (CTGF) that causes Af-Art dilatation in response to an increase in NaCl transport in the connecting tubule via the epithelial sodium channel (ENaC). Resetting of TGF post-UNx can allow systemic pressure to be transmitted to the glomerulus and cause renal damage, but the mechanism behind this resetting is unclear. Since CTGF is an Af-Art dilatory mechanism, we hypothesized that CTGF is increased after UNx, and contributes to TGF resetting. To test this hypothesis, we performed UNx in Sprague Dawley (SD) rats. Twenty-four hours after surgery, we performed micropuncture of individual nephrons and measured stop-flow pressure (PSF). PSF is an indirect measurement of PGC. Maximal TGF response at 40nl/min was 8.9 ± 1.24 mmHg in sham-UNx rats and 1.39 ± 1.02 mmHg in UNx rats indicating TGF resetting after UNx. When CTGF was inhibited with the ENaC blocker Benzamil (1μM/L), the TGF response was 12.29 ± 2.01 mmHg in UNx rats and 13.03 ± 1.25 mmHg in sham-UNx rats, indicating restoration of the TGF responses in UNx. We conclude that enhanced CTGF contributes to TGF resetting after UNx.
Increased expression of PDGF receptor-β (PDGFRβ) has been shown in the diabetic renal proximal tubules. The core molecular network used by high glucose to induce proximal tubular epithelial cell collagen I (α2) expression is poorly understood. We hypothesized that activation of PDGFRβ by high glucose increases collagen I (α2) production via Akt/mTORC1 signaling pathway in proximal tubular epithelial cells. Using biochemical and molecular biological techniques, we investigated this hypothesis. We show that high glucose increases activating phosphorylation of the PDGFRβ, resulting in the phosphorylation of the phosphatidylinositol 3 kinase. A specific inhibitor JNJ-10198409 and siRNAs targeting PDGFRβ blocked this phosphorylation without having any effect on MEK/Erk1/2 activation. We also found that PDGFRβ regulates high glucose-induced Akt activation, its targets tuberin and PRAS40 phosphorylation, and finally, mTORC1 activation. Furthermore, inhibition of PDGFRβ suppressed high glucose-induced expression of collagen I (α2) in proximal tubular cells. Importantly, expression of constitutively active Akt or mTORC1 reversed these processes. As a mechanism, we found that JNJ and PDGFRβ knockdown inhibited high glucose-stimulated Hif1α expression. Furthermore, overexpression of Hif1α restored the expression of collagen I (α2) inhibited by PDGFRβ knockdown in high glucose-stimulated cells. Finally, we show increased phosphorylation of PDGFRβ and its association with Akt/mTORC1 activation, Hif1α expression and elevated collagen I (α2) levels in the renal cortex of mice with diabetes. Our results identify PDGFRβ as a driver in activating Akt/mTORC1 nexus for high glucose-mediated expression of collagen I (α2) in proximal tubular epithelial cells, which contributes to tubulo-interstitial fibrosis in diabetic nephropathy.
Approximately 30% of all cancer patients treated with cisplatin, a widely used, broad-spectrum chemotherapeutic agent, experience acute kidney injury (AKI). Almost all patients receiving cisplatin have magnesium (Mg) losses, which are proposed to aggravate AKI. Currently, there are no methods to successfully treat or prevent cisplatin-AKI. While Mg supplementation has been shown to reduce AKI in experimental models and several small clinical trials, the effects of Mg status on tumor outcomes in immunocompetent tumor-bearing mice and humans have not been investigated. The purpose of this study was to further examine the effects of Mg deficiency (±Mg supplementation) on cisplatin-mediated AKI and tumor killing in immunocompetent mice bearing CT26 colon tumors. Using a model where cisplatin alone (20mg/kg cumulative dose) produced minimal kidney injury, Mg deficiency significantly worsened cisplatin-mediated AKI, as determined by biochemical markers (blood urea nitrogen and plasma creatinine) and histological renal changes, as well as markers of renal oxidative stress, inflammation, and apoptosis. By contrast, Mg supplementation blocked cisplatin-induced kidney injury. Using LLC-PK1 renal epithelial cells, we observed that Mg deficiency or inhibition of Mg uptake significantly enhanced cisplatin-induced cytotoxicity, while Mg supplementation protected. However, neither Mg deficiency nor inhibition of Mg uptake impaired cisplatin-mediated killing of CT26 tumor cells in vitro. Mg deficiency was associated with significantly larger CT26 tumors in BALB/c mice when compared to normal-fed control mice and Mg deficiency significantly reduced cisplatin-mediated tumor killing in vivo. Finally, Mg supplementation did not compromise cisplatin's anti-tumor efficacy in vivo.
Partial nephrectomy aims to maintain renal function by nephron sparing; however, functional changes in the contralateral kidney remain unknown. We evaluate the functional change in the contralateral kidney using a diethylene triamine penta-acetic acid (DTPA) renal scan and determine factors predicting contralateral kidney function after partial nephrectomy. A total of 699 patients underwent partial nephrectomy, with a DTPA scan before and after surgery to assess the separate function of each kidney. Patients were divided into three groups according to initial contralateral glomerular filtration rate (GFR; group 1: <30 ml/min/1.73 m2, group 2: 30-45 ml/min/1.73 m2, and group 3: greater double equals45 ml/min/1.73 m2). Multiple regression analysis was used to identify the factors associated with increased GFR of the contralateral kidney over a 4 year postoperative period. Patients in group 1 had a higher mean age and hypertension history, worse American Society of Anesthesiologists score, and larger tumor size than in the other two groups. The ipsilateral GFR changes at 4 years after partial nephrectomy were –18.9%, –3.6%, and 3.9% in groups 1, 2, and 3, respectively, while the contralateral GFR changes were 10.8%, 25.7%, and 38.8%. Age (Beta: –0.105, 95% confidence interval [CI]: –0.213; –0.011 p<0.05) and preoperative contralateral GFR (Beta: –0.256, 95% CI: –0.332; –0.050 p<0.01) were significant predictive factors for increased GFR of the contralateral kidney after 4 years. The contralateral kidney compensated for the functional loss of the ipsilateral kidney. The increase of GFR in contralateral kidney is more prominent in younger patients with decreased contralateral renal function.
Podocytes are highly differentiated epithelial cells wrapping glomerular capillary to form the filtration barrier in kidneys. As such, podocyte injury or dysfunction is a critical pathogenic event in glomerular disease. Autophagy plays an important role in the maintenance of the homeostasis and function of podocytes. However, it is less clear whether and how autophagy contributes to podocyte injury in glomerular disease. Here, we have examined the role of autophagy in adriamycin-induced nephropathy, a classical model of glomerular disease. We show that autophagy was induced by adriamycin in cultured podocytes in vitro and in podocytes in mice. In cultured podocytes, activation of autophagy with rapamycin led to the suppression of adriamycin-induced apoptosis, while inhibition of autophagy with chloroquine enhanced podocyte apoptosis during adriamycin treatment. To determine the role of autophagy in vivo, we established an inducible podocyte-specific Autophagy-related gene 7 knockout mouse model (Podo-Atg7-KO). Compared to wild-type littermates, Podo-Atg7-KO mice showed higher levels of podocyte injury, glomerulopathy, and proteinuria during adriamycin treatment. Together, these observations support an important role of autophagy in protecting podocytes under the pathological conditions of glomerular disease, suggesting the therapeutic potential of autophagy induction.
Hypertension is one of the most prevalent diseases worldwide, and a major risk factor for renal failure and cardiovascular disease. The role of albuminuria, a common feature of hypertension and robust predictor of cardiorenal disorders, remains incompletely understood. The goal of this study was to investigate the mechanisms leading to albuminuria in the kidney of a rat model of hypertension, the Dahl salt-sensitive (SS) rat. To determine the relative contributions of the glomerulus and proximal tubule (PT) to albuminuria, we applied intravital two-photon-based imaging to investigate the complex renal physiological changes that occur during salt-induced hypertension. Following a high salt diet, SS rats exhibited elevated blood pressure, increased glomerular sieving of albumin (GSCalb=0.0686), relative permeability to albumin (+16%) and impaired volume hemodynamics (-14%). Serum albumin, but not serum globulins or creatinine, concentration was decreased (-0.54g/dL), which was concomitant with increased filtration of albumin (3.7 vs 0.8 g per day normal diet). Pathologically, hypertensive animals had significant tubular damage as indicated by increased prevalence of granular casts, expansion and necrosis of PT epithelial cells (+2.20score/image), progressive augmentation of red blood cell velocity (+269µm/s) and micro vessel diameter (+4.3µm), and increased vascular injury (+0.61leakage/image). Therefore, development of salt-induced hypertension can be triggered by fast and progressive pathogenic remodeling of PT epithelia, which can be associated with changes in albumin handling. Collectively, these results indicate that both the glomerulus and the PT contribute to albuminuria and dual treatment of glomerular filtration and albumin reabsorption may represent an effective treatment of salt-sensitive hypertension.
We develop a pseudo-3D model of oxygen transport for the renal cortex of the rat, incorporating both the axial and radial geometry of the pre-glomerular circulation and quantitative information regarding the surface areas and transport from the vasculature and renal corpuscles. The computational model was validated by simulating four sets of published experimental studies of renal oxygenation in rats. Under the control conditions, the predicted cortical tissue oxygen tension (PtO2) or microvascular oxygen tension (µPO2) were within ± 1 standard error of the mean value (SEM) observed experimentally. The predicted PtO2 or µPO2 in response to ischemia-reperfusion injury, acute hemodilution, blockade of nitric oxide synthase, or uncoupling mitochondrial respiration, were within ± 2 SEM observed experimentally. We performed a sensitivity analysis of the key model parameters to assess their individual or combined impact on the predicted PtO2 and µPO2. The model parameters were: (i) the major determinants of renal oxygen delivery (DO2; arterial blood PO2 (PaO2), hemoglobin concentration (Hb) and renal blood flow (RBF)), (ii) the major determinants of renal oxygen consumption (VO2; glomerular filtration rate (GFR) and the efficiency of oxygen utilization for sodium reabsorption (β)) and (iii) peritubular capillary surface area (PCSA). Reductions in PCSA were found to profoundly increase the sensitivity of PtO2 and µPO2 to the major the determinants of DO2 and VO2. The increasing likelihood of hypoxia with decreasing PCSA provides a potential explanation for the increased risk of acute kidney injury in some experimental animals and for patients with acute and chronic kidney disease.
Deleterious effects of purinergic P2X1 and P2X7 receptors (P2XR) in angiotensin II (AngII)-dependent hypertension include increased renal vascular resistance, and impaired autoregulation and pressure natriuresis. However, their specific effects on the determinants of glomerular hemodynamics remain incompletely delineated. To investigate the P2XR contributions to altered glomerular hemodynamics in hypertension, the effects of acute blockade of P2X1R, P2X7R and P2X4R with NF449, A438079 and PSB12054 respectively, were evaluated in AngII-infused rats (435 ng/kg/min). P2X1R or P2X7R blockade reduced afferent (6.85 ± 1.05 vs. 2.37 ± 0.20 Dyn.s.cm-5) and efferent (2.85 ± 0.38 vs. 0.99 0.07 Dyn.s.cm-5) arteriolar resistances, leading to increases in glomerular plasma flow (75.82 ± 5.58 vs. 206.7 ± 16.38 nl/min), ultrafiltration coefficient (0.0198 ± 0.0024 vs. 0.0512 ± 0.0046 nl/min/mmHg) and single nephron glomerular filtration rate (22.73 ± 2.02 vs. 51.56 ± 3.87 nl/min) to near normal values. Blockade of P2X4R did not elicit effects in hypertensive rats. In normotensive Sham rats, only the P2X1R antagonist increased plasma flow and single nephron glomerular filtration rate, whereas the P2X4R antagonist induced glomerular vasoconstriction consistent with evidence that P2X4R stimulation increases release of nitric oxide, from endothelial cells. Mean arterial pressure remained unchanged in both hypertensive and normotensive groups. Western Blot analysis showed overexpression of P2X1R, P2X7R and P2X4R protein in the hypertensive rats. While it has generally assumed that the altered glomerular vascular resistances in AngII hypertension are due to AT1 receptor-mediated vasoconstriction these data indicate a predominant P2X1R and P2X7R control of glomerular hemodynamics in AngII hypertension.
AQP2 trafficking is regulated by phosphorylation and dephosphorylation of serine residues in the AQP2 c-terminus. Vasopressin (VP) binding to its receptor (V2R) leads to a cascade of events that result in the phosphorylation of serine 256 (S256), S264 and S269, but dephosphorylation of S261. To identify which phosphatase is responsible for VP-induced S261 dephosphorylation, we pretreated cells with different phosphatase inhibitors before VP stimulation. Only sanguinarine, a specific protein phosphatase 2C (PP2C) inhibitor, abolished VP-induced S261 dephosphorylation, but not inhibitors of PP1, PP2A (okadaic acid) or PP2B (cyclosporine). However, both sanguinarine and VP significantly increased phosphorylation of ERK, a kinase that can phosphorylate S261; inhibition of ERK by PD98059 partially decreased baseline S261 phosphorylation. These data support a role of ERK in S261 phosphorylation, but suggest that upon VP treatment, increased phosphatase activity overcomes the increase in ERK activity, resulting in overall dephosphorylation of S261. We also found that sanguinarine abolished VP-induced S261 dephosphorylation in cells expressing mutated AQP2 S256A, suggesting that the phosphorylation state of S261 is independent of S256. Sanguinarine alone did not induce AQP2 membrane trafficking, nor did it inhibit VP-induced AQP2 membrane accumulation in cells and kidney tissues, suggesting that S261 does not play an observable role in acute AQP2 membrane accumulation. In conclusion, PP2C activity is required for S261 AQP2 dephosphorylation upon VP stimulation, and this occurs independent of S256 phosphorylation. Understanding the pathways involved in modulating PP2C will help obtain a deeper understanding of the role of S261 in cellular events involving AQP2.
ABSTRACT: INTRODUCTION: In experimental models of diabetes, augmented sodium-glucose cotransport-2 (SGLT2) activity diminishes sodium (Na+) delivery at the macula densa. As a result, less vasoconstrictive adenosine is generated, leading to afferent arteriolar vasodilatation and hyperfiltration. The measurement and significance of urinary adenosine in humans has not been extensively examined in states of renal hemodynamic impairment, like that of diabetes. OBJECTIVE: Our aim was to validate a method for urine adenosine quantification in humans and perform an exploratory post-hoc analysis to determine whether urinary adenosine levels change dynamically in response to natriuresis in patients with type 1 diabetes (T1D) before and after treatment with the SGLT2 inhibitor (SGLT2i) empagliflozin. We hypothesized that SGLT2i, which reduces renal hyperfiltration through increased Na+ delivery to the macula densa, would increase urinary adenosine excretion. METHODS: Urine adenosine corrected for creatinine was measured using our validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method in 40 healthy participants and 40 patients with T1D. In the T1D cohort, measurements were performed during clamped euglycemic and hyperglycemic conditions, prior to and following 8 weeks of SGLT2i therapy. RESULTS: Urinary adenosine was detectable in healthy subjects (0.32± 0.11µmol/mmol Cr) and patients with T1D. In response to SGLT2i, urine adenosine increased during clamped hyperglycemia (0.40±0.11 vs 0.45±0.12 µmol/mmol Cr, p=0.005). Similar trends were observed during clamped euglycemia (p=0.08). CONCLUSIONS: SGLT2i increases urinary adenosine excretion under clamped hyperglycemic conditions in patients with T1D. The potentially protective role of SGLT2i against glomerular hyperfiltration and its mediation by adenosine in diabetes merits further study.
Animals living in desert environments are forced to survive despite severe heat, intense solar radiation, and both acute and chronic dehydration. These animals have evolved phenotypes that effectively address these environmental stressors. To begin to understand the ways in which the desert-adapted rodent P. eremicus survives, we performed an experiment where we subjected reproductively mature adults to 72 hours of water deprivation, during which they lost on average 23% of their body weight. The animals reacted via a series of changes in the kidney, which included modulating expression of genes responsible for reducing the rate of transcription and maintaining water and salt balance. Extracellular matrix turnover appeared to be decreased, and apoptosis was limited. Serum creatinine and other biomarkers of kidney injury were not elevated, which is different than the canonical human response, suggesting that changes in gene expression related to acute dehydration may effectively prohibit widespread kidney damage in the cactus mouse.
Chronic kidney disease (CKD) causes loss of lean body mass by multiple mechanisms. This study examines whether autophagy-mediated proteolysis contributes to CKD-induced muscle wasting. We tested autophagy in the muscle of CKD mice with plantaris muscle overloading to mimic resistance exercise or with acupuncture plus low frequency electrical stimulation (Acu/LFES) treatment. In CKD muscle, Bnip3, Beclin-1, LC3II mRNAs and proteins were increased compared with control muscle, indicating autophagosome-lysosome formation induction. Acu/LFES suppressed the CKD-induced upregulation of autophagy. However, overloading increased autophagy-related proteins in normal and CKD muscle. Serum from uremic mice induces autophagy formation but did not increase the myosin degradation or actin break-down in cultured muscle satellite cells. We examined mitochondrial biogenesis, copy number, and ATP production in cultured myotubes, and found all three aspects to be decreased by uremic serum. Inhibition of autophagy partially reversed this decline in cultured myotubes. In CKD mice, the mitochondrial copy number, biogenesis marker Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1 ), mitochondrial transcription factors A (TFAM) and mitochondrial fusion marker Mitofusin-2 (Mfn2) are decreased. Both muscle overloading and Acu/LFES increased mitochondrial copy number, and reversed the CKD-induced decreases in PGC-1 , TFAM and Mfn2. We conclude that the autophagy is activated in the muscle of CKD mice. However, myofibrillar protein is not directly broken down through autophagy. Instead, CKD-induced upregulation of autophagy leads to dysfunction of mitochondria and decrease of ATP production.
To assess the physiological significance of arterial-to-venous (AV) oxygen shunting, we generated a new pseudo-three-dimensional computational model of oxygen diffusion from intrarenal arteries to cortical tissue and veins. The model combines the eleven branching levels (known as 'Strahler' orders) of the pre-glomerular renal vasculature in the rat, with an analysis of an extensive dataset obtained using light microscopy to estimate oxygen mass transfer coefficients for each Strahler order. Further, the AV shunting model is now set within a global oxygen transport model that includes transport from arteries, glomeruli, peritubular capillaries and veins to tissue. While a number of lines of evidence suggest AV shunting is significant, most importantly our AV oxygen shunting model predicts AV shunting is small under normal physiological conditions (~0.9% of total renal oxygen delivery; range 0.4% to 1.4%), but increases during renal ischemia, glomerular hyperfiltration (~2.1% of total renal oxygen delivery; range 0.84% to 3.36%) and some cardiovascular disease states (~3.0% of total renal oxygen delivery; range 1.2% to 4.8%). Under normal physiological conditions, blood PO2 is predicted to fall by ~16 mmHg from the root of the renal artery to glomerular entry, with AV oxygen shunting contributing ~40% and oxygen diffusion from arteries to tissue contributing ~60% of this decline. Arterial PO2 is predicted to fall most rapidly from Strahler order 4, under normal physiological conditions. We conclude that AV oxygen shunting normally has only a small impact on renal oxygenation, but may exacerbate renal hypoxia during renal ischemia, hyperfiltration and some cardiovascular disease states.
Purpose: Here, we tested whether combined contrast-enhanced magnetic resonance imaging (CCE-MRI), using a mixture Gadolinium and iron-oxide based contrast agents, can segment the bladder wall from the bladder lumen. CCE-MRI relies on the differences in particle size and contrast mechanisms of two agents for improved image contrast. Methods: Under isoflurane anesthesia, T1-weighted imaging of adult female Sprague-Dawley rat bladder was performed using standard turbo spin echo sequences at 7 Tesla, before and after transurethral instillation of 0.3 mL of single contrast (CE-MRI) or combined contrast mixture (CCE-MRI) composed of 0.4-64 mM of gadolinium chelate (Gd-DTPA/Gadavist) and 5 mM ferumoxytol. Bladder wall contrast was assessed in control group exposed to saline and in bladder injury group exposed to 0.5 mL of protamine sulfate (10 mg/mL) for 30min. Results: CCE-MRI following instillation of 0.4-4 mM Gd-DTPA and 5 mM ferumoxytol mixture achieved segmentation between the bladder lumen and bladder wall. Hyperintensity in the bladder wall combined with hypointensity in the lumen is consistent with the increased diffusion of the dissolved Gd-DTPA and localization of the larger nanoparticles of ferumoxytol in the lumen. The normalized hyperintense signal in the bladder wall increased from 0.46 ± 0.07 in control group to 0.73 ± 0.14 in protamine sulfate exposed group (p < 0.0001). Conclusions: CCE-MRI following instillation of contrast mixture identifies bladder wall changes likely associated with bladder injury with improved image contrast.
Monophosphoryl lipid A (MPLA) is a detoxified derivative of LPS that induces tolerance to LPS and augments host resistance to bacterial infections. Previously we demonstrated that LPS inhibits HCO3- absorption in the medullary thick ascending limb (MTAL) through a basolateral TLR4-MyD88-ERK pathway. Here we examined whether pretreatment with MPLA would attenuate LPS inhibition. MTALs from rats were perfused in vitro with MPLA (1 µg/ml) in bath and lumen or bath alone for 2 hr, then LPS was added to (and MPLA removed from) the bath solution. Pretreatment with MPLA eliminated LPS-induced inhibition of HCO3- absorption. In MTALs pretreated with MPLA plus a phosphoinositide 3-kinase (PI3K) or Akt inhibitor, LPS decreased HCO3- absorption. MPLA increased Akt phosphorylation in dissected MTALs. The Akt activation was eliminated by a PI3K inhibitor and in MTALs from TLR4-/- or TRIF-/- mice. The effect of MPLA to prevent LPS inhibition of HCO3- absorption also was TRIF-dependent. Pretreatment with MPLA prevented LPS-induced ERK activation; this effect was dependent on PI3K. MPLA alone had no effect on HCO3- absorption and MPLA pretreatment did not prevent ERK-mediated inhibition of HCO3- absorption by aldosterone, consistent with MPLA's low toxicity profile. These results demonstrate that pretreatment with MPLA prevents the effect of LPS to inhibit HCO3- absorption in the MTAL. This protective effect is mediated directly through MPLA stimulation of a TLR4-TRIF-PI3K-Akt pathway that prevents LPS-induced ERK activation. These studies identify detoxified TLR4-based immunomodulators as novel potential therapeutic agents to prevent or treat renal tubule dysfunction in response to bacterial infections.
Kidney repair following injury involves the reconstitution of a structurally and functionally intact tubular epithelium. Growth factors and their receptors, such as EGFR, are important in the repair of renal tubules. Exosomes are cell-produced small (~100nm in diameter) vesicles that contain and transfer proteins, lipids, RNAs and DNAs between cells. In this study, we examined the relationship between exosome production and EGFR activation, and the potential role of exosome in wound healing. EGFR activation occurred shortly after scratch wounding in renal tubular cells. Wound repair after scratching was significantly promoted by EGF and suppressed by EGFR receptor inhibitor gefitinib. Interestingly, scratch wounding induced a significant increase of exosome production. The exosome production was decreased by EGF and increased by gefitinib, suggesting a suppressive role of EGFR signaling in exosome production. Conversely, inhibition of exosome release by GW4869 and manumycin-A markedly increased EGFR activation and promoted wound healing. Moreover, exosomes derived from scratch-wounding cells could inhibit wound healing. Collectively, the results indicate that wound healing in renal tubular cells is associated with EGFR activation and exosome production. While EGFR activation promotes wound healing, released exosomes may antagonize EGFR activation and wound healing.
Preemptive treatment with mesenchymal stem cells (MSCs) can attenuate cisplatin-induced acute kidney injury (AKI). However, it is uncertain whether MSC treatment after the development of renal dysfunction prevents AKI progression, or if their immunomodulatory properties contribute to MSC therapy. In this study, human umbilical cord blood (hUCB)-derived MSCs were used to compare the effects and mechanisms of early and late MSC therapy in a murine model. After cisplatin injection into C57BL/6 mice, hUCB-MSCs were administered on day 1 (early treatment) or day 3 (late treatment). With early treatment, cisplatin nephrotoxicity was attenuated as evidenced by decreased blood urea nitrogen (BUN), reduced apoptosis, and tubular injury scores on day 3. Early treatment resulted in downregulation of intrarenal MCP-1 and IL-6 expression and upregulation of IL-10 and VEGF expression. Flow cytometric analysis showed similar populations of infiltrated immune cells in both groups; however, regulatory T cell (Treg) infiltration was 2.5-fold higher in the early treatment group. The role of Tregs was confirmed by the blunted effect of early treatment on renal injury after Treg depletion. In contrast, late treatment (at a time when BUN levels were 2-fold higher than baseline levels) showed no renoprotective effects on day 6. Neither the populations of intrarenal infiltrating immune cells (including Tregs) nor cytokine expression levels were affected by late treatment. Our results suggest that early MSC treatment attenuates renal injury by Treg induction and immunomodulation, whereas late treatment (after the development of renal dysfunction) dose not prevent AKI progression or alter the intrarenal inflammatory micromilieu.
We aim to examine the role of PGF2α receptor (FP), a highly expressed prostaglandin receptor in the DCT, in regulating basolateral 40 pS K channel. The single channel studies demonstrated that PGF2α had a biphasic effect on the 40 pS K channel in the DCT: PGF2α stimulated at low concentrations (less than 500 nM) while at high concentrations (above 1µM) it inhibited the 40 pS K channels. Moreover, neither 13,14-dihydro-15-keto-PGF2α (a metabolite of PGF2α) nor PGE2 was able to mimic the effect of PGF2α on the 40 pS K channel in the DCT. The inhibition of PKC had no significant effect on the 40 pS K channel, however, it abrogated the inhibitory effect of 5 µM PGF2α on the K channel. Moreover, stimulation of PKC inhibited the 40 pS K channel in the DCT, suggesting that PKC mediates the inhibitory effect of PGF2α on the 40 pS K channel. Conversely, the stimulatory effect of PGF2α on the 40 pS K channel was absent in the DCT treated with DPI, a NADPH oxidase (NOX) inhibitor. Also, adding 100 µM H2O2 mimicked the stimulatory effect of PGF2α and increased the 40 pS K channel activity in DCT. Moreover, the stimulatory effect of 500 nM PGF2α and H2O2 was not additive, suggesting the role of superoxide-related species in mediating the stimulatory effect of PGF2α on the 40 pS K channel. The inhibition of Src family tyrosine protein kinase (SFK) not only inhibited the 40 pS K channel in the DCT but also completely abolished the stimulatory effects of PGF2α and H2O2 on the 40 pS K channel. We conclude that PGF2α at low doses stimulates the basolateral 40 pS K channel by NOX- and SFK-dependent mechanism, while at high concentrations it inhibits the K channel by a PKC-dependent pathway.
The goals of this study were to (i) develop a computational model of solute transport and oxygenation in the kidney of the female spontaneously hypertensive rat (SHR), and (ii) apply that model to investigate sex differences in nitric oxide (NO) levels in SHR and their effects on medullary oxygenation and oxidative stress. To accomplish these goals, we first measured NO synthase (NOS) NOS1 and NOS3 protein expression levels in the renal microvessels of male and female SHR. We found that the expression of both NOS1 and NOS3 is higher in the renal vasculature of females compared to males. To predict the implications of that finding on medullary oxygenation and oxidative stress levels, we developed a detailed computational model of the female SHR kidney. The model was based on a published male kidney model, and represents solute transport and the biochemical reactions among O2, NO, and superoxide (O2-) in the renal medulla. Model simulations conducted using both male and female SHR kidney models predicted significant radial gradients in interstitial fluid oxygen tension (PO2) and NO and O2- concentration in the outer medulla and upper inner medulla. The models also predicted that increases in endothelial NO-generating capacity, even when limited to specific vascular segments, may substantially raise medullary NO and oxygen tension levels. Other potential sex differences in SHR, including O2- production rate and anti-oxidant capacity, are predicted to significantly impact oxidative stress levels but effects on NO concentration and PO2 are limited.
Biological soft tissues are viscoelastic because they display time-independent pseudo-elasticity and time-dependent viscosity. Upon an imposed ramp increase then decrease in strain, the resultant stress changes, termed loading and unloading respectively, produce nonlinear stress-strain curves and a reversible reduction in the stress-strain work area that identifies viscosity. However, there is evidence that bladder may also display plasticity; an increase in strain that is unrecoverable unless work is done by the muscle. In the present study, an electronic lever was used to induce controlled changes in stress and strain to determine whether rabbit detrusor smooth muscle (rDSM) is best described as viscoelastic or viscoelastic-plastic. Using sequential ramp loading and unloading cycles, stress-strain and stiffness-stress analyses revealed that rDSM displayed reversible viscoelasticity, and that the viscous component was responsible for establishing a high stiffness at low stresses that increased only modestly with increasing stress compared to the large increase produced when the viscosity was absent and only pseudo-elasticity governed tissue behavior. The study also revealed that rDSM underwent softening correlating with plastic deformation and creep that was reversed slowly when tissues were incubated in a Ca2+-containing solution. Together, the data support a model of DSM as a viscoelastic-plastic material, and plasticity is by motor protein activation. This model explains the mechanism of intrinsic bladder compliance as "slipping" crossbridges, predicts that wall tension is dependent not only on vesicle pressure and radius but also on actomyosin crossbridge activity, and identifies a novel molecular target for compliance regulation both physiologically and therapeutically.
We assessed effects of acute volume reductions induced by administration of diuretics in rats. Direct block Na+ transport produced changes in urinary electrolyte excretion. Adaptations to these effects appeared as alterations in the expression of protein for the distal nephron Na+ transporters NCC and ENaC. Two hours after a single injection of furosemide (6 mg/kg) or hydrochlorothiazide (30 mg/kg) Na+ and K+ excretion increased but no changes in the content of activated forms of NCC (phosphorylated on residue T53) or ENaC (cleaved -subunit) were detected. In contrast, amiloride (0.6 mg/kg) evoked a similar natriuresis that coincided with decreased pT53NCC and increased cleaved ENaC. Alterations in post-translational membrane protein processing correlated with an increase in plasma K+ of 0.6 to 0.8 mM. Decreased pT53NCC occurred within one hour after amiloride injection, while changes in ENaC were slower and were blocked by the mineralocorticoid receptor antagonist spironolactone. Increased ENaC cleavage correlated with elevation of the surface expression of the subunit as assessed by in situ biotinylation. Na depletion induced by 2 hours of furosemide or HCTZ treatment increases total NCC expression without affecting ENaC protein. However restriction of Na intake for 10 hours (during the day) or 18 hours (overnight) increased the abundance of both total NCC and of cleaved α and ENaC. We conclude that the kidneys respond acutely to hyperkalemic challenges by decreasing the activity of NCC while increasing that of ENaC. They respond to hypovolemia more slowly, increasing Na+ reabsorptive capacities of both of these transporters.
Tubulointerstitial injury (TII) plays a crucial role in the progression of diabetic nephropathy (DN), but lack of specific and sensitive biomarkers for monitoring TII in DN management. This study is to investigate whether urinary decoy receptor 2 (uDcR2) could serve as a novel noninvasive biomarker for assessing TII in DN. We recruited 311 type 2 diabetics and 139 DN patients which diagnosed by renal biopsy. uDcR2 levels were measured by ELISA and renal DcR2 expression was detected immunohistochemically. Associations between uDcR2 and renal DcR2, renal functional parameters were evaluated. ROC curve analyzed AUC of uDcR2 for assessing TII. Double staining was undertaken for renal DcR2 with proximal and distal tubular markers, senescent markers p16, p21, and SA-β-gal, and fibrotic markers collagen I and IV. We found DcR2 was primarily expressed in renal proximal tubules; uDcR2 levels were elevated per albuminuria stratum and correlated with renal functional parameters in diabetics, and associated with percentage of tubular DcR2 and TII score in DN. The uDcR2 had an AUC of 0.909 for assessing TII in DN by ROC analysis. Almost all tubular DcR2 was co-expressed with p16 and p21, and nearly more than half of tubular DcR2 was positive for SA-β-gal, primarily in collagen I- and IV-positive regions of DN. Our results indicate uDcR2 could potentially serve as a novel biomarker for TII and may reflect senescence of renal proximal tubular cells in DN pathogenesis.
Removal of renal mass stimulates anatomical and functional adaptations in the surviving nephrons, including elevation in single-nephron glomerular filtration rate (SNGFR) and tubular hypertrophy. A goal of this study is to assess the extent to which the concomitant increases in filtered load and tubular transport capacity preserve homeostasis of water and salt. To accomplish that goal, we developed computational models to simulate solute transport and metabolism along nephron populations in a uninephrectomized (UNX) rat and a 5/6-nephrectomized (5/6-NX) rat. Model simulations indicate that nephrectomy-induced SNGFR increase and tubular hypertrophy go a long way to normalize excretion, but alone are insufficient to fully maintain salt balance. We then identified increases in the protein density of \Red{Na$^+$/K$^+$-ATPase,} Na$^+$/K$^+$-2Cl$^-$ cotransporter (NKCC2), Na$^+$-Cl$^-$ cotransporter (NCC), and epithelial Na$^+$ channel (ENaC) such that the UNX and 5/6-NX models predict urine flow and urinary Na$^+$ and K$^+$ excretions that are similar to sham levels. The models predict that in the UNX and 5/6-NX kidneys fractional water and salt reabsorption is similar to sham along the initial nephron segments (i.e., from the proximal tubule to the distal convoluted tubule), with a need to further reduce Na$^+$ reabsorption and increase K$^+$ secretion primarily along the connecting tubules and collecting ducts to achieve balance. Additionally, the models predict that, given the substantially elevated filtered and thus transport load among each of the surviving nephrons, oxygen consumption per nephron segment in a UNX or 5/6-NX kidney increases substantially. But due to the reduced nephron population, whole-animal renal oxygen consumption is lower. The efficiency of tubular Na$^+$ transport in the UNX and 5/6-NX kidneys is predicted to be similar to sham.
Dietary protein restriction has multiple benefits in kidney disease. Because protein intake is a major determinant of endogenous acid production, it is important that net acid excretion change in parallel during changes in dietary protein intake. Dietary protein restriction decreases endogenous acid production and ¬decreases urinary ammonia excretion, a major component of net acid excretion. Glutamine synthetase (GS) catalyzes the reaction of NH4+ and glutamate, which regenerates the essential amino acid glutamine and decreases net ammonia generation. Because renal proximal tubule GS expression increases during dietary protein restriction, this could contribute to the decreased ammonia excretion. The current study's purpose was to determine proximal tubule GS's role in the renal response to protein restriction. We generated mice with proximal tubule-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Cre-negative (Control) and PT-GS-KO mice in metabolic cages were provided 20% protein diet for 2 days and were then changed to low protein (6%) diet for the next 7 days. Additional PT-GS-KO mice were maintained on 20% protein diet. Dietary protein restriction caused a rapid decrease in urinary ammonia excretion in both genotypes, but PT-GS-KO blunted this adaptive response significantly. This occurred despite no significant genotype-dependent differences in urinary pH or in serum electrolytes. There were no significant differences between Control and PT-GS-KO mice in expression of multiple other proteins involved in renal ammonia handling. We conclude that proximal tubule glutamine synthetase expression is necessary for the appropriate decrease in ammonia excretion during dietary protein restriction.
Acute kidney injury (AKI) causes severe morbidity, mortality, and chronic kidney disease (CKD). Mortality is particularly marked in the elderly and with pre-existing CKD. Oxidative stress is a common theme in models of AKI induced by ischemia/reperfusion (I/R) injury. We recently characterized an intracellular isoform of matrix metalloproteinase-2 (MMP-2) induced by oxidative stress-mediated activation of an alternate promoter in the first intron of the MMP-2 gene. This generates an N-terminal truncated MMP-2 isoform (NTT-MMP-2) that is intracellular and associated with mitochondria. The NTT-MMP-2 isoform is expressed in kidneys of 14 month old mice and in a mouse model of coronary atherosclerosis and heart failure with CKD. We recently determined that NTT-MMP-2 is induced in human renal transplants with delayed graft function and correlated with tubular cell necrosis. To determine mechanism(s) of action, we generated proximal tubule cell-specific NTT-MMP-2 transgenic mice. While morphologically normal at the light microscopic level at 4 months, ultrastructural studies revealed foci of tubular epithelial cell necrosis, the mitochondrial permeability transition and mitophagy. To determine if NTT-MMP-2 expression enhances sensitivity to I/R injury, we performed unilateral I/R to induce mild tubular injury in wild type mice. In contrast, expression of the NTT-MMP-2 isoform resulted in a dramatic increase in tubular cell necrosis, inflammation and fibrosis. NTT-MMP-2 mice had enhanced expression of innate immunity genes and release of danger-associated molecular pattern molecules. We conclude that NTT-MMP-2 "primes" the kidney to enhanced susceptibility to I/R injury via induction of mitochondrial dysfunction. NTT-MMP-2 may be a novel AKI treatment target.
Pathophysiology of chronic kidney disease (CKD) is driven by alterations in surviving nephrons to sustain renal function with ongoing nephron loss. Oxygen supply-demand mismatch, due to hemodynamic adaptations, with resultant hypoxia, plays an important role in the pathophysiology in early CKD. We sought to investigate the underlying mechanisms of this mismatch. We utilized the subtotal nephrectomy (STN) model of CKD to investigate the alterations in renal oxygenation linked to sodium (Na) transport and mitochondrial function in the surviving nephrons. Oxygen delivery was significantly reduced in STN kidneys due to lower renal blood flow. Fractional oxygen extraction was significantly higher in STN. Tubular Na reabsorption was significantly lower per mole of oxygen consumed in STN. We hypothesized that decreased mitochondrial bioenergetic capacity may account for this and uncovered significant mitochondrial dysfunction in early STN kidney- higher oxidative metabolism without an attendant increase in ATP levels, elevated superoxide levels, and alterations in mitochondrial morphology. We further investigated the effect of activation of hypoxia inducible factor 1-α (HIF-1α), a master regulator of cellular hypoxia response. We observed significant improvement in renal blood flow, GFR and tubular Na reabsorption per mole of oxygen consumed with HIF-1α activation. Importantly, HIF-1α activation significantly lowered mitochondrial oxygen consumption and superoxide production and increased mitochondrial volume density. In conclusion, we report significant impairment of renal oxygenation and mitochondrial function at the early stages of CKD and demonstrate the beneficial role of HIF-1α activation on renal function and metabolism.
Megalin is a multiligand, endocytic receptor important for the normal, proximal tubule reabsorption of filtered proteins, hormones, enzymes, essential nutrients, and nephrotoxins. Megalin dysfunction has been associated with acute as well as chronic kidney diseases. Tubular proteinuria has been observed following unilateral ureteral obstruction (UUO) suggesting megalin dysfunction; however, the pathophysiological mechanism has not been resolved. In order to identify potential regulators of megalin expression, we examined renal microRNAs (miRNAs) expression and observed an upregulation of microRNA-148b (miR-148b) in obstructed mouse kidneys 7 days after UUO which was associated with a significant reduction in proximal tubule megalin expression and accumulation of megalin ligands. By in silico miRNA target prediction analysis, we identified megalin messenger RNA (mRNA) as a potential target of miR-148b and confirmed using a dual-luciferase reporter assay that miR-148b targeted the 3'-untranslated region of the megalin gene. Transfection of LLC-PK1 cells with miR-148b mimic reduced endogenous megalin mRNA and protein levels in a concentration-dependent manner, while transfection with miR-148b inhibitor resulted in an increase. Our findings suggest that miR-148b directly downregulates megalin expression and that miR-148b negatively regulates megalin expression in UUO-induced kidney injury. Furthermore, the identification of a miRNA regulating megalin expression may allow for targeted interventions to modulate megalin function and proximal tubule uptake of proteins as well as other ligands.
Adult rats exposed to maternal separation (MatSep) are normotensive but display lower glomerular filtration rate and increased renal neuroadrenergic drive. The aim of this study was to determine the renal alpha-adrenergic receptor density and the renal vascular responsiveness to adrenergic stimulation in male rats exposed to MatSep. In addition, baroreflex sensitivity was assessed to determine a component of neural control of the vasculature. Using tissue collected from 4-month-old MatSep and control rats, alpha 1-adrenergic receptors (α1-ARs) were measured in renal cortex and isolated renal vasculature using receptor binding assay, and the α-AR subtype gene expression was determined by RT-PCR. Renal cortical α1-AR density was similar between MatSep and control tissues (Bmax=44±1 vs. 42±2 fmol/mg protein, respectively); however, MatSep reduced α1-AR density in renal vasculature (Bmax=47±4 vs. 62±4 fmol/mg protein, p<0.05, respectively). In a separate group of rats, the pressor, bradycardic and renal vascular constrictor responses to acute norepinephrine injection (NE, 0.03-0.25 μg/μl) were determined in anesthetized rats. MatSep rats showed attenuated NE-induced renal vasoconstriction compared to control rats (p <0.05). A third group of rats was infused at steady state with the α1 agonist phenylephrine (PE, 10 μg/min, i.v.) and vasodilator sodium nitroprusside (SNP, 5 μg/min, i.v.). The difference between the HR/MAP slopes was indicative of reduced baroreflex sensitivity in MatSep vs. control rats (-0.45±0.04 vs. -0.95±0.07 bpm/mmHg, p<0.05). These data support the notion that reduced α-adrenergic receptor expression and function in the renal vasculature could develop secondary to MatSep-induced overactivation of the renal neuroadrenergic tone.
Bladder wall fibrosis is a major complication of ketamine-induced cystitis (KC), but the underlying pathogenesis is poorly understood. The aim of the present study was to elucidate the mechanism of ketamine-induced fibrosis in association with epithelial-to-mesenchymal transition (EMT) mediated by transforming growth factor-β1 (TGF-β1). Sprague-Dawley rats were randomly distributed into four groups, which received saline, ketamine, ketamine combined with a TGF-β receptor (TβR) inhibitor (SB505124) for 16 weeks, or 12 weeks of ketamine and 4 weeks of abstinence. In addition, the profibrotic effect of ketamine was confirmed in SV-40 immortalized human uroepithelial (SV-HUC-1) cells. The ketamine-treated rats displayed voiding dysfunction and decreased bladder compliance. Bladder fibrosis was accompanied by the appearance of a certain number of cells expressing both epithelial and mesenchymal markers, indicating that epithelial cells might undergo EMT upon ketamine administration. Meanwhile, the expression level of TGF-β1 was significantly up-regulated in the urothelium of bladders in ketamine-treated rats. Treatment of SV-HUC-1 cells with ketamine increased the expression of TGF-β1 and EMT-inducing transcription factors, resulting in the down-regulation of E-cadherin and up-regulation of fibronectin and α-smooth muscle actin. Administration of SB505124 inhibited EMT and fibrosis both in vitro and vivo. In addition, withdrawal from ketamine did not lead to recovery of bladder urinary function or decreased fibrosis. Taken together, our study shows for the first time that EMT might contribute to bladder fibrosis in KC. TGF-β1 may have an important role in bladder fibrogenesis via an EMT mechanism.
Urethral smooth muscle (USM) contributes to urinary continence by contracting during the urine storage phase, which is mainly mediated by activation of post-junctional α1-adrenoceptors. Males and females show differences in the functioning of the lower urinary tract and the most common urinary tract symptoms (LUTS). LUTS in men typically occur in association with bladder outlet obstruction, whereas in women urinary urge-incontinence symptoms are more common. Therefore, this study aimed to evaluate sex differences in α1-adrenoceptor subtype expression and their importance in proximal urethra contraction in mouse (C57BL6/J) and marmoset (Callithrix jacchus). Contractile responses to phenylephrine, noradrenaline, potassium chloride (KCl) and electrical-field stimulation (EFS) were evaluated. Phenylephrine, noradrenaline, KCl and EFS produced markedly greater contractions in male mice and marmoset USM compared with females. The sex differences remained unchanged by L-NAME (NOS inhibitor), atropine (muscarinic antagonist) and PPADS (P2X1 purinoceptor antagonist). Additionally, selective α1A- (but not α1B- and α1D) adrenoceptor antagonists significantly reduced phenylephrine-induced USM contractions. qRT-PCR for α1A, B and C-adrenoceptor subtypes revealed a marked presence of α1A adrenoceptor subtype in male USM, but not females. Male mouse urethra also exhibited a higher tyrosine hydroxylase mRNA expression. Histomorphometric analysis showed a greater USM area in male than female mice. In conclusion, male mouse and marmoset proximal USM shows strong α1A adrenoceptor-induced contractions and abundant α1A adrenoceptor expression, whereas α1A adrenoceptor-mediated mechanisms are much less important in females. The differential expression of α1-adrenoceptors in the proximal urethra may contribute to the higher incidence of urinary incontinence in women and obstructed voiding in men.
The gene encoding the aquaporin-2 water channel is regulated transcriptionally in response to vasopressin. In the renal collecting duct, vasopressin stimulates the nuclear translocation and phosphorylation (at Ser552) of β-catenin, a multifunctional protein that acts as a transcriptional co-regulator in the nucleus. The purpose of this study was to identify β-catenin interacting proteins that may be involved in transcriptional regulation in rat inner medullary collecting duct (IMCD) cells using both experimental and computational approaches. We used a standard chromatin immunoprecipitation procedure coupled to mass spectrometry (ChIP-MS) in a nuclear fraction isolated from rat IMCD suspensions. Over four biological replicates, we reproducibly identified 43 β-catenin binding proteins, including several known β-catenin binding partners as well as novel interacting proteins. Multiple proteins involved in transcriptional regulation were identified (Taf1, Jup, Tdrd3, Cdh1, Cenpj and several histones). Many of the identified β-catenin binding partners were found in prior studies to translocate to the nucleus in response to vasopressin. There was only one DNA-binding transcription factor (TF), specifically Taf1, part of the RNA-polymerase II pre-initiation complex. To identify sequence-specific TFs that may interact with β-catenin, Bayes' Theorem was used to integrate data from several information sources. The analysis identified several TFs with potential binding sites in the Aqp2 gene promoter that could interact with β-catenin in the regulation of Aqp2 gene transcription, specifically Jun, Junb, Jund, Atf1, Atf2, Mef2d, Usf1, Max, Pou2f1 and Rxra. The findings provide information necessary for modeling the transcriptional response to vasopressin.
Serum fibroblast growth factor 23 (FGF23) increases progressively in chronic kidney disease (CKD) and is associated with increased mortality. FGF23 is synthesized in osteoblasts and osteocytes; however, the factors regulating its production are not clear. Patients with CKD have decreased renal acid excretion leading to metabolic acidosis (MET). During MET, acid is buffered by bone with release of mineral calcium (Ca) and phosphate (P). MET increases intracellular Ca signaling and cyclooxygenase 2 (COX2)-induced prostaglandin production in the osteoblast leading to decreased bone formation and increased bone resorption. We found that MET directly stimulates FGF23 in mouse bone organ cultures and primary osteoblasts. We hypothesized that MET increases FGF23 through similar pathways that lead to bone resorption. Neonatal mouse calvariae were incubated in neutral (NTL, pH=7.44, Pco2=38mmHg, [HCO3-]=27mM) or acid (MET, pH=7.18, Pco2=37mmHg, [HCO3-]=13mM) medium without or with 2-APB (50 μM), an inhibitor of intracellular Ca signaling or NS-398 (1 μM), an inhibitor of COX2. Each agent significantly inhibited MET stimulation of medium FGF23 protein and calvarial FGF23 RNA as well as bone resorption at 48h. To exclude the potential contribution of MET-induced bone P release, we utilized primary calvarial osteoblasts. In these cells each agent inhibited MET stimulation of FGF23 RNA expression at 6h. Thus, stimulation of FGF23 by MET in mouse osteoblasts utilizes the same initial signaling pathways as MET-induced bone resorption. Therapeutic interventions directed toward correction of MET, especially in CKD, have the potential to not only prevent bone resorption but also lower FGF23 and perhaps decrease mortality.
The natriuretic hormone CCK exhibits its gene transcripts in total kidney extracts. To test the possibility of CCK acting as an intrarenal mediator of sodium excretion, we examined mouse kidneys by: (i) an in situ hybridization technique for CCK mRNA in animals fed a normal or a high-sodium diet; (ii) immuno-electron microscopy for the CCK peptide, (iii) an in situ hybridization method and immunohistochemistry for the CCK-specific receptor CCKAR; (iv) confocal image analysis of receptor-mediated Ca2+ responses in isolated renal tubules; and (v) metabolic cage experiments for the measurement of urinary sodium excretion in high salt-fed mice either treated or untreated with the CCKAR antagonist lorglumide. Results showed the CCK gene to be expressed intensely in the inner medulla and moderately in the inner stripe of the outer medulla, with the expression in the latter being enhanced by high sodium intake. Immunoreactivity for the CCK peptide was localized to the rough endoplasmic reticulum of the medullary interstitial cells in corresponding renal regions, confirming it as a secretory protein. Gene transcripts, protein products, and the functional activity for CCKAR were consistently localized to the late proximal tubule segments (S2 and S3) in the medullary rays, and the outer stripe of the outer medulla. Lorglumide significantly diminished natriuretic responses of mice to a dietary sodium load without altering the glomerular filtration rate. These findings suggest that the medullary interstitial cells respond to body fluid expansion by CCK release for feedback regulation of the late proximal tubular reabsorption.
Collagen IV (Col IV) is a major component of expanded glomerular extracellular matrix in diabetic nephropathy and Smad1 is a key molecule regulating Col IV expression in mesangial cells (MCs). The present study was conducted to determine if Smad1 pathway and Col IV protein abundance were regulated by store-operated Ca2+ entry (SOCE). In cultured human MCs, pharmacological inhibition of SOCE significantly increased the total amount of Smad1 protein. Activation of SOCE blunted high glucose-increased Smad1 protein content. Treating human MCs with angiotensin II at 1 µM for 15 min, or high glucose for 3 days, or TGF-β1 at 5 ng/ml for 30 min increased the level of phosphorylated Smad1. However, the phosphorylation of Smad1 by those stimuli was significantly attenuated by activation of SOCE. Knocking down Smad1 reduced, but expressing Smad1 increased the amount of Col IV protein. Furthermore, activation of SOCE significantly attenuated high glucose-induced Col IV protein production and blockade of SOCE substantially increased the abundance of Col IV. To further verify those in vitro findings, we downregulated SOCE specifically in MCs in mice using siRNA against Orai1 (the channel protein mediating SOCE) delivered by the targeted nanoparticle delivery system. Immunohistochemical examinations showed that expression of both Smad1 and Col IV proteins were significantly greater in the glomeruli with positively-transfected Orai1 siRNA compared to the glomeruli from the mice without Orai1 siRNA treatment. Taken together, our results indicate that SOCE negatively regulates the Smad1 signaling pathway and inhibits Col IV protein production in MCs.
Calcium (Ca2+) and Magnesium (Mg2+) reabsorption along the renal tubule is dependent on distinct trans- and paracellular pathways. Our understanding of the molecular machinery involved is increasing. Ca2+ and Mg2+ reclamation in kidney is dependent on a diverse array of proteins, which are important for both forming divalent cation permeable pores and channels, but also for generating the necessary driving forces for Ca2+ and Mg2+ transport. Alterations in these molecular constituents lead to profound effects on tubular Ca2+ and Mg2+ handling. Diuretics are used to treat a large range of clinical conditions, but most commonly for the management of blood pressure and fluid balance. The pharmacological targets of diuretics generally directly facilitate sodium (Na+) transport, but also indirectly affect renal Ca2+ and Mg2+ handling, i.e. by establishing a prerequisite electrochemical gradient. It is therefore not surprising that substantial alterations in divalent cation handling can be observed following diuretic treatment. The effects of diuretics on renal Ca2+ and Mg2+ handling are reviewed in the context of the current understanding of basal molecular mechanisms of Ca2+ and Mg2+ transport. Acetazolamide, osmotic diuretics, NHE3 inhibitors and antidiabetic SGLT blocking compounds, target the proximal tubule, where paracellular Ca2+ transport predominates. Loop-diuretics and ROMK inhibitors block thick ascending limb transport, a segment with significant paracellular Ca2+ and Mg2+ transport. Thiazides target the distal convoluted tubule, however, their effect on divalent cation transport is not limited to that segment. Finally, potassium-sparing diuretics, which inhibit electrogenic Na+ transport at distal sites, can also affect divalent cation transport.
Deficiency of cyclooxygenase-2 (COX-2) activity in the early postnatal time course causes impairment of kidney development leading to kidney insufficiency. We hypothesize that impaired NaCl reabsorption during the first days of life is a substantial cause for nephrogenic defects observed in COX-2-/- mice and that salt supplementation corrects these defects. Daily injections of 0.8 mg/g/d NaCl for the first 10 days after birth ameliorated impaired kidney development in COX-2-/- pups resulting in an increase in glomerular size and reduction of immature superficial glomeruli. However, impaired renal subcortical growth was not corrected. Increasing renal tubular flow by volume load or injections of KCl did not relieve the renal histomorphological damage. Administration of torsemide and spironolacton also affected nephrogenesis resulting in diminished glomeruli and cortical thinning. Treatment of COX-2-/- pups with NaCl/DOCA caused a stronger mitigation of glomerular size and also induced a slight but significant growth of cortical tissue mass. After birth renal mRNA expression of NHE3, NKCC2, ROMK, NCCT, ENaC, and Na+/K+-ATPase increased relative to day P2 in wild type mice. However, in COX-2-/- mice a significantly lower expression was observed for NCCT, while DOCA/NaCl treatment significantly increased NHE3 and ROMK expression. Regarding long-term effects of postnatal NaCl/DOCA injections improved kidney function with normalization of pathologically enhanced creatinine and urea plasma levels and albumin excretion was observed. In summary we present evidence that a salt supplementation during the COX-2 dependent time frame of nephrogenesis partly reverses renal morphological defects in COX-2-/- mice and improves kidney function.
Abstract Background: Renin-angiotensin system (RAS) plays a critical role in the progression of renal fibrosis. Angiotensin II type 1 receptor (AT1R) belongs to the B family of G protein coupled receptors (GPCRs) family. β-arrestins are known as negative regula-tors of GPCRs. Recently, β-arrestins have been found to regulate multiple intracellu-lar signaling pathways independent of G proteins. In this study we investigated the role of β-arrestins in regulating extracellular matrix (ECM) synthesis in renal fibrosis. Methods: Rat kidney fibroblast cell line (NRK-49F) was treated with β-arrestin bi-ased agonist SII ([1-sar, 4, 8-ile]-angiotensin II), which does not initiate AT1R-G pro-tein signaling. The cells were transfected with recombinant adenoviruses expressing β-arrestin2 gene or siRNA targeting β-arrestin2. Unilateral ureteral obstruction (UUO) model was used in vivo. Results: The mRNA and protein levels of β-arrestin2, not β-arrestin1, were signifi-cantly up-regulated in the UUO kidney tissues. SII induced the binding of β-arrestin2 with AT1R. SII increased the synthesis of collagen I and fibronectin in NRK-49F, which were abolished when pretreated with candesartan (AT1 receptor blocker). Transfection of siRNA targeting β-arrestin2 decreased the effects of SII on the ECM synthesis. Overexpression of β-arrestin2 enhanced SII-stimulated ECM synthesis. SII induced ERK1/2 phosphorylation in NRK-49F. Transfection of siRNA targeting β-arrestin2 inhibited ERK phosphorylation. Overexpression of β-arrestin2 increased ERK1/2 phosphorylation. Conclusions: Our study firstly showed that AT1R-β-arrestin2 pathway signaling play an important role in renal fibrosis, although it was previously believed that AT1R-G proteins pathway play a major role. Targeting β-arrestin2 may be potential therapeutic agents for renal fibrosis.
Chronic kidney disease (CKD) patients have exercise intolerance associated with increased cardiovascular mortality. Previous studies demonstrate that blood pressure (BP) and sympathetic nerve responses to handgrip exercise are exaggerated in CKD. These patients also have decreased nitric oxide (NO) bioavailability and endothelial dysfunction which could potentially lead to an impaired ability to vasodilate during exercise. We hypothesized that CKD patients have exaggerated BP responses during maximal whole body exercise and that endothelial dysfunction correlates with greater exercise pressor responses in these patients. Brachial artery flow-mediated dilation (FMD) was assessed before maximal treadmill exercise in 56 participants: 38 CKD (56.7±1.2 years old, 38 males) and 21 controls (52.8±1.8 years old, 20 males). During maximal treadmill exercise, the slope-of-rise in systolic BP (+10.32 mmHg/stage vs. +7.75 mmHg/stage, p<0.001), mean arterial pressure (+3.50 mmHg/stage vs. +2.63 mmHg/stage, p=0.004), and heart rate (+11.87 mmHg/stage vs. +10.69 mmHg/stage, p=0.031) was significantly greater in CKD compared to controls. Baseline FMD was significantly lower in CKD (2.76±0.42% vs. 5.84±0.97%, p=0.008). Lower FMD values were significantly associated with a higher slope of rise in systolic BP (+11.05 mmHg/stage vs. 8.71 mmHg/stage, p=0.003) during exercise in CKD, as well as poorer exercise capacity measured as peak oxygen uptake (VO2 peak; 19.47±1.47 ml min-1 kg-1 vs. 24.57±1.51 ml min-1 kg1, p<0.001). These findings demonstrate that low FMD in CKD correlates with augmented BP responses during exercise and lower peak VO2, suggesting that endothelial dysfunction may contribute to exaggerated exercise pressor responses and poor exercise capacity in CKD patients.
Changes in hemodynamics and blood pressure occur shortly before and after childbirth regardless of the mode of delivery. This study aimed to test the hypothesis that parturition induces a temporal increase in podocyturia monitored by podocyte-specific protein podocin mRNA expression levels (Pod-mRNA). A total of 105 urine specimens, consisting of 43 and 62 from 18 and 20 otherwise healthy women with vaginal (VD) and elective cesarean deliveries (ECS), respectively, were studied. Determination of urine protein and creatinine concentrations and quantitative analyses of Pod-mRNA, nephrin mRNA (Nep-mRNA), synaptopodin mRNA (Syn-mRNA), and aquaporin 2 mRNA expression (AQP2-mRNA) were performed using RT-PCR in pelleted urine samples. Levels of mRNA expression were corrected by urine creatinine concentration. Podocyturia increased significantly concomitant with significantly decreased Nep:Pod-mRNA ratio (NPR) in the urine collected immediately before or after childbirth regardless of the delivery mode compared to urine collected before commencement of labor or on postpartum day 3 or later. Podocyturia was significantly negatively correlated with NPR (correlation coefficient [r] = -0.614/-0.750 for VD/ECS women, respectively) as well as Syn:Pod-mRNA ratio. Systolic blood pressure exceeded 140 mmHg during labor in 50% of VD women and mean arterial pressure was significantly positively correlated with podocyturia during labor in VD women (r = 0.733). Thus, parturition induces a transient increase in urine podocytes with reduced Nep- and Syn-mRNA expressions. Glomerular podocytes with reduced Nep-mRNA and Syn-mRNA levels were suggested to be likely to detach from the glomerular basement membrane around childbirth.
The thiazide-sensitive NaCl cotransporter (NCC), located apically in distal convoluted tubule epithelia, regulates the fine-tuning of renal sodium excretion. Three isoforms of NCC are generated through alternative splicing of the transcript, of which the third isoform has been the most extensively investigated in pathophysiological conditions. The aim of this study was to investigate the effect of different anti-hypertensive treatments on the abundance and phosphorylation of all three NCC isoforms in urinary extracellular vesicles (uEVs) of essential hypertensive patients. In uEVs isolated from patients (n=23) before and after hydrochlorothiazide or valsartan treatment the abundance and phosphorylation of the NCC isoforms was determined. Additionally, clinical biochemistry and blood pressure of the patients was assessed. Our results show that NCC detected in human uEVs has a glycosylated and oligomeric structure, comparable to NCC present in human kidney membrane fractions. Despite the inhibitory action of hydrochlorothiazide on NCC activity, immunoblot analysis of uEVs showed significantly increased abundance of NCC isoforms 1 and 2 (NCC1/2), total NCC (NCC1-3), and the phosphorylated form of total NCC (pNCC1-3-T55/T60) in essential hypertensive patients treated with hydrochlorothiazide, but not with valsartan. This study highlights that NCC1/2, NCC1-3, and pNCC1-3-T55/T60 are upregulated by hydrochlorothiazide, and the increase in NCC abundance in uEVs of essential hypertensive patients correlates with the blood pressure response to hydrochlorothiazide.
About 50% of the Na+ reabsorbed in thick ascending limbs traverses the paracellular pathway. Nitric oxide (NO) reduces the permselectivity of this pathway via cGMP, but its effects on absolute Na+ (PNa+) and Cl- (PCl-) permeabilities are unknown. To address this, we measured the effect L-arginine (0.5mmol/L; NO synthase substrate) and cGMP (0.5mmol/L) on PNa+ and PCl- calculated from transepithelial resistance (Rt) and PNa+/PCl-. Rt was 7722±1554 ohm-cm in the control period and 6318±1757 ohm-cm after L-arginine treatment (p<0.05). PNa+/PCl- was 2.0±0.2 in the control period and 1.7±0.1 after L-arginine (p<0.04). Calculated PNa+ and PCl- were 3.52±0.2 x10-5 cm/sec and 1.81±0.10 x10-5 cm/sec respectively in the control period. After L-arginine they were 6.65±0.69 x10-5 cm/sec (p<0.0001 vs control) and 3.97±0.44 x10-5 cm/sec (p<0.0001), respectively. NOS inhibition with L-NAME (5mmol/L) prevented L-arginine's effect on Rt. Next we tested the effect of cGMP. Rt in the control period was 7592±1470 ohm-cm and 4796±847 ohm-cm after dibutyryl-cGMP (0.5mmol/L; db-cGMP) treatment (p<0.04). PNa+/PCl- was 1.8±0.1 in the control period and 1.6±0.1 after db-cGMP (p<0.03). PNa+ and PCl- were 4.58±0.80 x10-5 cm/sec and 2.66±0.57 x10-5 cm/sec, respectively, for the control period, and 9.48±1.63 x10-5 cm/sec (p<0.007) and 6.01±1.05 x10-5 cm/sec (p<0.005), respectively, after db-cGMP. We modeled NO's effect on luminal Na+ concentration along the thick ascending limb. We found that NO's effect on the paracellular pathway reduces net Na+ reabsorption, and that the magnitude of this effect is equal to that due to NO's inhibition of transcellular transport.
Transforming growth factor-alpha (TGFA) has been shown to play a role in experimental chronic kidney disease (CKD) associated with nephron reduction, while its role in diabetic kidney disease (DKD) is unknown. We have previously reported the development of a neutralizing antibody to human/mouse TGFA. This antibody was used to determine the role of TGFA in two models of renal disease, the remnant surgical reduction model and the uninephrectomized (uniNx) db/db DKD model. In addition, the role of TGFA in driving DKD was examined using an AAV approach to overexpress TGFA in a DKD model. In vivo blockade of TGFA attenuated kidney disease progression in both non-diabetic 129S6 nephron reduction and type 2 diabetic uniNx db/db models, whereas overexpression of TGFA in uniNx db/db model accelerated renal disease. Therapeutic activity of the TGFA antibody was enhanced with renin angiotensin system (RAS) inhibition with further improvement in renal parameters. In addition, we determined that urine and serum TGFA are increased in human DKD. These findings suggest a pathologic role for TGFA in DKD and support the possibility that therapeutic administration of neutralizing antibodies could provide a novel treatment for the disease.
BACKGROUND: Hyperuricemia has been reported to affect renal hemodynamics. In a recent study, both low and high levels of serum uric acid (SUA) were found to be associated with loss of kidney function. Objective: The goal of this study was to evaluate the relationship between SUA levels and intrarenal hemodynamic parameters in healthy subjects, utilizing plasma clearance of para-aminohippurate (CPAH) and inulin (Cin). Subjects and Methods: Glomerular hemodynamics were evaluated by CPAH and Cin in 48 healthy subjects (54.6 ± 13.4 years). Intrarenal hemodynamic parameters, including efferent (Re) and afferent (Ra) arteriolar resistance, were calculated using Gomez's formulae. Relationships of SUA levels with these intrarenal hemodynamic parameters were examined. Results: In quadratic regression analysis, SUA levels had a significant, inverse U-shaped relationship with Cin (p < 0.0001, R2 = 0.350) and CPAH (p = 0.0093, R2 = 0.188), and a U-shaped relationship with Ra (p = 0.0011, R2 = 0.262). In multiple regression analysis with normal (3.0 to 6.0 mg/dL) and mildly low or high (<3.0 or >6.0 mg/dL) SUA levels entered as dummy variables of 0 and 1, respectively, mildly low or high SUA levels were significantly and independently associated with Ra (β = 0.230, p = 0.0403) after adjustment for several factors (R2 = 0.597, p < 0.0001). Conclusions: Both mild hyperuricemia and mild hypouricemia are significantly associated with increased Ra; albeit weakly. The increase in Ra in subjects with mild hyperuricemia or hypouricemia may cause renal hemodynamic abnormalities, possibly leading to a decline in renal function.
Thickening of the glomerular basement membrane (GBM) and expansion of the mesangial matrix are hallmarks of diabetic nephropathy (DN) generally considered to emerge from different sites of overproduction; GBM components from podocytes and mesangial matrix from mesangial cells. Re-evaluation of 918 biopsies with DN revealed strong evidence that these mechanisms are connected to each other, wherein excess GBM components fail to undergo degradation and are deposited in the mesangium. These data do not exclude that also mesangial cells synthesize components that contribute to the accumulation of matrix in the mesangium. Light, electron microscopic, immunofluorescence and in-situ hybridization studies clearly show that the thickening of the GBM is not only due to overproduction of components of the mature GBM (α3 and α5 chains of collagen IV and agrin) by podocytes, but also to resumed increased synthesis of the α1 chain of collagen IV and of perlecan by endothelial cells usually seen during embryonic development. We hypothesize that these abnormal production mechanisms are caused by different processes; overproduction of mature GBM-components by the diabetic milieu and regression of endothelial cells to an embryonic production mode by decreased availability of mediators from podocytes. Keywords: Turnover of GBM components Production of mesangial matrix Resumption of embryonic GBM synthesis
Chronic kidney diseases (CKD) generally lead to renal fibrosis. Despite great progresses have been made in identifying molecular mediators of fibrosis, the mechanism that governs renal fibrosis remains unclear and so far no effective therapeutic anti-fibrosis strategy is available. Here we demonstrated that a switch of metabolism from oxidative phosphorylation to aerobic glycolysis (Warburg effect) in renal fibroblasts was the primary feature of fibroblast activation during renal fibrosis, and that suppressing renal fibroblast aerobic glycolysis could significantly reduce renal fibrosis. Both gene and protein assay showed that the expression of glycolysis enzymes were upregulated in mouse kidneys with unilateral ureter obstruction (UUO) surgery or in TGFβ1-treated renal interstitial fibroblasts. Aerobic glycolysis flux, indicated by glucose uptake and lactate production, was increased in mouse kidney with UUO nephropathy or TGFβ1-treated renal interstitial fibroblasts and positively correlated with fibrosis process. In line with this, we found that increasing aerobic glycolysis can remarkedly induce myofibroblasts activation while aerobic glycolysis inhibitors shikonin and 2-deoxyglucose (2DG) attenuate UUO-induced mouse renal fibrosis and TGFβ1-stimulated myofibroblast activation. Furthermore, mechanistic study indicated that shikonin inhibits renal aerobic glycolysis via reducing phosphorylation of pyruvate kinase type M2 (PKM2), a rate-limiting glycolytic enzyme associated with cell reliance on aerobic glycolysis. In conclusion, our findings demonstrate the critical role of aerobic glycolysis in renal fibrosis and provide the treatment with aerobic glycolysis inhibitors as a potential anti-fibrotic strategy.
Altered expression of nephrin underlies the pathophysiology of proteinuria in both congenital and acquired nephrotic syndrome. However, the epigenetic mechanisms of nephrin gene regulation remain elusive. Here, we show that Wolf-Hirschhorn syndrome candidate 1-like 1 long form (WHSC1L1-L) is a novel epigenetic modifier of nephrin gene regulation. WHSC1L1-L was associated with histone H3K4 and H3K36 in human embryonic kidney cells. WHSC1L1-L gene was expressed in the podocytes and functional protein product was detected in these cells. WHSC1L1-L was found to bind nephrin but not other podocyte specific gene promoters, leading to its inhibition/suppression, abrogating the stimulatory effect of WT1 and NF-kB. Gene knockdown of WHSC1L1-L in primary cultured podocytes accelerated the transcription of nephrin but not CD2AP. An in vivo zebrafish study involving the injection of Whsc1l1 mRNA into embryos demonstrated an apparent reduction of nephrin mRNA but not podocin and CD2AP mRNA. Immunohistochemistry showed that both WHSC1L1-L and nephrin emerged at the S-shaped body stage in glomeruli. Immunofluorescence and confocal microscopy displayed WHSC1L1 to colocalize with trimethylated H3K4 in the glomerular podocytes. Chromatin immunoprecipitation assay revealed the reduction of the association of trimethylated H3K4 at the nephrin promoter regions. Finally, nephrin mRNA was upregulated in the glomerulus at the early proteinuric stage of mouse nephrosis, which was associated with the reduction of WHSC1L1. In conclusion, our results demonstrate that WHSC1L1-L acts as a histone methyltransferase in podocytes and regulates nephrin gene expression, which may in turn contribute to the integrity of the slit diaphragm of the glomerular filtration barrier.
The direct renin inhibitor aliskiren has been shown to retain and persist in medullary collecting ducts even after treatment was discontinued, suggesting a new mechanism of action for this drug. The purpose of the present study was to investigate whether aliskiren regulates renal aquaporin expression in the collecting ducts and improves urinary concentrating defect induced by lithium in mice. The mice were either fed with normal chow or LiCl diet (40mM/kg dry food/day for 4 days and 20mM/kg dry food/day for last 3 days) for seven days. Some mice were intraperitoneally injected with aliskiren (50mg/kg BW/day in saline). Aliskiren significantly increased protein abundance of AQP2 in the kidney inner medulla in mice. In inner medulla collecting duct cell suspension, aliskiren markedly increased AQP2 and pS256-AQP2 protein abundance which was significantly inhibited either by adenylyl cyclase inhibitor MDL-12330A or by PKA inhibitor H89, indicating an involvement of the cAMP-PKA signaling pathway in aliskiren-induced increased AQP2 expression. Aliskiren treatment improved urinary concentrating defect in lithium-treated mice, and partially prevented the decrease of AQP2 and pS256-AQP2 protein abundance in inner medulla of the kidney. In conclusion, the direct renin inhibitor aliskiren upregulates AQP2 protein expression in inner medullary collecting duct principal cells and prevents lithium-induced nephrogenic diabetes insipidus (NDI) likely via cAMP-PKA pathways.
We previously reported a sex-specific effect of antenatal treatment with betamethasone (Beta) on sodium (Na+) excretion in adult sheep whereby treated males but not females had an attenuated natriuretic response to Ang-(1-7). The present study determined the Na+ uptake and nitric oxide (NO) response to low dose Ang-(1-7) (1 pM) in renal proximal tubule cells (RPTC) from adult male and female sheep antenatally exposed to Beta or vehicle. Data were expressed as % of basal uptake or area under the curve (AUC) for Na+ or % of control for NO. Male Beta RPTC exhibited greater Na+ uptake than male vehicle cells (433±28% vs. 330±26%; p<0.05); however, Beta exposure had no effect on Na+ uptake in the female cells (255±16% vs. 255±14%; p>0.05). Ang-(1-7) significantly inhibited Na+ uptake in RPTC from vehicle male (214±11%) and from both vehicle (190±14%) and Beta (209±11%) females, but failed to attenuate Na+ uptake in Beta male cells. Beta exposure also abolished stimulation of NO by Ang-(1-7) in male, but not female RPTC. Both the Na+ and NO responses to Ang-(1-7) were blocked by Mas receptor antagonist [D-Ala7]-Ang-(1-7). We conclude that the tubular Ang-(1-7)-Mas-NO pathway is attenuated in males and not females by antenatal Beta exposure. Moreover, since primary cultures of RPTC retain both the sex and Beta-induced phenotype of the adult kidney in vivo they appear to be an appropriate cell model to examine the effects of fetal programming on Na+ handling by the renal tubules.
Damage to endothelial cells contributes to acute kidney injury (AKI) by leading to impaired perfusion. Endothelial colony-forming cells (ECFCs) are endothelial precursor cells with high proliferative capacity, pro-angiogenic activity, and in vivo vessel forming potential. We hypothesized that ECFCs may ameliorate the degree of AKI and/or promote repair of the renal vasculature following ischemia/reperfusion (I/R). Rat pulmonary microvascular ECs (PMVEC) with high proliferative potential were compared with pulmonary artery ECs (PAEC) with low proliferative potential in rats subjected to renal I/R. PMVEC administration reduced renal injury and hastened recovery as indicated by serum creatinine and tubular injury scores, while PAEC did not. Vehicle-treated control animals showed consistent reductions in renal medullary blood flow (MBF) within 2 hours of reperfusion, while PMVEC protected against loss in MBF as measured by laser Doppler. Interestingly, PMVEC mediated protection occurred in the absence of homing to the kidney. Conditioned medium (CM) from human cultured cord blood ECFC also conveyed beneficial effects against I/R injury and loss of MBF. Moreover, ECFC-CM significantly reduced the expression of adhesion molecules such as ICAM-1 and p-selectin, and decreased the number of differentiated lymphocytes typically recruited into the kidney following renal ischemia. Taken together, these data suggest that ECFC secrete factors that preserve renal function post ischemia, in part, by preserving microvascular function.
The Na+/K+/2Cl- cotransporter (NKCC2) on the loop of Henle is the site of action of furosemide. Since outer medullary potassium channel (ROMK) inhibitors prevent reabsorption by NKCC2, we tested the hypothesis that ROMK inhibition with a novel, selective ROMK inhibitor (Compound C) blocks tubuloglomerular feedback (TGF) and reduces vascular resistance. Loop perfusion of either ROMK inhibitor or furosemide caused dose-dependent blunting of TGF, but the response to furosemide was 10-fold more sensitive (IC50= 10-6M for furosemide and IC50=10-5M for Compound C). During systemic infusion, both diuretics inhibited TGF but ROMK inhibitor was 10-fold more sensitive (Compound C: 63% inhibition; furosemide: 32% inhibition). Despite blockade of TGF, one hour of constant systemic infusion of both diuretics reduced the glomerular filtration rate (GFR) and renal blood flow (RBF) by 40-60% and increased renal vascular resistance (RVR) by 100-200%. Neither diuretic altered blood pressure or hematocrit. Proximal tubule hydrostatic pressures (PPT) increased transiently with both diuretics (Compound C: 56% increase; furosemide: 70% increase) but returned to baseline. ROMK inhibitor caused more natriuresis (3400% increase vs 1600% increase) and calciuresis (1200% increase vs 800% increase) but less kaliuresis (33% increase vs 167% increase) than furosemide. In conclusion, blockade of ROMK or Na+/K+/2Cl- transport inhibits TGF yet increases renal vascular resistance. The renal vasoconstriction was independent of volume depletion, blood pressure, TGF or PPT.
Lymphatic vessels are vital for the trafficking of immune cells from the interstitium to draining lymph nodes during inflammation. Hypertension is associated with renal infiltration of activated immune cells and inflammation, however it is unknown how renal lymphatic vessels change in hypertension. We hypothesized that renal macrophage infiltration and inflammation would cause increased lymphatic vessel density in hypertensive rats. Spontaneously hypertensive rats (SHR) that exhibit hypertension and renal injury (SHR-A3 strain) had significantly increased renal lymphatic vessel density and macrophages at 40 weeks of age compared to Wistar-Kyoto (WKY) controls. SHR rats that exhibit hypertension but minimal renal injury (SHR-B2 strain) had significantly less renal lymphatic vessel density compared to WKY rats. The signals for lymphangiogenesis, VEGF-C and its receptor VEGF-R3, and pro-inflammatory cytokine genes increased significantly in the kidneys of SHR-A3 rats but not in SHR-B2 rats. Fischer 344 rats exhibit normal blood pressure but develop renal injury as they age. Kidneys from 24-month and/or 20-month old Fischer rats had significantly increased lymphatic vessel density, macrophage infiltration, VEGF-C and VEGF-R3 expression, and pro-inflammatory cytokine gene expression compared to 4-month old controls. These data together demonstrate that renal immune cell infiltration and inflammation causes lymphangiogenesis in hypertension and aging associated renal injury.
Modulation of the epithelial Na+ channel (ENaC) activity in the collecting duct (CD) is an important mechanism for normal Na+ homeostasis. ENaC activity is inversely related to dietary Na+ intake, in part, due to inhibitory paracrine purinergic regulation. Evidence suggests that H+,K+-ATPase activity in the CD also influences Na+ excretion. We hypothesized that renal H+,K+-ATPases affect Na+ reabsorption by the CD by modulating ENaC activity. ENaC activity in HKα1 H+,K+-ATPase knockout (HKα1-/-) mice was uncoupled from Na+ intake. ENaC activity on a high Na+ diet was greater in the HKα1-/- mice compared to WT mice. Moreover, dietary Na+ content did not modulate ENaC activity in the HKα1 /- mice as it did in WT mice. Purinergic regulation of ENaC was abnormal in HKα1-/- mice. In contrast to WT where urinary [ATP] was proportional to dietary Na+ intake, urinary [ATP] did not increase in response to a high Na+ diet in the HKα1-/- and was significantly lower than in the WT . HKα1-/- mice fed a high Na+ diet had greater Na+ retention compared to WT mice and had an impaired dipsogenic response. These results suggest an important role for the HKα1 subunit in the regulation of purinergic signaling in the CD. They are also consistent with HKα1-containing H+,K+-ATPases as important components for the proper regulation of Na+ balance and the dipsogenic response to a high salt diet. Such observations suggest a previously unrecognized element in Na+ regulation in the CD.
A model of the rat nephron (Am. J. Physiol. 308:F1098, 2015) has been extended with addition of medullary vasculature. Blood vessels contain solutes from the nephron model, plus additional species from the model of Atherton et al. (Am. J. Physiol. 247:F61, 1984), representing hemoglobin buffering. In contrast to prior models of the urine concentrating mechanism, reflection coefficients for DVR are near zero. Model unknowns are initial proximal tubule pressures and flows, connecting tubule pressure, and medullary interstitial pressures and concentrations. The model predicts outer medullary (OM) interstitial gradients for Na+, K+, CO2, and NH4+, such that at OM-IM junction, the respective concentrations relative to plasma are 1.2, 3.0, 2.7, and 8.0; within IM, there is high urea and low HCO3-, with concentration ratios of 11 and 0.5 near the papillary tip. Quantitative similarities are noted between K+ and urea handling (medullary delivery and permeabilities). The model K+ gradient is physiologic, and the urea gradient is steeper due to restriction of urea permeability to distal collecting duct. Nevertheless, the predicted urea gradient is less than expected, suggesting reconsideration of proposals of an unrecognized reabsorptive urea flux. When plasma K+ is increased from 5.0 to 5.5 mM, Na+ and K+ excretion increase 2.3- and 1.3-fold. The natriuresis derives from a 3.3% decrease in proximal Na+ reabsorption, and occurs despite delivery-driven increases in Na+ reabsorption in distal segments; kaliuresis derives from a 30% increase in connecting tubule Na+ delivery. Thus, this model favors the importance of proximal over distal events in K+-induced diuresis.
AIMS: To investigate whether blockade of calcium channels (CCs) or Angiotensin II Type 1 receptors 1 (AT1R) modulates renal responses to nitric oxide synthesis inhibition (NOSI) in humans. METHODS: Fourteen sodium replete healthy volunteers underwent 90 minute infusions of 3.0 μg.kg.min-1 NG-nitro-L-arginine methylester (L-NAME), on 3 occasions, preceded by 3 days of either placebo (PL), 10 mg Manidipine (MANI), or 50 mg losartan (LOS). At each phase, mean arterial pressure (MAP), glomerular filtration rate (GFR, inulin), renal blood flow (RBF, p-aminohippurate), urinary sodium (UNaV) and 8-isoprostane (U8-iso-PGF2αV - an oxidative stress marker) were measured. RESULTS: With PL+L-NAME, the following changes were observed: +6 % MAP (p<0.005 vs. baseline), -10 % GFR, -20 % RBF, -49% UNaV (p<0.001) and +120% U8-iso-PGF2αV (p<0.01). In contrast, MAP did not increase during LOS+L-NAME or MANI+L-NAME (p>0.05 vs. baseline), while renal changes were the same during LOS+L-NAME vs. PL+L-NAME (ANOVA p>0.05). However, during MANI+L-NAME, changes vs. baseline in GFR (-6 %), RBF (-12%) and UNaV (-34%) were blunted vs. PL+L-NAME and LOS+L-NAME (p<0.005), and the rise in U8-iso-PGF2αV was almost abolished (+37%, p>0.05 vs. baseline; p<0.01 vs. PL+L-NAME or LOS+L-NAME). CONCLUSION: Since MANI blunted L-NAME-induced renal hemodynamic changes, CCs participate in the renal responses to NOSI in healthy, sodium-replete humans, independent of changes in MAP and without apparent contribution of the AT1R. Because the rise in U8-iso-PGF2αV was essentially prevented during MANI+L-NAME, CC blockade may oppose the renal effects of NOSI in part by counteracting oxidative stress responses to acutely impaired renal NO bioavailability.
Zebrafish provide an excellent model in which to assess the role of the renin-angiotensin system in renal development, injury and repair. In contrast to mammals, zebrafish kidney organogenesis terminates with the mesonephros. Despite this, the basic functional structure of the nephron is conserved across vertebrates. The relevance of teleosts for studies relating to the regulation of the renin-angiotensin system was established by assessing the phenotype and functional regulation of renin-expressing cells in zebrafish. Transgenic fluorescent reporters for renin (ren), smooth muscle actin (acta2), and platelet derived growth factor receptor beta (pdgfrb) were studied to determine the phenotype and secretory ultrastructure of perivascular renin-expressing cells. Whole-kidney ren transcription responded to altered salinity, pharmacological renin-angiotensin system inhibition, and renal injury. Mesonephric ren-expressing cells occupied niches at the pre-glomerular arteries and afferent arterioles, forming intermittent epithelioid-like multi-cellular clusters exhibiting a granular secretory ultrastructure. In contrast, renin cells of the efferent arterioles were thin-bodied and lacked secretory granules. Renin cells expressed the perivascular cell markers acta2 and pdgfrb. Transcriptional responses of ren to physiological challenge support the presence of a functional renin-angiotensin system and are consistent with the production of active renin. The reparative capability of the zebrafish kidney was harnessed to demonstrate that ren transcription is a marker for renal injury and repair. Our studies demonstrate substantive conservation of renin regulation across vertebrates and ultrastructural studies of renin cells reveal at least two distinct morphologies of mesonephric perivascular ren-expressing cells.
Unbiased transcriptome profiling and functional genomics approaches have identified ubiquitin specific protease 40 (USP40) as a highly specific glomerular transcript. This gene product remains uncharacterized, and its biological function is completely unknown. Here, we showed that mouse and rat glomeruli exhibit specific expression of the USP40 protein, which migrated at 150 kDa and was exclusively localized in the podocyte cytoplasm of the adult kidney. Double-labeling immunofluorescence staining and confocal microscopy analysis of fetal and neonate kidney samples revealed that USP40 was also expressed in the vasculature, including in glomerular endothelial cells at the premature stage. USP40 in cultured glomerular endothelial cells and podocytes was specifically localized to the intermediate filament protein: nestin. In glomerular endothelial cells, immunoprecipitation confirmed actual protein-protein binding of USP40 with nestin, and USP40-siRNA transfection revealed significant reduction of nestin. In rat model of minimal change nephrotic syndrome, apparent reduction of USP40 in the diseased podocytes at the proteinuric stage, which was also associated with the reduction of nestin. Morphants lacking USP40 in zebrafish exhibited disorganized glomeruli with the reduction of the cell junction in the endothelium and foot process effacement in the podocytes. Permeability studies in these zebrafish morphants demonstrated a disruption of the selective glomerular permeability filter. These data indicate that USP40 is a novel protein that might play a crucial role in glomerulogenesis and the glomerular integrity after birth through the modulation of intermediate filament protein homeostasis.
Glomerular hypertension and hyperfiltration in early diabetes is associated with development and progression of diabetic kidney disease. The tubular hypothesis of diabetic hyperfiltration proposes that it is initiated by a primary increase in sodium (Na) reabsorption in the proximal tubule (PT) and the resulting tubuloglomerular feedback (TGF) response and lowering of Bowman space pressure (PBow). Here we utilized a mathematical model of the human kidney to investigate over acute and chronic timescales the mechanisms responsible for the magnitude of the hyperfiltration response. The model implicates that the primary hyperreabsorption of Na in the PT produces a Na imbalance that is only partially restored by the hyperfiltration induced by TGF and changes in PBow. Thus, secondary adaptations are needed to restore Na balance. This may include neurohumoral transport regulation and/or pressure-natriuresis (i.e., the decrease in Na reabsorption in response to increased renal perfusion pressure). We explored the role of each tubular segment in contributing to this compensation, and the consequences of impairment in tubular compensation. The simulations indicate that impaired secondary down-regulation of transport potentiated the rise in glomerular hypertension and hyperfiltration needed to restore Na balance at a given level of primary PT hyperreabsorption. Therefore, we propose for the first time that both the extent of primary PT hyperreabsorption and the degree of impairment of the distal tubular responsiveness to regulatory signals determines the level of glomerular hypertension and hyperfiltration in the diabetic kidney, thereby extending the tubule-centric concept of diabetic hyperfiltration and potential therapeutic approaches beyond the proximal tubule.
Polycystic Kidney Disease is a major cause of end-stage renal disease. The disease mechanisms are not well understood and the pathogenesis towards renal failure remains elusive. In this study, we present the first RNASeq analysis of a Pkd1-mutant mouse model in a combined meta-analysis with other published PKD expression profiles. We introduce the PKD signature, a set of 1515 genes that are commonly dysregulated in PKD studies. We show that the signature genes include many known and novel PKD-related genes and functions. Moreover, genes with a role in injury repair, as evidenced by expression data and/or automated literature analysis, were significantly enriched in the PKD signature, with 35% of the PKD signature genes being directly implicated in injury repair. NF-B signaling, epithelial-mesenchymal transition, inflammatory response, hypoxia, and metabolism were amongst the most prominent injury or repair-related biological processes with a role in the PKD etiology. Novel PKD genes with a role in PKD and in injury were confirmed in another Pkd1-mutant mouse model as well as in animals treated with a nephrotoxic agent. We propose that compounds that can modulate the injury-repair response, could be valuable drug candidates for PKD treatment.
Prolonged decreases in urinary bladder blood flow are linked to overactive and underactive bladder pathologies. However, the mechanisms regulating bladder vascular reactivity are largely unknown. To investigate these mechanisms, we examined myogenic and vasoactive properties of mouse bladder feed arterioles (BFAs). Unlike similar-sized arterioles from other vascular beds, BFAs failed to constrict in response to increases in intraluminal pressure (5-80 mmHg). Consistent with this lack of myogenic tone, arteriolar smooth muscle cell membrane potential was hyperpolarized (-72.8±1.4 mV) at 20 mmHg and unaffected by increasing pressure to 80 mmHg (-74.3±2.2 mV). In contrast, BFAs constricted to the thromboxane analog U-46619 (100 nM), the adrenergic agonist phenylephrine (10 µM), and KCl (60 mM). Inhibition of nitric oxide synthase or intermediate- and small-conductance Ca2+-activated K+ channels did not alter arteriolar diameter, indicating that the dilated state of BFAs is not attributable to overactive endothelium-dependent dilatory influences. Myocytes isolated from BFAs exhibited BaCl2 (100 µM)-sensitive K+ currents consistent with strong inward-rectifier K+ (KIR) channels. Notably, block of these KIR channels "restored" pressure-induced constriction and membrane depolarization. This suggests that these channels, in part, account for hyperpolarization and associated absence of tone in BFAs. Furthermore, smooth muscle-specific knockout of KIR2.1 caused significant myogenic tone to develop at physiological pressures. This suggests that: (1) the regulation of vascular tone in the bladder is independent of pressure, insomuch that pressure-induced depolarizing conductances cannot overcome KIR2.1-mediated hyperpolarization; and (2) that maintenance of bladder blood flow during bladder filling is likely controlled by neurohumoral influences.
The bladder urothelium is essentially quiescent but regenerates readily upon injury. The process of urothelial regeneration harkens back to the process of urothelial development whereby urothelial stem/progenitor cells must proliferate and terminally differentiate to establish all three urothelial layers. How the urothelium regulates the level of proliferation and the timing of differentiation to ensure the precise degree of regeneration is of significant interest in the field. Without a carefully-orchestrated process, urothelial regeneration may be inadequate, thereby exposing the host to toxins or pathogens, or excessive, thereby setting the stage for tumor development. This review describes our current understanding of urothelial regeneration. The current controversies surrounding the identity and location of urothelial progenitor cells which mediate urothelial regeneration are discussed and evidence for each model is provided. We emphasize the factors which have been shown to be crucial for urothelial regeneration, including local growth factors which stimulate repair and epithelial-mesenchymal crosstalk which ensures feedback regulation. Also highlighted is the emerging concept of epigenetic regulation of urothelial regeneration which additionally fine tunes the process through transcriptional regulation of cell cycle genes and growth and differentiation factors. Finally, we emphasize how several of these pathways and/or programs are often dysregulated during malignant transformation, further corroborating their importance in directing normal urothelial regeneration. Together, evidence in the field suggests that any attempt to exploit regenerative programs for the purposes of enhanced urothelial repair or replacement must take into account this delicate balance.
Primary cilia sense environmental conditions including osmolality, but whether cilia participate in osmotic response in renal epithelial cells is not known. Transient receptor potential (TRP) channels TRPV4 and TRPM3 are osmoresponsive. TRPV4 localizes to cilia in certain cell types, while renal subcellular localization of TRPM3 is not known. We hypothesized that primary cilia are required for maximal activation of the osmotic response of renal epithelial cells, and that ciliary TRPM3 and TRPV4 mediate that response. Ciliated (mIMCD-3 and 176-5) and non-ciliated (176-5) renal cells expressed Trpv4 and Trpm3. Ciliary expression of TRPM3 was observed in mIMCD-3 and 176-5 cells and in wildtype mouse kidney tissue. TRPV4 was identified in cilia and apical membrane of mIMCD-3 cells by electrophysiology, and in the cell body by immunofluorescence. Hyperosmolal stress at 500 mOsm/kg (via NaCl addition) induced the osmotic response genes Bgt1 and Akr1b3 in all ciliated cell lines. This induction was attenuated in non-ciliated cells. A TRPV4 agonist abrogated Bgt1 and Akr1b3 induction in ciliated and non-ciliated cells. A TRPM3 agonist attenuated Bgt1 and Akr1b3 induction in ciliated cells only. Knocking out TRPM3 attenuated Akr1b3 induction. Ciliated cells had greater viability under osmotic stress compared to non-ciliated cells. Akr1b3 induction was also less in non-ciliated than in ciliated cells when mannitol was used to induce hyperosmolal stress. These findings suggest that primary cilia are required for the maximal osmotic response in renal epithelial cells and that TRPM3 is involved in this mechanism. TRPV4 appears to modulate the osmotic response independent of cilia.
Grem1, an antagonist of bone morphogenetic proteins, plays a key role in embryogenesis. A highly specific temporospatial gradient of Grem1 and BMP signalling is critical to normal lung, kidney and limb development. Grem1 levels are increased in renal fibrotic conditions including acute kidney injury, diabetic nephropathy, chronic allograft nephropathy and immune glomerulonephritis. A small number of grem1-/- whole body knockout mice on a mixed genetic background (8 %) are viable, with a single, enlarged left kidney and grossly normal histology. Grem1-/- mice displayed mild renal dysfunction at 4 wk, which recovered by 16 wk. Tubular epithelial specific targeted deletion of Grem1 (Grem1-TEC-/-) mice displayed a milder response in both the acute injury and recovery phase of the folic acid model. Grem1-TEC-/- mice had smaller increases in indices of kidney damage compared to wild-type. In the recovery phase of the folic acid model, associated with renal fibrosis, Grem1-TEC-/- mice displayed reduced histological damage and an attenuated fibrotic gene response compared to wild-type controls. Together, these data demonstrated that Grem1 expression in the tubular epithelial compartment plays a significant role in the fibrotic response to renal injury in vivo.
Mononuclear phagocytes are the most common cells in the kidney associated with immunity and inflammation. While the presence of these cells in the kidney has been known for decades, the study of mononuclear phagocytes in the context of kidney function and dysfunction is still at an early stage. The purpose of this review is to summarize the current knowledge regarding classification of these cell in the mouse kidney and to identify relevant questions that would further advance the field and potentially lead to new opportunities for treatment of acute kidney injury and other kidney diseases.
It is well-recognized that Murine Double Minute gene 2 (MDM2) plays a critical role in cell proliferation and inflammatory processes during tumorigenesis. Also it is reported that MDM2 is expressed in glomeruli and involved in podocyte injury. However, whether MDM2 is implicated in renal fibrosis remains unclear. Here we investigated the role of MDM2 in tubulointerstitial fibrosis (TIF). By immunohistochemical staining and western blotting we confirmed that MDM2 is up-regulated in tubulointerstitial compartment in TIF patients and Unilateral Urethral Obstruction (UUO) mice, which mainly originates from myofibroblasts. Consistently, in vitro MDM2 is increased in TGF-β1 treated fibroblasts, one of the major sources of collagen-producing myofibroblasts during TIF, along with fibroblast activation. Importantly, genetic deletion of MDM2 significantly attenuates fibroblast activation. Then we analyzed the possible downstream signaling of MDM2 during fibroblast activation. p53-dependent pathway is the classic downstream signaling of MDM2 and Nutlin-3 is a small molecular inhibitor of MDM2-p53 interaction. To our surprise, Nutlin-3 could not ameliorate fibroblast activation in vitro and TIF in UUO mice. However we found Notch1 signaling is attenuated during fibroblast activation, which could be markedly rescued by MDM2 knocking down. Over-expression of intracellular domain of Notch1 (NICD) by plasmid could obviously minimize fibroblast activation induced by TGF-β1. In addition, the degradation of NICD is strikingly suppressed by PYR-41, an inhibitor of ubiquitin-activating enzyme E1, and proteasome inhibitor MG132. Taken together, our findings provide the first evidence that MDM2 is involved in fibroblast activation and TIF and which associates with Notch1 ubiquitination and proteasome degradation.
The preglomerular microcirculation of spontaneously hypertensive rats (SHR) is hypersensitive to angiotensin II, and studies show that this is likely due to enhanced convergent signaling between G-protein subunits αq (Gαq; released by angiotensin II) and G-protein subunits β (Gβ; released by Gi-coupled receptors) to active phospholipase C (PLC). Here we investigated the molecular basis for the enhanced convergent signaling between Gβ and Gαq in SHR preglomerular vascular smooth muscle cells (PGVSMCs). Because receptor for activated C kinase 1 (RACK1; scaffolding protein) organizes interactions between Gβ, Gαq and PLC, we included RACK1 in this investigation. Cell fractionation studies demonstrated increased levels of membrane (but not cytosolic) Gβ, Gαq, PLCβ3 and RACK1 in SHR PGVSMCs compared with Wistar-Kyoto rat PGVSMCs. In SHR PGVSMCs, co-immunoprecipitation demonstrated RACK1 binding to Gβ and PLCβ3, but only at cell membranes. Pertussis toxin (blocks Gβ) and U73122 (blocks PLC) reduced membrane RACK1; however, RACK1 knockdown (shRNA) did not affect membrane levels of Gβ, Gαq or PLCβ3. In a novel gel contraction assay RACK1 knockdown in SHR PGVSMCs attenuated contractions to angiotensin II and abrogated the ability of neuropeptide Y (signals via Gβ) to enhance angiotensin II-induced contractions. We conclude that in SHR PGVSMCs the enlarged pool of Gβ and PLCβ3 recruits RACK1 to membranes and RACK1 then organizes signaling. Consequently, knockdown of RACK1 prevents convergent signaling between angiotensin II and the Gi-pathway. This is the first study to implicate RACK1 in vascular smooth muscle cell contraction and suggests that RACK1 inhibitors could be effective cardiovascular drugs.
White adipose tissue plays an important role in the development of metabolic disturbance which is a common feature in patients with chronic kidney disease (CKD). The effect of CKD on white adipose tissue remains poorly appreciated. Here we evaluated the inflammatory potential of visceral white adipose tissue in a rat model of CKD. The results showed production of pro-inflammatory cytokines and infiltration of macrophage in the tissue were significantly increased in CKD rats than sham rats. Moreover, the primary adipocytes and stromal vascular fraction under the condition of CKD could trigger the inflammatory response in each other. Free fatty acid induced robust inflammatory response in ex vivo peritoneal-derived macrophages from CKD rats, which was associated with reduced activity of silent information regulator T1 (SIRT1). Improvement of SIRT1 activity by an activator could alleviate free fatty acid-induced inflammatory response in the macrophages and inflammation in the white adipose tissue. Moreover, oxidative stress occurred in the tissue and linked with the reduced activity of SIRT1 in macrophages and enhanced release of free fatty acid in the tissue. We thus identified CKD as a risk factor for chronic inflammation in white adipose tissue. These observations might open up new therapeutic strategies for metabolic disturbance in CKD via the modulation of adipose tissue-related pathways.
(5R)-5-Hydroxytriptolide (LLDT-8), a triptolide derivative with low toxicity, was previously reported to have strong immunosuppressive effects both in vitro and in vivo, but it remains unknown whether LLDT-8 has a therapy effect on systemic lupus erythematosus. In this study, we aimed to investigate the therapeutic effects of LLDT-8 on lupus nephritis in MRL/lpr mice, a model of systemic lupus erythematosus. Compared with vehicle group, different clinical parameters were improved upon LLDT-8 treatment: prolonged life-span of mice, decreased proteinuria, downregulated blood urea nitrogen and serum creatinine, reduced glomerular IgG deposits, and ameliorated histopathology. A decreased expression of the inflammatory cytokines IFN-, IL-17, IL-6, TNF-α was also observed in the kidney of LLDT-8 treated MRL/lpr mice. Moreover, infiltration of T cells into kidney was mitigated after LLDT-8 treatment, which was corresponding with decreased expression of related chemokines IP-10, Mig, and RANTES in kidney. The proportion of macrophage and neutrophil cells and related chemokines expression were also reduced in kidney of LLDT-8 treated mice. In human proximal tubule epithelial cell line and mouse mesangial cell line, consistent with our in vivo experiment results, LLDT-8 suppressed the expression of related chemokines and IL-6. In summary, LLDT-8 has a therapeutic benefit for lupus nephritis via suppressing chemokines expression and inhibiting immune cells infiltration in kidneys of MRL/lpr mice.
Diabetes Mellitus (DM) is a prevalent chronic disease. Type 1 diabetes mellitus (T1DM) is a metabolic disorder that is characterized by hyperglycemia in the context of absolute lack of insulin; whereas type 2 diabetes mellitus (T2DM) is due to insulin resistance-related relative insulin deficiency. Obesity is an established risk factor for T2DM. In comparison with T1DM, T2DM is more complex. The natural history of T2DM in most patients typically involves a course of obesity to impaired glucose tolerance, to insulin resistance and hyperglycemia. Diabetes causes some serious microvascular and macrovascular complications, such as retinopathy, nephropathy, neuropathy, angiopathy and stroke. Diabetes also causes uropathy, which is among the most common complications of DM. In addition, obesity itself affects lower urinary tract. Urological complications of obesity and diabetes (UCOD) affect quality of life substantially. The symptoms and pathophysiology of urological complications in T1DM and T2DM are not same. The aim of this perspective is to review the available data combined with the experience of our research teams who has spent a good part of last decade on studies of association between DM and lower urinary tract symptoms (LUTS) with the aim of bringing more focus to the future scientific exploration of UCOD. We focus on the most common seen urological complications, urinary incontinence (UI), bladder dysfunction, and LUTS, in obesity and diabetes. Knowledge of these associations will lead to a better understanding of the pathophysiology underlying UCOD and hopefully assist urologists in the clinical management of obese or diabetic patients with LUTS.
Histone deacetylase 6 (HDAC6) inhibition has been reported to protect against ischemic stroke and prolong survival after sepsis in animal models. However, it remains unknown whether HDAC6 inhibition offers a renoprotective effect after acute kidney injury (AKI). In this study, we examined the therapeutic effect of tubastatin A (TA), a highly selective inhibitor of HDAC6, on AKI in a murine model of glycerol (GL) injection-induced rhabdomyolysis. Following GL injection, the mice developed severe acute tubular injury as indicated by renal dysfunction, expression of neutrophil gelatinase-associated lipocalin (NGAL), an injury marker of renal tubules and increase of TUNEL positive tubular cells. These changes were companied by increased HDAC6 expression in the cytoplasm of renal tubular cells. Administration of TA significantly reduced serum creatinine and blood urea nitrogen levels as well as attenuated renal tubular damage in injured kidneys. HDAC6 inhibition also resulted in decreased expression of NGAL, reduced apoptotic cell and inactivated caspase-3 in the kidney after acute injury. Moreover, injury to the kidney increased phosphorylation of nuclear factor (NF)-B and expression of multiple cytokines/chemokines including tumor necrotic factor-α and interleukin-6, and monocyte chemoattractant protein-1, as well as macrophages infiltration. Treatment with TA attenuated all those responses. Collectively, these data indicate that HDAC6 contributes to the pathogenesis of rhabdomyolysis-induced AKI and suggest that HDAC6 inhibitors have therapeutic potential for AKI treatment.
Patients with stage 5 chronic kidney disease who are on hemodialysis (HD) remain in a chronic inflammatory state, characterized by the accumulation of uremic toxins that induce endothelial damage and cardiovascular disease (CVD). Our aim was to examine microvesicles (MVs), monocyte subpopulations, and angiopoietins to identify prognostic markers in HD patients with or without diabetes mellitus (DM). A total of 160 prevalent HD patients from 10 centers across Spain were obtained from the Biobank of the Nephrology Renal Network (REDinREN, Madrid): 80 patients with diabetes mellitus (DM) and 80 patients without DM who were matched for clinical and demographic criteria. MVs from plasma and several monocyte subpopulations (CD14++/CD16+, CD14+/CD16++) were analyzed by flow cytometry, and the plasma concentrations of angiopoietin (Ang)1 and Ang2 were quantified by ELISA. Data on cardiovascular disease were gathered over the 5.5 years after these samples were obtained. MV level, monocyte subpopulations (CD14+/CD16++ and CD14++/CD16+), and Ang2/Ang1 ratios increased in HD patients with DM compared with non-DM patients. Moreover, MV level above the median (264 MVs/µl) were associated independently with greater mortality. MVs, monocyte subpopulations, and Ang2/Ang1 ratio can be used as predictors for CVD. In addition, MV level have potential predictive value in the prevention of CVD in HD patients. These parameters undergo more extensive changes in patients with DM.
MicroRNAs (miRNAs) are noncoding RNAs that regulate post-transcriptional gene expression. In this study we characterized the circulating and urinary miRNA pattern associated with reduced glomerular filtration rate using Affymetrix GeneChip miR 4.0 in 28 patients with chronic kidney disease (CKD). Top miRNA discoveries from the human studies were validated in an Alb/TGF-β mouse model of CKD, and in rat renal proximal tubular cells (NRK52E) exposed to TGF-β1. Plasma and urinary levels of procollagen III N-terminal propeptide and collagen IV were elevated in patients with decreased estimated glomerular filtration rate (eGFR). Expression of 384 urinary and 266 circulatory miRNAs were significantly different between CKD patients with eGFR≥30 vs. <30 ml/min/1.73 m2. Pathway analysis mapped multiple miRNAs to TGF-β signaling-related mRNA targets. Specifically, Let-7a was significantly downregulated and miR-130a was significantly upregulated in urine of patients with eGFR<30; miR-1825 and miR-1281 were upregulated in both urine and plasma of patients with decreased eGFR; and miR-423 was significantly down regulated in plasma of patients with decreased eGFR. miRNA expression in urine and plasma of Alb/TGF-β mice generally resembled and confirmed most, although not all, of the observations from the human studies. In response to TGF-β1 exposure, rat renal proximal tubular cells overexpressed miR-1825 and down regulated miR-423. Thus, miRNA are associated with kidney fibrosis and specific urinary and plasma miRNA profile may have diagnostic and prognostic utility in CKD.
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the fourth most common cause of end-stage renal disease. The disease course can be highly variable and treatment options are limited. To identify new therapeutic targets and prognostic biomarkers of disease, we conducted parallel discovery microarray profiling in normal and diseased human PKD1 cystic kidney cells. A total of 1515 genes and 5 miRNA were differentially expressed by more than two-fold in PKD1 cells. Functional enrichment analysis identified 30 dysregulated signalling pathways including the epidermal growth factor (EGF) receptor pathway. In this paper, we report that the EGF family receptor, ErbB4, is a major factor driving cyst growth in ADPKD. Expression of ErbB4 in vivo was increased in human ADPKD and Pkd1 cystic kidneys, both transcriptionally and post-transcriptionally by mir-193b-3p. Ligand-induced activation of ErbB4 drives cystic proliferation and expansion suggesting a pathogenic role in cystogenesis. Our results implicate ErbB4 activation as functionally relevant in ADPKD, both as a marker of disease activity and as a new therapeutic target in this major kidney disease.
Aldosterone (Aldo) has been shown as an important contributor of podocyte injury. However, the underlying molecular mechanisms are still elusive. Recently, the pathogenic role of NLRP3 inflammasome in mediating renal tubular damage was identified while its role in podocyte injury still needs evidence. Thus the present study was undertaken to investigate the role of NLRP3 inflammasome in Aldo-induced podocyte damage. In vitro, exposure of podocytes to Aldo enhanced NLRP3, caspase-1 and IL-18 expressions in dose- and time-dependent manners, indicating an activation of NLRP3 inflammasome, which was significantly blocked by a mineralocorticoid receptor antagonist Eplerenone (EPL) or an antioxidant NAC. Silencing NLRP3 by a siRNA approach strikingly attenuated Aldo-induced podocyte apoptosis and nephrin protein downregulation in line with the blockade of caspase-1 and IL-18. In vivo, since day 5 of Aldo-infusion, NLRP3 inflammasome activation and podocyte injury evidenced by nephrin reduction occurred concurrently. More importantly, immunofluorescence analysis showed a significant induction of NLRP3 in podocytes of glomeruli following Aldo infusion. In the mice with NLRP3 gene deletion, Aldo-induced downregulation of nephrin and podocin, podocyte foot processes, and albuminuria was remarkably improved, indicating an amelioration of podocyte injury. Finally, we observed a striking induction of NLRP3 in glomeruli and renal tubules in line with an enhanced urinary IL-18 output in nephrotic syndrome patients with MCD or FSGS. Together, these results demonstrated an important role of NLRP3 inflammasome in mediating the podocyte injury induced by Aldo.
Changes in renal hemodynamics have a major impact on blood pressure (BP). Angiotensin (Ang) II has been shown to induce vascular dysfunction by interacting with phosphodiesterases (PDE)1 and PDE5. The predominant PDE isoform responsible for renal vascular dysfunction in hypertension is unknown. Here, we measured effects of PDE5-(sildenafil) or PDE1-(vinpocetine) inhibition on renal blood flow (RBF), BP and renal vascular function in normotensive and hypertensive mice. During acute short-term Ang II infusion, sildenafil decreased BP and increased RBF in C57BL/6 (WT) mice. In contrast, vinpocetine showed no effect on RBF and BP. Additionally, renal cGMP levels were significantly increased after acute sildenafil but not after vinpocetine infusion, indicating a predominant role of PDE5 in renal vasculature. Furthermore, chronic Ang II infusion (500ng/kg/min) increased BP and led to impaired NO-dependent vasodilation in kidneys of WT mice. Additional treatment with sildenafil (100mg/kg/d) attenuated Ang II-dependent hypertension and improved NO-mediated vasodilation. During chronic Ang II infusion urinary nitrite excretion, a marker for renal NO generation, was increased in WT mice, whereas renal cGMP generation was decreased and restored after sildenafil treatment suggesting a preserved cGMP signaling after PDE5 inhibition. To investigate the dependency of PDE5 effects on NO/cGMP signaling, we next analyzed eNOS-KO mice, a mouse model characterized by low vascular NO/cGMP levels. In eNOS-KO mice, chronic Ang II infusion increased BP but did not impaired NO-mediated vasodilation. Moreover, sildenafil did not influence BP or vascular function in eNOS-KO mice. These results highlight PDE5 as a key regulator of renal hemodynamics in hypertension.
Vasoconstriction plays an important role in the development of acute kidney injury in rhabdomyolysis. We hypothesized that myoglobin enhances the angiotensin II (Ang II) response in afferent arterioles by increasing superoxide and reducing nitric oxide (NO) bioavailability. Afferent arterioles of C57Bl6 mice were isolated perfused and vasoreactivity was analyzed using video microscopy. NO bioavailability, superoxide concentration in the vessels wall, and changes in cytosolic calcium were measured using fluorescence techniques. Myoglobin treatment (10-5M) did not change the basal arteriolar diameter during a 20min period compared to control conditions. L-NAME (10-4M) and L-NAME + myoglobin reduced diameters to 94.7% and 97.9% of the initial diameter, respectively. Myoglobin or L-NAME enhanced the Ang II induced constriction of arterioles compared to control (36.6% and 34.2%, respectively, vs. 65.9%). Norepinephrine responses were not influenced by myoglobin. Combined application of myoglobin and L-NAME further facilitated the Ang II response (7.0%). Myoglobin or L-NAME decreased the NO related fluorescence in arterioles similarly. Myoglobin enhanced the superoxide related fluorescence and tempol prevented this enhancement. Tempol also partly prevented the myoglobin effect on the Ang II response. Myoglobin increased the Fura-2 fluorescence ratio (intracellular calcium) during Ang II application (10-12 to 10-6M). The results suggest that the enhanced afferent arteriolar reactivity to Ang II is mainly due to a myoglobin induced increase in superoxide and associated reduction in the NO bioavailability. Signalling pathways for the augmented Ang II response include enhanced cytosolic calcium transients. In conclusion, myoglobin may contribute to the afferent arteriolar vasoconstriction in this rhabdomyolysis model.
Background: While urothelial signals, including sonic hedgehog (Shh), drive bladder mesenchyme differentiation, it is unclear which pathways within the mesenchyme are critical for its development. Studies have shown fibroblast growth factor receptor 2 (Fgfr2) is necessary for kidney and ureter mesenchymal development. Objective: Out objective was to determine the role of Fgfr2 in bladder mesenchyme. Methods: We used Tbx18cre mice to delete Fgfr2 in bladder mesenchyme (Fgfr2BM-/-). We performed three dimensional (3D) reconstructions, real time PCR (qPCR), in situ hybridization, immunolabeling, ELISAs, immunoblotting, void stain on paper (VSOP), ex vivo bladder sheet assays, and in vivo decerebrated cystometry. Results: Compared to controls, embryonic day (E) 16.5 Fgfr2BM-/- bladders have thin muscle layers with reduced alpha smooth muscle actin (αSMA) levels and thickened lamina propria with increased collagen expression that intrudes into muscle. From P1 to P30, Fgfr2BM-/- bladders demonstrate progressive muscle loss and increased collagen expression. Postnatal Fgfr2BM-/- bladder sheets exhibit decreased contractility and increased passive stretch tension vs. controls. In vivo cystometry revealed high baseline and threshold pressures and shortened intercontractile intervals in Fgfr2BM-/- bladders vs. controls. Mechanistically, while Shh expression appears normal, mRNA and protein readouts of hedgehog activity are increased in E16.5 Fgfr2BM-/- bladders vs. controls. Moreover, E16.5 Fgfr2BM-/- bladders exhibit higher levels of Cdo and Boc, hedgehog co-receptors that enhance sensitivity to Shh, vs. controls Conclusion: Fgfr2 is critical for bladder mesenchyme patterning by modulating hedgehog signaling.
Liver X Receptors (LXRs) including LXRα and LXRβ are nuclear receptor transcription factors and play an important role in lipid and glucose metabolism. It has been previously reported that mice lacking LXRβ but not LXRα develop a severe urine concentrating defect, likely via a central mechanism. Here we provide evidence that LXRβ regulates water homeostasis through increasing aquaporin 2 (AQP2) protein levels in renal collecting ducts. LXRβ-/- mice exhibited a reduced response to dDAVP stimulation, suggesting that the diabetes insipidus phenotype is of both central and nephrogenic origin. AQP2 protein abundance in the renal inner medulla was significantly reduced in LXRβ-/- mice but with little change in AQP2 mRNA levels. In vitro studies showed that AQP2 protein levels were elevated upon LXR agonist treatment in both primary cultured mouse inner medullary duct cells (mIMCD) and the mIMCD3 cell line with stably expressed AQP2. In addition, LXR agonists including TO901317 and GW3965 failed to induce AQP2 gene transcription but diminished its protein ubiquitination in primary cultured mIMCD cells, thereby inhibiting its degradation. Moreover, LXR activation-induced AQP2 protein expression was abolished by the protease inhibitor MG132 and the ubiquitination-deficient AQP2 (K270R). Taken together, the present study demonstrates that activation of LXRβ increases AQP2 protein levels in the renal collecting ducts via a posttranscriptional mechanism. As such, LXRβ represents a key regulator of body water homeostasis.
We have examined the pathogenic role of increased complement expression and activation during kidney fibrosis. Here we show that PDGFRβ positive pericytes isolated from mice subjected to obstructive or folic acid injury secrete C1q. This was associated with increased production of proinflammatory cytokines, extracellular matrix components, collagens and increased Wnt3a-mediated activation of Wnt/β-catenin signaling, that are hallmarks of myofibroblast activation. Real-time PCR, immunoblots, immunohistochemistry and flow cytometry analysis performed in whole kidney tissue confirmed increased expression of C1q, C1r, C1s as well as complement activation measured as increased synthesis of C3 fragments predominantly in the interstitial compartment. Flow studies localized increased C1q expression to PDGFRβ positive pericytes, as well as to CD45 positive cells. Although deletion of C1qA did not prevent kidney fibrosis, global deletion of C3 reduced macrophage infiltration, reduced synthesis of C3 fragments and reduced fibrosis. Clodronate mediated depletion of CD11bF4/80 high macrophages in UUO mice also reduced complement gene expression and reduced fibrosis. Our studies demonstrate local synthesis of complement by both PDGFRβ positive pericytes and CD45 positive cells in kidney fibrosis. Inhibition of complement activation represents a novel therapeutic target to ameliorate fibrosis and progression of chronic kidney disease.
The hypoxanthine-xanthine oxidase (XO) axis is considered to be a key driver of transplantation related ischemia reperfusion (I/R) injury. Whereas interference with this axis effectively quenches I/R injury in preclinical models, there is limited efficacy of XO inhibitors in clinical trials. In this context, we considered clinical evaluation of a role for the hypoxanthine-XO axis in human I/R to be relevant. Patients undergoing renal allograft transplantation were included (n=40) and classified based on duration of ischemia (short; intermediate; prolonged). Purine metabolites excreted by the reperfused kidney (arteriovenous differences) were analysed by UPLCMS/MS method and tissue XO activity was assessed by in situ enzymography. We confirmed progressive hypoxanthine accumulation (P<0.006) during ischemia, using kidney transplantation as a clinical model of I/R. Yet, arteriovenous concentration differences of uric acid and in situ enzymography of XO did not indicate significant XO activity in ischemic and reperfused kidney grafts. Furthermore, we tested a putative association between hypoxanthine accumulation and renal oxidative stress by assessing renal malondialdehyde and isoprostane levels, and allantoin formation during the reperfusion period. Absent release of these markers is not consistent with an association between ischemic hypoxanthine accumulation and postreperfusion oxidative stress. On basis of these data for the human kidney we hypothesize that the role for the hypoxanthine-XO axis in clinical I/R injury is less than commonly thought, and as such the data provides an explanation for the apparent limited clinical efficacy of XO inhibitors.
The aim of this analysis was to examine sex-based differences in renal segmental resistances in healthy controls (HC) and patients with type 1 diabetes (T1D). We hypothesized that hyperfiltration - an early hemodynamic abnormality associated with diabetic nephropathy (DN) - would disproportionately affect women with T1D, thereby attenuating protection against the development of renal complications. Glomerular hemodynamic parameters were evaluated in HC (n=30) and in normotensive, normoalbuminuric patients with T1D and either baseline normofiltration (n=36, T1D-N, glomerular filtration rate [GFR] 90-134 ml/min/1.73m2) or hyperfiltration (n=32, T1D-H, GFR≥135 ml/min/1.73m2) during euglycemic conditions (4-6 mmol/L). Gomez's equations were used to derive efferent (RE) and afferent (RA) arteriolar resistances, glomerular hydrostatic pressure (PGLO) from inulin (GFR) and paraaminohippurate (effective renal plasma flow - ERPF) clearances, plasma protein and estimated ultrafiltration coefficients (KFG). Female TID-H had higher RE (1985±487 vs. 1381±296 dyne•sec•cm-5, p<0.001) and filtration fraction (FF, 0.20±0.047 vs. 0.16±0.03 p<0.05) and lower ERPF (876±245 vs. 1111±298 ml/min/1.73m2 p<0.05) compared to male T1D-H patients. Overall, T1D-H patients had higher PGLO and lower RA vs. HC subjects, although there were no sex-based differences. In conclusion, female T1D-H patients had higher RE and FF and lower ERPF than their male counterparts with no associated sex differences in RA. Prospective intervention studies should consider sex as a modifier of renal hemodynamic responses to renal protective therapies.
The textbook account of whole-body acid-base balance in terms of endogenous acid production, renal net acid excretion and gastrointestinal alkali absorption which is the only comprehensive model around, has never been applied in clinical practice or been formally validated. In order to improve understanding of acid-base modeling, we managed to write up this conventional model as an expression solely on urine chemistry. Renal net acid excretion and endogenous acid production was already formulated in terms of urine chemistry, and we could from the literature also see gastrointestinal alkali absorption in terms of urine excretions. With few assumptions it was possible to see that this expression of net acid balance was arithmetically identical to minus urine charge, whereby under the development of acidosis, urine was predicted to acquire a net negative charge. The literature already mentions unexplained negative urine charges so we scrutinized a series of seminal papers and confirmed empirically the theoretical prediction that observed urine charge did acquire negative charge as acidosis developed. Hence we can conclude that the conventional model is problematic since it predicts what is physiologically impossible. Therefore, we need a new model for whole-body acid-base balance, which doesn't have impossible implications. Furthermore, new experimental studies are needed to account for charge imbalance in urine under development of acidosis.
The prorenin receptor (PRR) is a receptor for renin and prorenin, and an accessory subunit of the vacuolar proton pump H+-ATPase. Renal branching morphogenesis, defined as growth and branching of the ureteric bud (UB), is essential for mammalian kidney development. Previously, we demonstrated that conditional ablation of the PRR in the UB in PRRUB-/- mice causes severe defects in UB branching, resulting in marked kidney hypoplasia at birth. Here, we investigated UB transcriptome using whole-genome-based analysis of gene expression in UB cells FACS-isolated from PRRUB-/- and control kidneys at birth (P0) to determine the primary role of the PRR in terminal differentiation and growth of UB-derived collecting ducts. Three genes with expression in UB cells previously shown to regulate UB branching morphogenesis, including Wnt9b, β-catenin and Fgfr2, were upregulated, whereas the expression of Wnt11, Bmp7, Etv4 and Gfrα1 was downregulated. We next demonstrated that infection of immortalized UB cells with shPRR in vitro or deletion of the UB PRR in double-transgenic PRRUB-/-/BatGal+ mice, a reporter strain for β-catenin transcriptional activity, in vivo increases β-catenin activity in the UB epithelia. In addition to UB morphogenetic genes, the functional groups of differentially expressed genes within the downregulated gene set included genes involved in molecular transport, metabolic disease, aminoacid metabolism and energy production. Together, these data demonstrate that UB PRR performs essential functions during UB branching and collecting duct morphogenesis via control of hierarchy of genes that control UB branching and terminal differentiation of the collecting duct cells.
We have characterized the expression and secretion of the acute kidney injury biomarkers Insulin-Like Growth Factor Binding Protein 7 (IGFBP7) and Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) in human kidney epithelial cells in primary cell culture and tissue. We established cell culture model systems of primary kidney cells of proximal and distal tubule origin and observed that both proteins are indeed expressed and secreted in both tubule cell types in vitro. However, TIMP-2 is both expressed and secreted preferentially by cells of distal tubule origin, while IGFBP7 is equally expressed across tubule cell types yet preferentially secreted by cells of proximal tubule origin. In human kidney tissue, strong staining of IGFBP7 was seen in the luminal brush border region of a subset of proximal tubule cells, and TIMP-2 stained intracellularly in distal tubules. Additionally, while some tubular co-localization of both biomarkers was identified with the injury markers Kidney Injury Molecule-1 and Neutrophil Gelatinase-Associated Lipocalin, both biomarkers could also be seen alone, suggesting the possibility for differential mechanistic and/or temporal profiles of regulation of these early AKI biomarkers from known markers of injury. Lastly, an in vitro model of ischemia-reperfusion demonstrated enhancement of secretion of both markers early after reperfusion. This work provides a rationale for further investigation of these markers for their potential role in the pathogenesis of acute kidney injury.
Background: Recurrent heat stress and dehydration have recently been shown experimentally to cause chronic kidney disease (CKD). One potential mediator may be vasopressin, acting via the type 2 vasopressin receptor (V2 receptor). We tested the hypothesis that desmopressin accelerates CKD in mice subjected to heat stress and recurrent dehydration. Methods: Recurrent exposure to heat with limited water availability was performed in male mice over a 5 week period, with one group receiving desmopressin twice daily and the other group received vehicle. Two additional control groups were not exposed to heat or dehydration and received vehicle or desmopressin. The effects of the treatment on renal injury was assessed. Results: Heat stress and recurrent dehydration induced functional changes (albuminuria, elevated urinary NGAL), glomerular changes (mesangiolysis, matrix expansion) and tubulointerstitial changes (fibrosis, inflammation). Desmopressin also induced albuminuria, glomerular changes and tubulointerstitial fibrosis in normal animals, and also exacerbated injury in mice with heat stress nephropathy. Both heat stress and/or desmopressin were also associated with activation of the polyol pathway in the renal cortex, likely due to increased interstitial osmolarity. Conclusions: Our studies document both glomerular and tubulointerstitial injury and inflammation in heat stress nephropathy, and may be clinically relevant to the pathogenesis of Mesoamerican Nephropathy. Our data also suggest that vasopressin may play a role in the pathogenesis of the renal injury of heat stress nephropathy, likely via a V2-receptor dependent pathway.
Calcineurin dephosphorylates NFAT transcription factors, thereby facilitating T-cell mediated immune responses. Calcineurin inhibitors are instrumental for immunosuppression after organ transplantation, but may cause side effects including hypertension and electrolyte disorders. Kidneys were recently shown to display activation of the furosemide-sensitive Na-K-2Cl cotransporter (NKCC2) of the thick ascending limb and the thiazide-sensitive Na-Cl cotransporter (NCC) of the distal convoluted tubule upon calcineurin inhibition using cyclosporin A (CsA). An involvement of major hormones like angiotensin II or arginine vasopressin (AVP) has been proposed. To resolve this issue, the effects of CsA treatment in normal Wistar and AVP-deficient Brattleboro rats, and cultured renal epithelial cells endogenously expressing either NKCC2 or NCC, were studied. Acute administration of CsA to Wistar rats rapidly augmented phosphorylation levels of NKCC2, NCC, and their activating kinases (WNK and SPAK/OSR1), suggesting intraepithelial activating effects. Chronic CsA administration caused salt retention and hypertension, along with stimulation of renin, and suppression of renal cyclooxygenase 2, pointing to a contribution of endocrine and paracrine mechanisms at long-term. In Brattleboro rats, CsA induced activation of NCC, but not NKCC2, and parallel effects were obtained in cultured cells in the absence of AVP. Stimulation of cultured TAL cells with AVP agonist restored their responsiveness to CsA. Our results suggest that the direct epithelial effect of calcineurin inhibition is sufficient for the activation of NCC, whereas its effect on NKCC2 is more complex and requires concomitant stimulation by AVP.
Chronic kidney disease (CKD) will progress to end stage without treatment, but the decline of renal function may not be linear. Compared to GFR and proteinuria, new surrogate markers, such as KIM-1, NGAL, apoA-IV, and soluble urokinase receptor (suPAR), may allow potential intervention and treatment in the earlier stages of CKD, which could be useful for clinical trials. New omic-based technologies reveal potential new genomic and epigenomic mechanisms which appear different from those causing the initial disease. Various clinical studies also suggest that acute kidney injury (AKI) is a major risk for progressive CKD. To ameliorate the progression of CKD, the first step is optimizing renin-angiotensin-aldosterone system (RAAS) blockade. New drugs targeting endothelin, transforming growth factor-β (TGFβ), oxidative stress and inflammatory and cell-based regenerative therapy may have add-on benefit.
MicroRNAs (miRNAs) are essential for the maintenance of podocyte homeostasis. Emerging evidence demonstrated a protective role of miRNA-30a, a member of miR-30 family in podocyte injury. However, the role of other members of miR-30 family in podocyte injury is unclear. The present study was undertaken to investigate the contribution of miR-30e in the pathogenesis of podocyte injury induced by aldosterone (Aldo), as well as the underlying mechanism. Following Aldo treatment, miR-30e was dose-and time-dependently reduced. Notably, overexpression of miR-30e remarkably attenuated Aldo-induced apoptosis in podocytes. In agreement with this finding, miR-30e silencing led to significant podocyte apoptosis. Mitochondrial dysfunction (MtD) has been shown as an early event in Aldo-induced podocyte injury. Here we found that overexpressing miR-30e improved Aldo-induced MtD while miR-30e silencing resulted in MtD. Next, we found that miR-30e could directly target BCL2/adenovirus E1B interacting protein 3-like (BNIP3L) gene. Aldo remarkably enhanced BNIP3L expression in podocytes and silencing BNIP3L largely abolished Aldo-induced MtD and cell apoptosis. On the contrary, overexpression of BNIP3L induced MtD and apoptosis in podocytes. Taken together, these findings demonstrated that miR-30e protected mitochondria and podocytes against Aldo challenge by targeting BNIP3L.
The organization of the mammalian genome into gene subsets corresponding to specific functional classes has provided key tools for systems biology research. Here, we have created a web-accessible resource called the Mammalian Metabolic Enzyme Database (https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/MetabolicEnzymeDatabase.html) keyed to the biochemical reactions represented on iconic metabolic pathway wall charts created in the previous century. Overall, we have mapped 1647 genes to these pathways, representing approximately 7 percent of the protein-coding genome. To illustrate the use of the database, we apply it to the area of kidney physiology. In so doing, we have created an additional database (Database of Metabolic Enzymes in Kidney Tubule Segments: https://hpcwebapps.cit.nih.gov/ESBL/Database/MetabolicEnzymes/), mapping mRNA abundance measurements (mined from RNA-Seq studies) for all metabolic enzymes to each of 14 renal tubule segments. We carry out bioinformatics analysis of the enzyme expression pattern among renal tubule segments and mine various data sources to identify vasopressin-regulated metabolic enzymes in the renal collecting duct.
Using a transgenic cross, we evaluated features of preeclampsia, renal injury and the sFlt1/VEGF changes. Transgenic hAGT and hREN, or wild type (WT) C57Bl/6 mice were cross-bred: hAGT x hREN for preeclampsia (PRE) model and WT x WT for pregnant controls (WTP). Samples were collected for plasma VEGF, sFlt1 and urine albumin. Blood pressures (BP) were monitored by telemetry. Vascular reactivity was investigated by wire myography. Kidneys and placenta were immunostained for sFlt1 and VEGF. Eleven PRE and 9 WTP mice were compared. PRE more frequently demonstrated albuminuria, glomerular endotheliosis (80% vs. 11%; p=0.02), and placental necrosis (60% vs. 0%; p<0.01). PRE group demonstrated declining BPs with advancing gestation. Plasma sFlt1 increased across pregnancy in PRE, VEGF did not vary. IHC demonstrated the presence of sFlt1 in glomeruli, lymphatics and collecting tubules of PRE kidneys suggesting excretion. VEGF immunostaining was increased specifically in the glomeruli of PRE kidneys. Placenta in PRE showed marked immunostaining for sFlt1. We conclude that this transgenic model of preeclampsia recapitulates human preeclamptic state with high fidelity, and that, vascular adaptation to pregnancy is suggested by declining BPs and reduced vascular response to PE and increased response to Ach. Placental damage with resultant increased release of sFlt1, proteinuria, deficient spiral artery remodeling and glomerular endotheliosis were observed in this model of PRE. Increased VEGF binding to glomerular endothelial cells in this model of PRE is similar to human PRE and leads us to hypothesize that renal injury in preeclampsia may be mediated through local VEGF.
Chronic kidney disease (CKD) is associated with increased cardiovascular mortality, and vascular smooth muscle cell (VSMC) dysfunction plays a pivotal role in uremic atherosclerosis. Axl signaling is involved in vascular injury and is highly expressed in VSMC. Recent reports have shown that cilostazol, a phosphodiesterase type 3 inhibitor (PDE3), can regulate various stages of the atherosclerotic process. However, the role of cilostazol in uremic vasculopathy remains unclear. This study aimed to identify the effect of cilostazol in VSMCs in the experimental CKD and to investigate whether the regulatory mechanism occurs through Axl signaling. We investigated the effect of P-cresol and cilostazol on Axl signaling in A7r5 rat VSMCs and the rat, human CKD models. From in vivo CKD rats and patients, aortic tissue exhibited significantly decreased Axl expression after cilostazol treatment. P-cresol increased Axl, PCNA, FAK and MMP-2 expressions, decreased caspase-3 expression, and was accompanied with increased cell viability and migration. Cilostazol significantly reversed P-cresol-induced Axl, downstream gene expressions and cell functions. Along with the increased Axl expression, P-cresol activated PLC, Akt and ERK phosphorylation and cilostazol significantly suppressed the effect of P-cresol. Axl knockdown significantly reversed the expressions of P-cresol-induced Axl related gene expression and cell functions. Cilostazol with Axl knockdown have additive changes in downstream gene expression and cell functions in P-cresol culture. Both in vitro and in vivo experimental CKD models elucidate a new signal transduction of cilostazol-mediated protection against uremic toxin related VSMCs dysfunction and highlight the involvement of the Axl signaling and downstream pathways.
Diabetes mellitus is associated with decreased nitric oxide bioavailability thereby affecting renal blood flow regulation. Previous reports have demonstrated that cellular uptake of L-arginine is rate limiting for nitric oxide production, and that plasma L-arginine concentration is decreased in diabetes. We therefore investigated if regional renal blood flow regulation is affected by cellular L-arginine uptake in streptozotocin-induced diabetic rats. Rats were anesthetized with thiobutabarbital and left kidney was exposed. Total, cortical and medullary renal blood flow was investigated before and after renal artery infusion of increasing doses of either L-homoarginine to inhibit cellular uptake of L-arginine, or L-NAME to inhibit nitric oxide synthase. L-homoarginine infusion did not affect total or cortical blood flow in any of the groups, but caused a dose-dependent reduction in medullary blood flow. L-NAME decreased total, cortical and medullary blood flow in both groups. However, the reductions in medullary blood flow in response to both L-homoarginine and L-NAME were more pronounced in the control groups compared to the diabetic groups. Isolated cortical tubular cells displayed similar L-arginine uptake capacity whereas medullary tubular cells isolated from diabetic rats had increased L-arginine uptake capacity. Diabetics had reduced L-arginine concentrations in plasma and medullary tissue but increased L-arginine concentration in cortical tissue. In conclusion, the reduced L-arginine availability in plasma and medullary tissue in diabetes results in reduced nitric oxide-mediated regulation of renal medullary hemodynamics. Cortical blood flow regulation displays less dependency on extracellular L-arginine and the upregulated cortical tissue L-arginine may protect cortical hemodynamics in diabetes.
We have reported that the myogenic response of the renal afferent arteriole (Af-art) and middle cerebral artery (MCA), and autoregulation of renal and cerebral blood flow are impaired in Fawn Hood Hypertensive (FHH) rats. Transfer of a region of chromosome 1 containing gamma-adducin (Add3) from Brown Norway rats rescues the vascular dysfunction and the development of renal disease. To examine whether Add3 is a viable candidate gene altering renal and cerebral hemodynamics in FHH rats, we knocked down the expression of Add3 in rat Af-art and MCA cultured for 36-hours using a 27-mer Dicer-substrate short interfering RNA (DsiRNA). Control Af-arts constricted by 10 ± 1% in response to an elevation in pressure from 60 to 120 mm Hg but dilated by 4 ± 3% when treated with Add3 DsiRNA. Add3 DsiRNA had no effect on the vasoconstrictor response of the Af-art to NE (10-7 M). Add3 DsiRNA had a similar effect to attenuate the myogenic response in MCA. Peak potassium currents were 3-fold higher in smooth muscle cells isolated from Af-arts or MCAs transfected with Add3 DsiRNA than in non-transfected cells isolated from the same vessels. This is the first study demonstrating that Add3 plays a role in the regulation of potassium channel function and vascular reactivity. It supports the hypothesis that sequence variants in Add3, we previously identified in FHH rats, may play a causal role in the impaired myogenic response and autoregulation in renal and cerebral circulation.
Regulated dicarboxylate transport is critical for acid-base homeostasis, prevention of calcium nephrolithiasis, regulation of collecting duct sodium chloride transport and the regulation of blood pressure. Although luminal dicarboxylate reabsorption via NaDC1 (SLC13A2) is believed to be the primary mechanism regulating renal dicarboxylate transport, the specific localization of NaDC1 in the human kidney is currently unknown. This study's purpose was to determine NaDC1's expression in normal and neoplastic human kidneys. Immunoblot analysis demonstrated NaDC1 expression with an apparent molecular weight of ~61 kDa. Immunohistochemistry showed apical NaDC1 immunolabel in the proximal tubule of normal human kidney tissue; well-preserved proximal tubule brush border was clearly labeled. Apical NaDC1 expression was evident throughout the entire proximal tubule, including the initial proximal convoluted tubule, as identified by origination from the glomerular tuft, and extending through the terminal of the proximal tubule, the proximal straight tubule in the outer medulla. We confirmed proximal tubule localization by co-localization with the proximal tubule specific protein, NBCe1. NaDC1 immunolabel was not detected other than in the proximal tubule. In addition, NaDC1 immunolabel was not detected in tumors of presumed proximal tubule origin, clear cell and papillary renal cell carcinoma, or in tumors of non-proximal tubule origin, oncocytoma and chromophobe carcinoma. In summary: 1) in the human kidney, apical NaDC1 immunolabel is present throughout the entire proximal tubule, and is not detectable in other renal cells; and, 2) NaDC1 immunolabel is not present in renal tumors. These studies provide important information regarding NaDC1's role in human dicarboxylate metabolism.
The role of cannabinoid type 1 (CB1) receptors in tibial and pudendal neuromodulation of bladder overactivity induced by intravesical infusion of 0.5% acetic acid (AA) was determined in α-chloralose anesthetized cats. AA irritation significantly (p<0.01) reduced bladder capacity to 36.6±4.8% of saline control capacity. Tibial nerve stimulation (TNS) at 2 or 4 times threshold (T) intensity for inducing toe movement inhibited bladder overactivity and significantly (p<0.01) increased bladder capacity to 69.2±9.7% and 79.5±7.2% of saline control, respectively. AM 251 (a CB1 receptor antagonist) administered intravenously (i.v.) at 0.03 or 0.1 mg/kg significantly (p<0.05) reduced the inhibition induced by 2T or 4T TNS, respectively, without changing the pre-stimulation bladder capacity. However, intrathecal administration of AM 251 (0.03 mg) to L7 spinal segment had no effect on TNS inhibition. Pudendal nerve stimulation (PNS) also inhibited bladder overactivity induced by AA irritation, but AM 251 at 0.01-1 mg/kg (i.v.) had no effect on PNS inhibition or the pre-stimulation bladder capacity. These results indicate that CB1 receptors play an important role in tibial but not pudendal neuromodulation of bladder overactivity and the site of action is not within the lumbar L7 spinal cord. Identification of neurotransmitters involved in TNS or PNS inhibition of bladder overactivity is important for understanding the mechanisms of action underlying clinical application of neuromodulation therapies for bladder disorders.
The renal proximal tubules are a key functional component of the kidney and express the angiotensin precursor angiotensinogen; however, it is unclear the extent that tubular angiotensinogen reflects local synthesis or internalization. Therefore, the current study established the extent that angiotensinogen is internalized by proximal tubules and the intracellular distribution. Proximal tubules were isolated from the kidney cortex of male sheep by enzymatic digestion and a discontinuous Percoll gradient. Tubules were incubated with radiolabeled 125I-angiotensinogen for 2 hrs at 37°C in serum/phenol-free DMEM/F12 media. Approximately 10% of exogenous 125I-angiotensinogen was internalized by sheep tubules. Subcellular fractionation revealed that 21 ± 4% of the internalized 125I-angiotensinogen associated with the mitochondrial fraction with additional labeling evident in the nucleus [60 ± 7%], endoplasmic reticulum [4 ± 0.5%] and cytosol [15 ± 4%; n=4]. Subsequent studies determined whether mitochondria directly internalized 125I-angiotensinogen using isolated mitochondria from renal cortex and human HK-2 proximal tubule cells. Sheep cortical and HK-2 mitochondria internalized 125I-angiotensinogen at a comparable rate of [33 ± 9 vs. 21 ± 10 pmol/min/mg protein; n=3]. Lastly, unlabeled angiotensinogen (100 nM) competed for 125I-angiotensinogen uptake to a greater extent than human albumin in HK-2 mitochondria [60 ± 2% vs. 16 ± 13%; p<0.05, n=3]. Collectively, our data demonstrate angiotensinogen import and subsequent trafficking to the mitochondria in proximal tubules. We conclude that this pathway may constitute a source of the angiotensinogen precursor for the mitochondrial expression of angiotensin peptides.
Diabetic nephropathy (DN) is the leading cause of kidney failure in the world. To understand important mechanisms underlying this condition, and to develop new therapies, good animal models are required. In mouse models of type-1 diabetes, the DBA/2J strain has been shown to be more susceptible to develop kidney disease than other common strains. We hypothesized this would also be the case in type-2 diabetes. We studied db/db and wt DBA/2J mice and compared these with the db/db BLKS/J mouse, which is currently the most widely used type-2 DN model. Mice were analyzed from age 6 to 12 weeks for systemic insulin resistance, albuminuria and glomerular histopathological and ultra-structural changes. Body weight and non-fasted blood glucose were increased by 8-weeks in both genders, while systemic insulin resistance commenced by 6-weeks in female and 8-weeks in male db/db DBA/2J mice. The urinary albumin-to-creatinine ratio (ACR) was closely linked to systemic insulin resistance in both sexes and was increased ~50-fold by 12 weeks in the db/db DBA/2J cohort. Glomerulosclerosis, foot process effacement and glomerular basement membrane thickening were observed at 12-weeks of age in db/db DBA/2J mice. Compared to db/db BLKS/J mice, db/db DBA/2J mice had significantly increased levels of urinary ACR, but similar glomerular histopathological and ultrastructural changes. The db/db DBA/2J mouse is a robust model of early stage albuminuric DN and its levels of albuminuria correlate closely with systemic insulin resistance. This mouse model will be helpful in defining early mechanisms of DN and ultimately the development of novel therapies.
Lupus nephritis is a life-threatening complication of systemic lupus erythematosus (SLE). Various growth factors, cytokines, and chemokines are implicated in the development of SLE. However, the pathophysiological processes involved in the development of lupus nephritis still remain unclear. In this study, we examined the involvement of activin A, a member of TGF-β superfamily, in the progression of renal damage in lupus-prone MRL-lpr mice. Activin A was was not expressed in the kidneys of normal C57BL/6 mice but was detectable in perivascular infiltrating CD68-positive cells in the kidneys of MRL-lpr mice. Urinary activin A, which was also absent in normal C57BL/6 mice, was detectable in MRL-lpr mice from 16 weeks onwards. Urinary activin A levels were significantly correlated with the number of perivascular inflammatory cell layers, the number of crescentic glomeruli, and the percentage of EVG-positive fibrotic areas, but not with urinary protein levels or serum activin A. When activin action was blocked in vivo by the intraperitoneal administration of an activin antagonist, follistatin, the number of cresentic glomeruli, percentage of EVG-positive fibrotic areas, CD68-positive cell infiltration, and proteinuria were significantly reduced in a dose-dependent manner. These data suggest that infiltrating macrophage-derived activin A is involved in the progression of renal damage in MRL-lpr mice.
Intrarenal drug infusion plays an important role in renal experimental research. Laminar flow of the blood can cause streaming and inhomogeneous intrarenal distribution of infused drugs. We suggest a simple method to achieve a homogeneous intravascular distribution of drugs infused into the renal artery of anesthetized rats. The method employs a multiple sidehole catheter inserted into the renal artery, which enables an efficient drug mixing with the arterial blood. To verify the efficiency of this method we use laser speckle imaging and renal artery flowmetry. The results show that, compared to the conventional single-hole catheter, the multiple sidehole catheter provides a more uniform drug distribution and a homogenous vascular response on the surface of the kidney.
Unique experimental advantages, such as its embryonic/larval transparency, high-throughput nature and ease of genetic modification, underpin the rapid emergence of the zebrafish (Danio rerio) as a preeminent model in biomedical research. Particularly in the field of nephrology, the zebrafish provides a promising model for studying the physiological implications of human solute transport processes along consecutive nephron segments. However, while the zebrafish might be considered a valuable model for numerous renal ion transport diseases and functional studies of many channels and transporters, not all human renal electrolyte transport mechanisms and human diseases can be modeled in the zebrafish. With this review, we explore the ontogeny of zebrafish renal ion transport, its nephron structure and function and thereby demonstrate the clinical translational value of this model. By critical assessment of genomic and amino acid conservation of human proteins involved in renal ion handling (channels, transporters and claudins), kidney and nephron segment conservation, and renal electrolyte transport physiology in the zebrafish, we provide researchers and nephrologists with an indication of the possibilities and considerations of the zebrafish as a model for human renal ion transport. Combined with advanced techniques envisioned for the future, implementation of the zebrafish might expand beyond unraveling pathophysiological mechanisms that underlie distinct genetic or environmentally, i.e. pharmacological and life-style, induced renal transport deficits. Specifically, the ease of drug administration and the exploitation of improved genetic approaches might argue for the adoption of the zebrafish as model for preclinical personalized medicine for distinct renal diseases and renal electrolyte transport proteins.
T cells have been implicated in the pathogenesis of AKI as well as its progression to CKD. Previous studies suggest that Th17 cells participates during the AKI to CKD transition and inhibition of T cell activity by mycophenolate mofetil (MMF) or losartan attenuates the development of fibrosis following AKI. We hypothesized that T cell deficient rats may have reduced levels of IL-17 cytokine leading to decreased fibrosis following AKI. Renal I/R was performed on T cell deficient athymic rats (Foxn1rnu-/rnu-) and control euthymic rats (Foxn1rnu-/+) and CKD progression was hastened by unilateral nephrectomy at Day 33 and subsequent exposure to 4.0% sodium diet. Renal fibrosis developed in euthymic rats and was reduced by MMF treatment. Athymic rats exhibited a similar degree of fibrosis but this was unaffected by MMF treatment. FACS analysis demonstrated that the number of IL-17+ cells was similar between post ischemic athymic vs euthymic rats. The source of IL-17 production in euthymic rats was predominately from conventional T cells (CD3+/CD161-). In the absence of conventional T-cells in athymic rats, a compensatory pathway involving Natural Killer (NK) cells (CD3-/CD161+) was the primary source of IL-17. Blockade of IL-17 activity using IL-17Rc receptor significantly decreased fibrosis and neutrophil recruitment in both euthymic and athymic rats as compared to vehicle treated controls. Taken together, these data suggest that IL-17 secretion participates in the pathogenesis of AKI induced fibrosis possibly via the recruitment of neutrophils and that the source of IL-17 may be from either conventional T cells or NK cells.
Three decades ago a revolutionary idea ascribing to dysfunctional endothelia some manifestations of diabetes was born, the Steno hypothesis. Here I briefly outline the accomplishments made in the past 15 years to buttress this hypothesis. Those include development of novel technological platforms to examine microcirculatory beds, deeper understanding of patterns of microvascular derangement in diabetes, pathophysiology of nitric oxide synthesis and availability, nitrosative and oxidative stress in diabetes, premature senescence of endothelial cells and the role of sirtuin 1 and lysosomal dysfunction in this process, state of endothelial glycocalyx and endothelial progenitor cells in diabetes. These pathophysiologic findings may confer some therapeutic benefits.
Krüppel-like factors (KLFs) are a family of zinc-finger transcription factors critical to mammalian embryonic development, regeneration, and human disease. There is emerging evidence that KLFs play a vital role in key physiological processes in the kidney, ranging from maintenance of glomerular filtration barrier to tubulointerstitial inflammation to progression of kidney fibrosis. Seventeen members of the KLF family have been identified, and several have been well characterized in the kidney. Although they may share some overlap in their downstream targets, their structure and function remain distinct. This review highlights our current knowledge of KLFs in the kidney, which includes their pattern of expression and their function in regulating key biological processes. We will also critically examine the currently available literature on KLFs in the kidney and offer some key areas in need of further investigation.
Proteinuria is one of the primary risk factors for the progression of chronic kidney disease (CKD), and has been implicated in the induction of endoplasmic reticulum (ER) stress. We hypothesized that the suppression of ER stress with a low molecular weight chemical chaperone, 4-phenylbutyric acid (4-PBA), would reduce the severity of CKD and proteinuria in the Dahl salt-sensitive (SS) hypertensive rat. To induce hypertension and CKD, 12-week old male rats were placed on a high salt (HS) diet for 4-weeks with or without 4-PBA treatment. We assessed blood pressure and markers of CKD, including proteinuria, albuminuria, and renal pathology. Further, we determined if HS feeding resulted in an impaired myogenic response, subsequent to ER stress. 4-PBA treatment reduced salt-induced hypertension, proteinuria and albuminuria, and preserved myogenic constriction. Further, renal pathology was reduced with 4-PBA treatment, as indicated by lowered expression of pro-fibrotic markers and fewer intratubular protein casts. In addition, ER stress in the glomerulus was reduced, and the integrity of the glomerular filtration barrier was preserved. These results suggest that 4-PBA treatment protects against proteinuria in the SS rat by preserving the myogenic response, and by preventing ER stress, which led to a breakdown in the glomerular filtration barrier. As such, alleviating ER stress serves as a viable therapeutic strategy to preserve kidney function and to delay the progression of CKD in the animal model under study.
The early progression of diabetic nephropathy is notoriously difficult to detect and quantify prior to the occurrence of substantial histological damage. Recently, hyperpolarized [1-13C] pyruvate has demonstrated increased lactate production in the kidney early after the onset of diabetes, implying increased lactate dehydrogenase activity as a consequence of increased nicotinamide adenine dinucleotide substrate availability due to upregulation of the polyol pathway, i.e., pseudohypoxia. In this study, we investigated the role of oxidative stress in mediating these metabolic alterations using state-of-the-art hyperpolarized magnetic resonance (MR) imaging. Ten-week-old female Wistar rats were randomly divided into three groups: healthy controls, untreated diabetic (streptozotocin treatment to induce insulinopenic diabetes), and diabetic, receiving chronic antioxidant treatment with TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl) via the drinking water. Examinations were performed 2, 3, and 4 weeks after the induction of diabetes by using a 3T Clinical MR system equipped with a dual tuned 13C/1H-volume rat coil. The rats received intravenous hyperpolarized [1-13C] pyruvate and were imaged using a slice-selective 13C-IDEAL spiral sequence. Untreated diabetic rats showed increased renal lactate production compared to that shown by the controls. However, chronic TEMPOL treatment significantly attenuated diabetes-induced lactate production. No significant effects of diabetes or TEMPOL were observed on 13C-alanine levels, indicating an intact glucose-alanine cycle, or 13C-bicarbonate, indicating normal flux through the Krebs cycle. In conclusion, this study demonstrates that diabetes-induced pseudohypoxia, as indicated by an increased lactate-to-pyruvate ratio, is significantly attenuated by antioxidant treatment. This demonstrates a pivotal role of oxidative stress for renal metabolic alterations occurring in early diabetes.
AMP-activated kinase (AMPK) controls cell energy homeostasis by modulating ATP synthesis and expenditure. In vitro studies have suggested AMPK may also control key elements of renal epithelial electrolyte transport but in vivo physiological confirmation is still insufficient. We studied sodium renal handling and extracellular volume regulation in mice with genetic deletion of AMPK catalytic subunits. AMPK alpha-1 KO mice exhibit normal renal sodium handling and a moderate antidiuretic state. This is accompanied by higher urinary aldosterone excretion rates and reduced blood pressure. Plasma volume, however, was found to be increased compared to wild-type mice. Thus blood volume is preserved despite a significantly lower hematocrit. The lack of a defect in renal function in AMPK alpha-1 KO mice could be explained by a compensatory upregulation in AMPK alpha-2 subunit. Therefore we used the Cre-loxP system to knock down AMPK alpha-2 expression in renal epithelial cells. Combining this approach with the systemic deletion of AMPK alpha-1 we achieved reduced renal AMPK activity, accompanied by a shift to a moderate water and salt wasting phenotype. Thus we confirm the physiologically relevant role of AMPK in the kidney. Furthermore, our results indicate that in vivo AMPK activity stimulates renal sodium and water reabsorption.
Current animal models of hemorrhagic shock-induced acute kidney injury (HS-induced AKI) require extensive surgical procedures and constant monitoring of hemodynamic parameters. Application of these HS-induced AKI models in mice to produce consistent kidney injury is challenging. In the present study, we developed a simple and highly reproducible mouse model of HS-induced AKI by combining moderate bleeding and renal pedicles clamping, which was abbreviated as HS-AKI. HS was induced by retro-orbital bleeding of 0.4 ml blood in C57BL/6 mice. Mice were left in HS stage for 30 min, followed by renal pedicles clamping for 18 min at 36.8-37.0 0C. Mean arterial pressure (MAP) and heart rate were monitored with pre-implanted radio transmitters throughout the experiment. The acute response in renal blood flow (RBF) triggered by HS were measured with transonic flow probe. Mice received sham operation, bleeding alone, and renal pedicles clamping alone served as respective controls. MAP was reduced from 77 ± 4 to 35 ± 3 mmHg after bleeding. RBF was reduced by 65% in the HS period. Plasma creatinine and Kim-1 levels were increased by more than 22-fold 24 hrs after reperfusion. GFR was declined by 78% of baseline 3 days after reperfusion. Histological examination revealed a moderate to severe acute tubular damage mostly at the cortex-medulla junction area, followed by the medullar and cortex regions. HS alone did not induce significant kidney injury, but synergistically enhanced pedicels clamping-induced AKI. This is a well-controlled, simple and reliable mouse model of HS-AKI.
Renin is the initiator and rate-limiting factor in the renin-angiotensin blood pressure regulation system. Whilst renin is not exclusively produced in the kidney, in non-murine species the synthesis and secretion of the active circulatory enzyme is confined almost exclusively to the dense core granules of juxtaglomerular (JG) cells, where prorenin is processed and stored for release via a regulated pathway. Despite its importance, the structural organization and regulation of granules within these cells is not well understood, in part due to the difficulty in culturing primary JG cells in vitro and the lack of appropriate cell lines. We have streamlined the isolation and culture of primary renin-expressing cells suitable for high-speed, high-resolution live imaging using a Percoll gradient-based procedure to purify cells from RenGFP+ transgenic mice. Fibronectin-coated glass coverslips proved optimal for the adhesion of renin-expressing cells and facilitated live cell imaging at the plasma membrane of primary renin cells using total internal reflection fluorescence microscopy (TIRFM). To obtain quantitative data on intracellular function, we stained mixed granule and lysosome populations with Lysotracker Red and stimulated cells using 100 nM isoproterenol. Analysis of membrane-proximal acidic granular organelle dynamics and behavior within renin-expressing cells revealed the existence of two populations of granular organelles with distinct functional responses following isoproterenol stimulation. The application of high-resolution techniques for imaging JG and other specialized kidney cells provides new opportunities for investigating renal cell biology.
Clinical recommendations limit menopausal hormone therapy to a few years, yet the impact of a shorter treatment duration on cardiovascular health is unknown. We hypothesized that both short- and long-term estradiol (E2) treatment exerts positive and lasting effects on blood pressure, vascular reactivity, and renal health. This study was designed to mimic midlife menopause followed by E2 treatment that either followed or exceeded the current clinical recommendations. Female Long Evans retired breeders were ovariectomized (OVX) at 11 months of age and randomized into three groups: 80d vehicle (Veh>Veh), 40d E2 + 40d vehicle (E2>Veh), and 80d E2 (E2>E2). In comparison to Veh>Veh, both the E2>Veh and E2>E2 groups had lower systolic blood pressure and enhanced mesenteric relaxation in response to estrogen receptor α stimulation. Despite the reduced blood pressure, E2>E2 induced renal and cardiac hypertrophy, reduced glomerular filtration, and increased proteinuria. Interestingly, kidneys from E2>Veh rats had significantly fewer tubular casts than both other groups. In conclusion, long-term E2 lowered blood pressure but exerted detrimental effects on kidney health in midlife OVX Long Evans rats, while short-term E2 lowered blood pressure and reduced renal damage. These findings highlight that the duration of hormone therapy may be an important factor for renal health in aging postmenopausal women.
Mice transgenic for genomic segments harboring PHAII (pseudohypoaldosteronism type II) mutant Wnk4 (with-No-Lysine kinase 4) (TgWnk4PHAII) have hyperkalemia which is currently believed to be the result of high activity of Na-Cl cotransporter (NCC). This leads to decreasing Na+ delivery to the distal nephron segment including late distal convoluted tubule (DCT) and connecting tubule (CNT). Since epithelial Na+ channel (ENaC) and renal outer medullary K+ channel (ROMK or Kir4.1) are expressed in the late DCT and play an important role in mediating K+ secretion, the aim of the present study is to test whether ROMK and ENaC activity in the DCT/CNT are also compromised in the mice expressing PHAII mutant Wnk4. Western blot analysis shows that the expression of βENaC and ENaC subunits but not αENaC subunit was lower in TgWnk4PHAII mice than that in wild type (WT) and TgWnk4WT mice. Patch-clamp experiments detected amiloride-sensitive Na+ currents and TPNQ-sensitive K+ currents in DCT2/CNT, suggesting the activity of ENaC and ROMK. However, both Na+ and ROMK currents in DCT2/CNT of TgWnk4PHAII mice were significantly smaller than those in WT and TgWnk4WT mice. In contrast, the basolateral K+ currents in the DCT were similar among three groups, despite higher NCC expression in TgWnk4PHAII mice than those of WT and TgWnk4WTmice. An increase in dietary K+ intake significantly increased both ENaC and ROMK currents in the DCT2/CNT of all three groups. However, high K+ (HK) intake-induced stimulation of Na+ and K+ currents was smaller in TgWnk4PHAII mice than those in WT and TgWnk4WT mice. We conclude that ENaC and ROMK channel activity in DCT2/CNT are inhibited in TgWnk4PHAII mice and that Wnk4PHAII-induced inhibition of ENaC and ROMK may contribute to the suppression of K+ secretion in the DCT2/CNT in addition to increased NCC activity.
A Ca2+-activated non-selective cation channel (NSCCa) is found in principal cells of mouse cortical collecting duct (CCD). However, the molecular identity of this channel remains unclear. We used mpkCCDc14 cells, a mouse CCD principal cell line, to determine whether NSCCa represents the transient receptor potential (TRP) channel, the melastatin subfamily 4 (TRPM4). A Ca2+-sensitive single-channel current was observed in inside-out patches excised from the apical membrane of mpkCCDc14 cells. Like TRPM4 channels found in other cell types, this channel has an equal permeability for Na+ and K+ and has a linear current-voltage relationship with a slope conductance of approximately 23 pS. The channel was inhibited by a specific TRPM4 inhibitor, 9-phenanthrol. Moreover, the frequency of observing this channel was dramatically decreased in TRPM4 knockdown mpkCCDc14 cells. Unlike those previously reported in other cell types, the TRPM4 in mpkCCDc14 cells was unable to be activated by hydrogen peroxide (H2O2). Conversely, after treatment with H2O2, TRPM4 density in the apical membrane of mpkCCDc14 cells was significantly decreased. The channel in intact cell-attached patches was activated by ionomycin (a Ca2+ ionophore), but not by adenosine triphosphate (ATP) (a purinergic P2 receptor agonist). These data suggest that the NSCCa current previously described in CCD principal cells is actually carried through TRPM4 channels. However, the physiological role of this channel in CCD remains to be further determined.
Acute kidney injury (AKI) induced by clamping of renal vein or pedicle is more severe than clamping of artery, but the mechanism has not been clarified. In present study, we tested our hypothesis that increased proximal tubular pressure (Pt) during the ischemic phase exacerbates kidney injury and promotes the development of AKI. We induced AKI by bilateral clamping of renal arteries, pedicles or veins for 18 min at 37° C, respectively. Pt during the ischemic phase was measured with micropuncture. We found that higher Pt was associated with more severe AKI. To determine the role of Pt during the ischemic phase on the development of AKI, we adjusted the Pt by altering renal artery pressure. We induced AKI by bilateral clamping of renal veins, and the Pt was modulated by adjusting the renal artery pressure during the ischemic phase by constriction of aorta and mesenteric artery. When we decreased renal artery pressure from 85±5 to 65±8 mmHg, Pt decreased from 53.3±2.7 to 44.7±2.0 mmHg. Plasma creatinine decreased from 2.48±0.23 to 1.91±0.21 mg/dl at 24 hours after renal ischemia. When we raised renal artery pressure to 103±7 mmHg, Pt increased to 67.2±5.1 mmHg. Plasma creatinine elevated to 3.17±0.14 mg/dl 24 hours after renal ischemia. Changes in KIM-1, NGAL and histology were in the similar pattern as plasma creatinine. In summary, we found that higher Pt during the ischemic phase promoted the development of AKI, while lower Pt protected from kidney injury. Pt may be a potential target for treatment of AKI.
Within the CCD of the distal nephron of the rabbit, the BK (maxi K) channel mediates Ca2+- and/or stretch-dependent flow-induced K+ secretion (FIKS) and contributes to K+ adaptation in response to dietary K+ loading. An unresolved question is whether BK channels in intercalated cells (ICs) and/or principal cells (PCs) in the CCD mediate these K+ secretory processes. In support for a role for ICs in FIKS is the higher density of immunoreactive apical BKα (pore forming subunit) and functional BK channel activity than detected in PCs, and an increase in IC BKα expression in response to a high K+ diet. PCs possess a single apical cilium which has been proposed to serve as a mechanosensor; direct manipulation of cilia leads to increases in cell Ca2+ concentration, albeit of non-ciliary origin. Immunoperfusion of isolated and fixed CCDs isolated from control K+ fed rabbits with channel subunit-specific Abs revealed colocalization of immunodetectable BKα and β1 subunits in cilia as well as on the apical membrane of cilia-expressing PCs. Ciliary BK channels were more easily detected in rabbits fed a low K+ vs. high K+ diet. Single channel recordings of cilia revealed K+ channels with conductance and kinetics typical for the BK channel. The observations that (1) FIKS was preserved but (2) the high amplitude Ca2+ peak elicited by flow was reduced in microperfused CCDs subject to pharmacologic deciliation suggests that cilia BK channels do not contribute to K+ secretion in this segment, but that cilia serve as modulators of cell signaling.
The specific roles of NO synthases (NOSs) in bladder smooth muscle remain to be elucidated. We examined the roles of NOSs in β-adrenoceptor (AR)-mediated bladder relaxation. Male mice (C57BL6) deficient of neuronal NOS (nNOS-KO), endothelial NOS (eNOS-KO), neuronal/endothelial NOS (n/eNOS-KO), neuronal/endothelial/inducible NOS (n/e/iNOS-KO) and their controls (wild-type, WT) were used. Immunohistochemical analysis was performed in the bladder. Then, the responses to relaxing agents and the effects of several inhibitors on the relaxing responses were examined in bladder strips precontracted with carbachol. Immunofluorescence staining showed expressions of nNOS and eNOS in the urothelium and smooth muscle of the bladder. Isoproterenol-induced relaxations were significantly reduced in nNOS-KO mice and were further reduced in n/eNOS-KO and n/e/iNOS-KO mice compared with WT mice. The relaxation in n/e/iNOS-KO mice was almost the same with n/eNOS-KO mice. Inhibition of Ca2+-activated K+ (KCa) channel with charybdotoxin and apamin abolished isoproterenol-induced bladder relaxation in WT mice. Moreover, direct activation of KCa channel with NS1619 caused comparable extent of relaxations among WT, nNOS-KO and n/eNOS-KO mice. In contrast, NONOate (a NO donor) or hydrogen peroxide (H2O2) (another possible relaxing factor from eNOS) caused minimal relaxations, and catalase (H2O2 scavenger) had no inhibitory effects on isoproterenol-induced relaxations. These results indicate that both nNOS and eNOS are substantially involved in β-AR-mediated bladder relaxations in a NO- or H2O2-independent manner through activation of KCa channels.
Improving drug delivery to the kidney using renal-targeted therapeutics is a promising but under-developed area. We aimed to develop a kidney-targeting construct for renal-specific drug delivery. Elastin-like polypeptides (ELPs) are non-immunogenic protein-based carriers that can stabilize attached small molecule and peptide therapeutics. We modified ELP at its N-terminus with a cyclic, seven amino acid kidney-targeting peptide (KTP) and at its C-terminus with a cysteine residue for tracer conjugation. Comparative in vivo pharmacokinetics and biodistribution in rat and swine models and in vitro cell binding studies using human renal cells were performed. KTP-ELP had longer plasma half-life than ELP in both animal models and similarly accumulated in kidneys at levels 5-fold higher than untargeted ELP, showing renal levels 15- to over 150-fold higher than in other major organs. Renal fluorescence histology demonstrated high accumulation of KTP-ELP in proximal tubules and vascular endothelium. Furthermore, a 14-day infusion of a high dose of ELP or KTP-ELP did not affect body weight, glomerular filtration rate, albuminuria, or induce renal tissue damage compared to saline-treated controls. In vitro experiments showed higher binding of KTP-ELP to human podocytes, proximal tubule epithelial, and glomerular microvascular endothelial cells than untargeted ELP. These results show the high renal selectivity of KTP-ELP, support the notion that the construct is not species specific, and demonstrate that it does not induce acute renal toxicity. The plasticity of ELP for attachment of any class of therapeutics unlocks the possibility of applying ELP technology for targeted treatment of renal disease in future studies.
The paracellular pathways in renal tubular epithelia such as the proximal tubules, which reabsorb the largest fraction of filtered solutes and water and are leaky epithelia, are important routes for transepithelial transport of solutes and water. Movement occurs passively via an extracellular route through the tight junction between cells. The characteristics of paracellular transport vary among different nephron segments with leaky or tighter epithelia. Claudins expressed at tight junctions form pores and barriers for paracellular transport. Claudins are from a multigene family, comprising at least 27 members in mammals. Multiple claudins are expressed at tight junctions of individual nephron segments in a nephron segment-specific manner. Over the last decade there have been advances in our understanding of the structure and functions of claudins. This paper is a review of our current knowledge of claudins, with special emphasis on their physiological roles in proximal tubule paracellular solute and water transport.
Microsomal prostaglandin E2 synthase-1 (mPGES-1), an inducible enzyme that converts prostaglandin H2 to prostaglandin E2 (PGE2), plays an important role in a variety of inflammatory diseases. We investigated the contribution of mPGES-1 to renal fibrosis and inflammation in unilateral ureteral obstruction (UUO) for 7 days using wild type (WT) and mPGES-1 knockout (KO) mice. UUO induced increased mRNA and protein expression of mPGES-1 and cyclooxygenase-2 in WT mice. UUO was associated with increased renal PGE2 content and upregulated PGE2 receptor (EP) 4 expression in obstructed kidneys of both WT and KO mice; EP4 expression levels were higher in KO mice with UUO than that in WT mice. Protein expression of NLRP3 inflammasomes components ASC and interleukin-1β was significantly increased in obstructed kidneys of KO mice compared with that in WT mice. mRNA expressions of fibronectin, collagen III, and transforming growth factor-β1 (TGF-β1) were significantly higher in obstructed kidneys of KO mice than that in WT mice. In mPGES-1 KO mice, protein expression of fibronectin and collagen III was markedly increased in obstructed kidneys when compared with WT mice, which was associated with increased phosphorylation of protein kinase B (AKT). EP4 agonist CAY10598 attenuated increased expression of collagen I and fibronectin induced by TGF-β1 in inner medullary collecting duct 3 cells. Moreover, CAY10598 prevented the activation of NLRP3 inflammasomes induced by angiotensin II in human proximal tubule cells (HK2). In conclusion, these findings suggested that mPGES-1 exerts a potentially protective effect against renal fibrosis and inflammation induced by UUO in mice.
Mutations in the renal sodium-dependent phosphate co-transporters NPT2a and NPT2c have been reported in patients with renal stone disease and nephrocalcinosis. Oral phosphate supplementation is currently thought to reduce risk by reversing the hypercalciuria, but the exact mechanism remains unclear and the relative contribution of modifiers of mineralization such as osteopontin (Opn) to the formation of renal mineral deposits in renal phosphate wasting disorders has not been studied. We observed a marked decrease of renal gene expression and urinary excretion of Opn in Npt2a-/- mice, a mouse model of these disorders, at baseline. Following supplementation with phosphate Opn gene expression was restored to WT levels in Npt2a-/- mice, however, urine excretion of the protein remained low. To further investigate the role of Opn we used a double-knockout strategy, which provides evidence that loss of Opn worsens the nephrocalcinosis and nephrolithiasis observed in these mice on high phosphate diet. These studies suggest that impaired Opn gene expression and urinary excretion in Npt2a-/- mice may be an additional risk factor for nephrolithiasis, and normalizing urine Opn levels may improve the therapy of phosphaturic disorders.
Phosphorylation of the aquaporin-2 (AQP2) water channel at four COOH-terminal serines plays a central role in the regulation of water permeability of the renal collecting duct. The level of phosphorylation at these sites is determined by a balance between phosphorylation by protein kinases and dephosphorylation by phosphatases. The phosphatases that dephosphorylate AQP2 have not been identified. Here, we use large-scale data integration techniques to identify serine-threonine phosphatases likely to interact with AQP2 in renal collecting duct principal cells. As a first step, we have created a comprehensive list of 38 S/T phosphatase catalytic subunits present in the mammalian genome. Then we used Bayes' theorem to integrate available information from large-scale data sets from proteomic and transcriptomic studies in order to rank the known S/T phosphatases with regard to the likelihood that they interact with AQP2 in renal collecting duct cells. To broaden the analysis, we have generated new proteomic data (LC-MS/MS) identifying 4538 distinct proteins including 22 S/T phosphatases in cytoplasmic fractions from native inner medullary collecting duct cells from rats. The official gene symbols corresponding to the top-ranked phosphatases (common names in parentheses) were: Ppp1cb (PP1-β), Ppm1g (PP2C), Ppp1ca (PP1-α), Ppp3ca (PP2-B or calcineurin), Ppp2ca (PP2A-α), Ppp1cc (PP1-), Ppp2cb (PP2A-β), Ppp6c (PP6C) and Ppp5c (PP5). This ranking correlates well with results of prior reductionist studies of ion and water channels in renal collecting duct cells.
Previously, we documented that activation of protein kinase C- (PKC-) mediates mitochondrial dysfunction in cultured renal proximal tubule cells (RPTC). This study tested whether deletion of the PKC- decreases dysfunction of renal cortical mitochondria and improves kidney function after renal ischemia. PKC- levels in mitochondria of ischemic kidneys increased 24h after ischemia. Complex I- and complex II-coupled state 3 respirations were reduced 44% and 27%, respectively, in wild-type (WT) but unchanged and increased in PKC--deficient (KO) mice after ischemia. Respiratory control ratio coupled to glutamate/malate oxidation decreased 50% in WT, but not in KO mice. Activities of complexes I, III, and IV were decreased 59, 89, and 61%, respectively, in WT but not in KO ischemic kidneys. Proteomics revealed increases in levels of ATP synthase (α-subunit), complexes I and III, cytochrome oxidase, α-ketoglutarate dehydrogenase, and thioredoxin-dependent peroxide reductase after ischemia in KO, but not in WT animals. PKC- deletion prevented ischemia-induced increases in oxidant production. Plasma creatinine levels increased 12-fold in WT and 3-fold in KO ischemic mice. PKC- deletion reduced tubular necrosis, brush border loss, and distal segment damage in ischemic kidneys. PKC- activation in hypoxic RPTC in primary culture exacerbated, whereas PKC- inhibition reduced decreases in: 1) complex I- and complex II-coupled state 3 respirations, and 2) activities of complexes I, III, and IV. We conclude that PKC- activation mediates: 1) dysfunction of complexes I and III of the respiratory chain, 2) oxidant production, 3) morphological damage to the kidney, and 4) decreases in renal functions after ischemia.
Autosomal dominant polycystic kidney disease (ADPKD) is the most common life-threatening monogenic renal disease. ADPKD results from mutations in either of two proteins: polycystin-1 (also known as PC1 or PKD1) or transient receptor potential cation channel, subfamily P, member 2 (TRPP2, also known as polycystin-2, PC2, or PKD2). Each of these proteins is expressed in the primary cilium that extends from many renal epithelial cells. Existing evidence suggests that the cilium can promote renal cystogenesis, while PC1 and TRPP2 counter this cystogenic effect. To better understand the function of TRPP2, we investigated its electrophysiological properties in the native ciliary membrane. We recorded directly from the cilia of mIMCD-3 cells, a murine cell line of renal epithelial origin. In one-third of cilia examined, a large-conductance channel was observed. The channel was not permeable to Cl– but conducted cations with permeability ratios PK:PCa:PNa of 1:0.55:0.14. The single-channel conductance ranged from 97 pS in typical physiological solutions to 189 pS in symmetrical 145 mM KCl. Open probability of the channel was very sensitive to membrane depolarization or increasing cytoplasmic free Ca2+ in the low micromolar range, with the open probability increasing in either case. Knocking out TRPP2 by CRISPR/Cas9 genome editing eliminated the channel current, establishing it as TRPP2-dependent. Possible mechanisms for activating the TRPP2-dependent channel in the renal primary cilium are discussed.
STAT3 is a transcription factor implicated in renal fibrotic injury, but the role of STAT3 in mesenchymal stem cell (MSC)-induced renoprotection during renal fibrosis remains unknown. We hypothesized that MSCs protect against obstruction-induced renal fibrosis by downregulating STAT3 activation and STAT3-induced matrix metalloproteinase 9 (MMP-9) expression. Male Sprague-Dawley rats underwent renal arterial injection of vehicle or MSCs (1 x 106 per rat) immediately prior to sham operation or induction of unilateral ureteral obstruction (UUO). The kidneys were harvested after 4 weeks and analyzed for collagen I and III gene expression, collagen deposition (Masson's trichrome), fibronectin, α-SMA, active STAT3 (pSTAT3), MMP-9, and tissue inhibitors of matrix metalloproteinases 1 (TIMP-1), expression. In a separate arm, the STAT3 inhibitor, S3I-201 (10mg/kg), vs. vehicle was administered to rats intraperitoneally (IP) just after induction of UUO and daily for 14 days thereafter. The kidneys were harvested after 2 weeks and analyzed for pSTAT3 and MMP-9 expression, and collagen and fibronectin deposition. Renal obstruction induced a significant increase in collagen, fibronectin, α-SMA, pSTAT3, MMP-9 and TIMP-1 expression, while exogenously administered MSCs significantly reduced these indicators of obstruction-induced renal fibrosis. STAT3 inhibition with S3I-201 significantly reduced obstruction-induced MMP-9 expression and tubulointerstitial fibrosis. These results demonstrate that MSCs protect against obstruction-induced renal fibrosis, in part, by decreasing STAT3 activation and STAT3-dependent MMP-9 production.
Within the past decade tremendous efforts have been made to understand the mechanism behind aquaporin-2 (AQP2) water channel trafficking and recycling, in order to open a path towards effective diabetes insipidus therapeutics. A recent study has shown that Integrin-Linked Kinase (ILK) conditional-knockdown mice developed polyuria along with decreased expression of AQP2. To understand whether ILK also regulates AQP2 trafficking in kidney tubular cells, we performed in vitro analysis using LLCPK1 cells stably expressing rat AQP2 (LLC-AQP2 cells). Upon treatment of LLC-AQP2 cells with ILK inhibitor cpd22 and ILK-siRNA, we observed increased accumulation of AQP2 in the perinuclear region, without any significant increase in the rate of endocytosis. This perinuclear accumulation did not occur in cells expressing a serine-256-aspartic acid mutation that retains AQP2 in the plasma membrane. We then examined clathrin-mediated endocytosis after ILK inhibition using rhodamine-conjugated transferrin. Despite no differences in overall transferrin endocytosis, the endocytosed transferrin also accumulated in the perinuclear region where it colocalized with AQP2. These accumulated vesicles also contained the recycling endosome marker Rab11. In parallel, the usual vasopressin-induced AQP2 membrane accumulation was prevented after ILK inhibition; however, ILK inhibition did not measurably affect AQP2 phosphorylation at serine-256 or its dephosphorylation at serine-261. Instead, we found that inhibition of ILK increased F-actin polymerization. When F-actin was depolymerized with latrunculin, the perinuclear located AQP2 dispersed. We conclude that ILK is important in orchestrating dynamic cytoskeletal architecture during recycling of AQP2, which is necessary for its subsequent entry into the exocytotic pathway.
Acquired renal scarring occurs in a subset of patients following febrile urinary tract infections and is associated with hypertension, proteinuria, and chronic kidney disease. Limited knowledge of histopathology, immune cell recruitment and gene expression changes during pyelonephritis restricts the development of therapies to limit renal scarring. Here, we address this knowledge gap using immunocompetent mice with vesicoureteral reflux. Transurethral inoculation of uropathogenic Escherichia coli in C3H/HeOuJ mice leads to renal mucosal injury, tubulointerstitial nephritis, and cortical fibrosis. The extent of fibrosis correlates most significantly with inflammation at 7 and 28 days post infection. The recruitment of neutrophils and inflammatory macrophages to infected kidneys is proportional to renal bacterial burden. Transcriptome analysis reveals molecular signatures associated with renal ischemia-reperfusion injury, immune cell chemotaxis, and leukocyte activation. This murine model recapitulates the cardinal histopathologic features observed in humans with acquired renal scarring following pyelonephritis. The integration of histopathology, quantification of cellular immune influx, and unbiased transcriptional profiling begins to define potential mechanisms of tissue injury during pyelonephritis in the context of an intact immune response. The clear relationship between inflammatory cell recruitment and fibrosis supports the hypothesis that acquired renal scarring arises as a consequence of excessive host inflammation and suggests that immunomodulatory therapies should be investigated to reduce renal scarring in patients with pyelonephritis.
Kidney collecting duct is an important renal tubular segment for regulation of body water homeostasis and urine concentration. Water reabsorption in the collecting duct principal cells is controlled by vasopressin, a peptide hormone which induces the osmotic water transport across the collecting duct epithelia through regulation of water channel proteins aquaporin-2 (AQP2) and aquaporin-3 (AQP3). In particular, vasopressin induces both intracellular translocation of AQP2-bearing vesicles to the apical plasma membrane and transcription of Aqp2 gene to increase AQP2 protein abundance. The signaling pathways, including AQP2 phosphorylation, RhoA phosphorylation, intracellular calcium mobilization, and actin depolymerization, play a key role in the translocation of AQP2. This review summarizes recent data demonstrating the regulation of AQP2, as the underlying molecular mechanisms for the homeostasis of water balance in the body.
Chronic kidney disease (CKD) is associated to an increased risk of death, CKD progression and acute kidney injury (AKI) even from early stages, when glomerular filtration rate (GFR) is preserved. The link between early CKD and these risks is unclear, since there is no accumulation of uremic toxins. However, pathological albuminuria and kidney inflammation are frequent features of early CKD and the production of kidney protective factors may be decreased. Indeed, Klotho expression is already decreased in CKD category G1 (normal GFR). Klotho has anti-aging and nephroprotective properties and decreased Klotho levels may contribute to increase the risk of death, CKD progression and AKI. In this review, we discuss the downregulation by mediators of inflammation of molecules with systemic and/or renal local protective functions, exemplified by Klotho and peroxisome proliferator-activated receptor gamma coactivador-1α (PGC-1α), a transcription factor that promotes mitochondrial biogenesis. Cytokines such as TWEAK, TNFα or TGFβ1 produced locally during kidney injury or released from inflammatory sites at other organs may decrease kidney expression of Klotho and PGC-1α or lead to suboptimal recruitment of these nephroprotective proteins. Transcription factors (e.g. Smad3, NF-B) and epigenetic mechanisms (e.g. histone acetylation or methylation) contribute to downregulate the expression of Klotho and/or PGC-1α, while histone crotonylation promotes PGC-1α expression. NFBiz facilitates the repressive effect of NF-B on Klotho expression. A detailed understanding of these mediators may contribute to develop novel therapeutic approaches to prevent CKD progression and its negative impact on mortality and AKI.
In the setting of normal kidney function, iron deficiency is associated with increased FGF23 production and cleavage, altering circulating FGF23 levels. Our objective was to determine how chronic kidney disease (CKD) and dietary iron intake affect FGF23 production and metabolism in wild type (WT) and hepcidin knockout (HKO) mice. For eight weeks, the mice were fed diets that contained adenine (to induce CKD) or no adenine (control group), with either low iron (4 ppm) or standard iron (335 ppm) concentrations. The low iron diet induced iron deficiency anemia in both the WT and HKO mice. Among the WT mice, in both the control and CKD groups, low iron compared to standard iron diet increased bone Fgf23 mRNA expression, C-terminal FGF23 (cFGF23) levels, and FGF23 cleavage as manifested by a lower percentage intact FGF23 (iFGF23). Independent of iron status, CKD was associated with inhibition of FGF23 cleavage. Similar results were observed in the HKO control and CKD groups. Dietary iron content was more influential on FGF23 parameters than the presence or absence of hepcidin. In the CKD mice (WT and HKO, total n=42), independent of the effects of serum phosphate, iron deficiency was associated with increased FGF23 production but also greater cleavage, whereas worse kidney function was associated with increased FGF23 production but decreased cleavage. Therefore, in both the WT and HKO mouse models, dietary iron content and CKD affected FGF23 production and metabolism.
Carboxyl-terminus of AQP2 (AQP2c) undergoes post-translational modifications, including phosphorylation and ubiquitination, for the regulation of aquaporin-2 (AQP2) translocation and protein abundance. We aimed to identify novel proteins interacting with AQP2c. Recombinant AQP2c protein was made in E. coli BL21 (DE3) by exploiting the pET32 TrxA fusion system. Lysates of rat kidney inner medullary collecting duct (IMCD) tubule suspensions interacted with rat AQP2c bound to Ni2+-resin were subjected to LC-MS/MS proteomic analysis. Potential interacting proteins were identified, including vacuolar protein sorting-associated protein 35 (Vps35). Co-immunoprecipitation assay demonstrated that Vps35 interacted with AQP2c. Immunohistochemistry of rat kidney revealed that AQP2 and Vps35 were partly co-localized at the intracellular vesicles in the collecting duct cells. The role of Vps35 in the dDAVP-induced AQP2 regulation was examined in mpkCCDc14 cells. Cell surface biotinylation assay demonstrated that dDAVP-induced apical translocation of AQP2 was significantly decreased under the siRNA-mediated Vps35 knockdown. dDAVP-induced AQP2 up-regulation was less prominent in the cells with Vps35 knockdown. Moreover, AQP2 protein abundance was decreased to a greater extent during the withdrawal period after dDAVP stimulation under the Vps35 knockdown, which was significantly inhibited by chloroquine (a blocker of the lysosomal pathway) treatment, but not by MG132 (a proteasome inhibitor). Immunocytochemistry demonstrated that internalized AQP2 was more associated with lysosomal marker (LAMP-1) in the primary cultured IMCD cells under the Vps35 knockdown. Taken together, Vps35 interacts with AQP2c and depletion of Vps35 is likely to be associated with decreased AQP2 trafficking and increased lysosomal degradation of AQP2 protein.
Renal Na+-Cl- cotransporter (NCC) is expressed in early distal convoluted tubule (DCT) 1 and late DCT (DCT2). NCC activity can be stimulated by aldosterone, and the mechanism is assumed to depend on the enzyme, 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which inactivates glucocorticoids that would otherwise occupy aldosterone receptors. Because 11β-HSD2 in rat may only be abundantly expressed in DCT2 cells and not in DCT1 cells, it has been speculated that aldosterone specifically stimulates NCC activity in DCT2 cells. In mice, however, it is debated if 11β-HSD2 is expressed in DCT2 cells. The present study examined whether aldosterone-administration in mice stimulates NCC abundance and phosphorylation in DCT2 cells but not in DCT1 cells. B6/C57 male mice were administered 100 µg aldosterone (kg body weight)-1 (24 h)-1 for 6 days and euthanized during isoflurane inhalation. Western blotting of whole kidney homogenate showed that aldosterone administration stimulated NCC and pT58-NCC abundances (P < 0.001). In DCT1 cells, confocal microscopy detected no effect of the aldosterone administration on NCC and pT58-NCC abundances. By contrast, NCC and pT58-NCC abundances were stimulated by aldosterone administration in the middle of DCT2 (P < 0.001 and 0.01, respectively) and at the junction between DCT2 and CNT (P < 0.001 and 0.05, respectively). In contrast to rat, immunohistochemistry in mouse showed no/very weak 11β-HSD2 expression in DCT2 cells. Collectively, long-term aldosterone administration stimulates mouse NCC and pT58-NCC abundances in DCT2 cells and presumably not in DCT1 cells.
Aims: The relationship between the renal renin-angiotensin aldosterone system (RAAS) and cardiorenal pathophysiology is unclear. Our aims were to assess (1) levels of urinary RAAS components and (2) the association between RAAS components and HbA1c, urine albumin/creatinine ratio (ACR), estimated glomerular filtration rate (eGFR) and blood pressure in otherwise healthy adolescents with type 1 diabetes mellitus (TID) vs. healthy controls (HC). Methods: Urinary angiotensinogen and ACE2 levels, activity of ACE and ACE2, blood pressure (BP), HbA1c, ACR and eGFR were measured in 65 HC and 194 T1D from the Adolescent Type 1 Diabetes Cardio-Renal Intervention Trial (AdDIT). Results: Urinary levels of all RAAS components were higher in T1D vs. HC (p<0.0001). Higher HbA1c was associated with higher urinary angiotensinogen, ACE2, and higher activity of ACE and ACE2 (p<0.0001, p=0.0003, p=0.003 and p=0.007 respectively) in T1D. Higher ACR (within the normal range) was associated with higher urinary angiotensinogen (p<0.0001) and ACE activity (p=0.007), but not with urinary ACE2 activity or ACE2 levels. These observations were absent in HC. Urinary RAAS components were not associated with BP or eGFR in T1D or HC. Conclusions: Otherwise healthy adolescents with T1D exhibit higher levels of urinary RAAS components compared to HC. While levels of all urinary RAAS components correlate with HbA1c in T1D, only urinary angiotensinogen and ACE activity correlate with ACR, suggesting that these factors reflect an intermediary pathogenic link between hyperglycemia and albuminuria within the normal range.
Pregnancy is characterized by avid renal sodium retention and plasma volume expansion in the presence of decreased blood pressure. Decreased maternal blood pressure is a consequence of reduced systemic vascular tone which results from an increased production of vasodilators (nitric oxide (NO), prostaglandins, and relaxin) and decreased vascular responsiveness to the potent vasoconstrictor (angiotensin II (Ang II)). The kidneys participate in this vasodilatory response resulting in marked increases in renal plasma flow and glomerular filtration rate (GFR) during pregnancy. In women, sodium retention drives plasma volume expansion (~40%) and is necessary for perfusion of the growing uterus and fetus. For there to be avid sodium retention in the presence of the potent natriuretic influences of increased NO and elevated GFR, there must be modifications of the tubules to prevent salt wasting. The purpose of this review is to summarize these adaptations.
The aim was to quantify the glomerular capillary surface area, the segmental tubular radius, length, and area of single nephrons in mouse and rat kidneys. Multiple 2.5-µm-thick serial Epon sections were obtained from three mouse and three rat kidneys for three-dimensional reconstruction of the nephron tubules. Micrographs were aligned for each kidney, and 359 nephrons were traced and their segments localized. 30 mouse and 30 rat nephrons were selected for further investigation. The luminal radius of each segment was determined by two methods. The luminal surface area was estimated from the radius and length of each segment. High resolution micrographs were recorded for five rat glomeruli, and the capillary surface area determined. The capillary volume and surface area were corrected for glomerular shrinkage. A positive correlation was found between glomerular capillary area and proximal tubule area. The thickest part of the nephron, i.e. the proximal tubule, was followed by the thinnest part of the nephron, i.e. the descending thin limb, and the diameters of the seven identified nephron segments share the same rank in the two species. The radius and length measurements from mouse and rat nephrons generally share the same pattern; rat tubular radius/mouse tubular radius ratio 1.47, and rat tubular length/mouse tubular length ratio 2.29, suggesting relatively longer tubules in the rat. The detailed tables of mouse and rat glomerular capillary area and segmental radius, length, and area values may be used to enhance understanding of the associated physiology, including existing steady-state-models of the urine-concentrating mechanism.
The goal of this study was to investigate water and solute transport, with a focus on sodium transport (T$_{\rm Na}$) and metabolism along individual nephron segments under differing physiological and pathophysiological conditions. To accomplish this goal, we developed a computational model of solute transport and oxygen consumption (Q$_{\rm O2}$) along different nephron populations of a rat kidney. The model represents detailed epithelial and paracellular transport processes along both the superficial and juxtamedullary nephrons, with the loop of Henle of each model nephron extending to differing depths of the inner medulla. We used the model to assess how changes in T$_{\rm Na}$ may alter Q$_{\rm O2}$ in different nephron segments, and how shifting the T$_{\rm Na}$ sites alters overall kidney Q$_{\rm O2}$. Under baseline conditions, the model predicted a whole-kidney T$_{\rm Na}$/Q$_{\rm O2}$, which denotes the number of moles of Na$^+$ reabsorbed per moles of O$_2$ consumed, of about 15, with T$_{\rm Na}$ efficiency predicted to be significantly greater in cortical nephron segments than medullary segments. The T$_{\rm Na}$/Q$_{\rm O2}$ ratio was generally similar among the superficial and juxtamedullary nephron segments, except for the proximal tubule, where T$_{\rm Na}$/Q$_{\rm O2}$ was $\sim$20\% higher in superficial nephrons, due to the larger luminal flow along the juxtamedullary proximal tubules and the resulting flow-induced, higher transcellular transport. Moreover, the model predicted that an increase in single-nephron glomerular filtration rate (SNGFR) \Red{does not significantly affect} T$_{\rm Na}$/Q$_{\rm O2}$ in the proximal tubules but generally increases T$_{\rm Na}$/Q$_{\rm O2}$ along downstream segments. The latter result can be attributed to the generally higher luminal [Na$^+$], which raises paracellular T$_{\rm Na}$. Consequently, vulnerable medullary segments, such as the S3 segment and medullary thick ascending limb, may be relatively protected from flow-induced increases in Q$_{\rm O2}$ under pathophysiological conditions.
Sodium and its associated anions are the major determinant of extracellular fluid volume, and renal Na+ reabsorption plays a crucial role in long-term blood pressure control. The goal of this study was to investigate the extent to which inhibitors of transepithelial Na$^+$ transport (T$_{\rm Na}$) along the nephron alter urinary solute excretion and T$_{\rm Na}$ efficiency, and how those effects may vary along different nephron segments. We used the multi-nephron model developed in the companion study [28], which represents detailed transcellular and paracellular transport processes along the nephrons of a rat kidney. We simulated the inhibition of the Na+/H+ exchanger (NHE3), the bumetanide-sensitive Na+-K+-2C- transporter (NKCC2), the Na+-C- cotransporter (NCC), and the amiloride-sensitive Na$^+$ channel (ENaC). Under baseline conditions, NHE3, NKCC2, NCC and ENaC reabsorb 36, 22, 4 and 7\%, respectively, of filtered Na$^+$. The model predicted that NHE3 inhibition substantially reduced proximal tubule T$_{\rm Na}$ and oxygen consumption (Q$_{\rm O2}$). Whole-kidney T$_{\rm Na}$ efficiency, reflected by the number of moles of Na$^+$ reabsorbed per moles of O$_2$ consumed (denoted by T$_{\rm Na}$/Q$_{\rm O2}$), decreased by ~20% with 80% inhibition of NHE3. NKCC2 inhibition simulations predicted a substantial reduction in thick ascending limb T$_{\rm Na}$ and Q$_{\rm O2}$; however, the effect on whole-kidney T$_{\rm Na}$/Q$_{\rm O2}$ was minor. Tubular K+ transport was also substantially impaired, resulting in elevated urinary K$^+$ excretion. The most notable effect of NCC inhibition was to increase the excretion of Na+, K+, and Cl-; its impact on whole-kidney T$_{\rm Na}$ and its efficiency was minor. ENaC inhibition was predicted to have opposite effects on the excretion of Na+ (increased) and K+ (decreased), and to have only a minor impact on whole-kidney T$_{\rm Na}$ and T$_{\rm Na}$/Q$_{\rm O2}$. Overall, model predictions agree well with measured changes in Na+ and K+ excretion in response to diuretics and Na+ transporter mutations.
Insulin resistance (IR) is an early metabolic alteration in CKD patients, being apparent when the glomerular filtration rate is still within the normal range and becoming almost universal in those who reach the ESKF. The skeletal muscle represents the primary site of IR in CKD and post-receptor alterations are recognized as the main defect underlying IR in this condition. Estimates of IR based on fasting insulin concentration are easier but may not be adequate in CKD patients because renal insufficiency reduces insulin catabolism. The hyperinsulinemic euglycemic clamp is the gold standard for the assessment of insulin sensitivity. The etiology of IR in CKD is multifactorial in nature and may be secondary to disturbances which are prominent in renal diseases, including chronic inflammation, oxidative stress, vitamin D deficiency, adipokines derangement and altered gut microbiome. IR contributes to renal disease progression by worsening renal hemodynamics by various mechanisms, including activation of the sympathetic nervous system, sodium retention and downregulation of the natriuretic peptide system. IR has been solidly associated with intermediate mechanisms leading to cardiovascular (CV) disease in CKD including left ventricular hypertrophy, vascular dysfunction and atherosclerosis. However, it remains unclear whether IR is an independent predictor of mortality and CV complications in CKD. Because IR is a modifiable risk factor and its reduction may lower CV morbidity and mortality, unveiling the molecular mechanisms responsible for the pathogenesis of CKD-related insulin resistance is of importance for the identification of novel therapeutic targets aimed at reducing the high CV risk of this condition.
Nitric oxide (NO) inhibits collecting duct (CD) Na+ and water reabsorption. Mice with CD-specific knockout (KO) of NO synthase 1 (NOS1) have salt-sensitive hypertension. In contrast, the role of NOS3 in CD salt and water reabsorption is unknown. Mice with CD NOS3 KO were generated with loxP-flanked exons 9-12 (encodes the calmodulin binding site) of the NOS3 gene and the aquaporin-2 promoter-Cre transgene. There were no differences between control and CD NOS3 KO mice, irrespective of sex, in food intake, water intake, urine volume, urinary Na+ or K+ excretion, plasma renin concentration, blood pressure or pulse during 7 days of normal (0.3%), high (3.17%) or low (0.03%) Na+ intake. Blood pressure was similar between genotypes during DOCA-high salt. CD NOS3 KO did not alter urine volume or urine osmolality after water deprivation. In contrast, CD NOS3 KO male, but not female, mice had lower urine volume and higher urine osmolality over the course of 7 days of water loading as compared to control mice. Male, but not female, CD NOS3 KO mice had reduced urinary nitrite+nitrate excretion compared to controls after 7 days of water loading. Urine AVP and AVP-stimulated cAMP accumulation in isolated inner medullary CD were similar between genotypes. Western analysis did not reveal a significant effect of CD NOS3 KO on renal aquaporin expression. In summary, these data suggest that CD NOS3 may be involved in the diuretic response to a water load in a gender-specific manner; the mechanism of this effect remains to be determined.
Activation of the thick ascending limb (TAL) Na+-K+-2Cl--cotransporter (NKCC2) by the antidiuretic hormone arginine-vasopressin (AVP) is an essential mechanism of renal urine concentration and contributes to extracellular fluid and electrolyte homeostasis. AVP effects in the kidney are modulated by locally and/or by systemically produced epoxyeicosatrienoic acid derivates (EET). The relation between AVP and EET metabolism has not been determined. Here we show that chronic treatment of AVP-deficient Brattleboro rats with the AVP V2 receptor analog desmopressin (dDAVP; 5ng/h, 3d) significantly lowered renal EET levels (-56 ± 3% for 5,6-EET, -50 ± 3.4% for 11,12-EET, and -60 ± 3.7% for 14,15-EET). The abundance of the principal EET-degrading enzyme soluble epoxide hydrolase (sEH) was increased at the mRNA (+160 ± 37%) and protein levels (+120 ± 26%). Immunohistochemistry revealed dDAVP-mediated induction of sEH in connecting tubules and cortical and medullary collecting ducts, suggesting a role of these segments in the regulation of local interstitial EET signals. Incubation of murine kidney cell suspensions with 1 µM 14,15-EET for 30 min reduced phosphorylation of NKCC2 at the AVP-sensitive threonine residues T96 and T101 (-66 ± 5%; p<0.05) while 14,15-DHET had no effect. Concomitantly, isolated perfused cTAL pretreated with 14,15-EET showed a 30% lower transport current under high and a 70% lower transport current under low symetric chloride concentrations. In sum, we have shown that activation of AVP signaling stimulates renal sEH biosynthesis and enzyme activity. The resulting reduction of EET tissue levels may be instrumental for increased NKCC2 transport activity during AVP-induced antidiuresis.
There is increasing evidence that the permeability of the glomerular filtration barrier (GFB) is partly regulated by a balance between the bioavailability of nitric oxide (NO) and that of reactive oxygen species (ROS). It has been postulated that normal or moderately elevated NO levels protect the GFB from permeability increases, while ROS, through reducing the bioavailability of NO, have the opposite effect. We tested the tentative antagonism between NO and ROS on glomerular permeability in anaesthetized Wistar rats, in which the left ureter was cannulated for urine collection, while simultaneously blood access was achieved. Rats were systemically infused with either L-NAME or L-NAME together with the superoxide scavenger Tempol, or together with L-arginine or the NO-donor DEA-NONOate, or the cGMP agonist 8-Bromo-cGMP. To measure glomerular sieving coefficients () to Ficoll, rats were infused with fluorescein isothiocyanate (FITC)-Ficoll 70/400 (mol.radius 10-80Å). Plasma and urine samples were analyzed by high performance size exclusion chromatography (HPSEC) for determination of for Ficoll repeatedly during up to 2 hours. L-NAME increased for Ficoll70Å from 2.27±1.30·10-5 to 8.46±2.06· 10-5 (n=6; p<0.001) in 15 min. Tempol abrogated these increases in glomerular permeability and an inhibition was also seen with L-arginine and the cGMP agonist, 8-Bromo-cGMP. In conclusion acute NOS inhibition in vivo by L-NAME caused rapid increases in glomerular permeability, which could be reversed by either a ROS antagonist or by activating the guanylyl cyclase-cGMP pathway. The data strongly suggest a protective effect of NO in maintaining normal glomerular permeability in vivo.
Background: Fabry Nephropathy is a major cause of morbidity and premature death in patients with Fabry disease (FD) a rare X-linked lysosomal storage disorder.Gb3, the main substrate of α-gal A, progressively accumulates within cells in a variety of tissues. Establishment of cell models have been useful as a tool for testing hypotheses of disease pathogenesis. Methods: We applied CRISPR/Cas9 genome editing technology to the GLA gene to develop human kidney cell models of FD in human immortalized podocytes, which are the main affected renal cell type. Results: Our podocytes lack detectable α-gal A activity and have increased levels of Gb3. To explore different pathways that could have distinct patterns of activation under conditions of α-gal A deficiency, we used a high-throughput antibody array to perform phosphorylation profiling of CRISPR/Cas9-edited and control podocytes. Changes in both total protein levels and in phosphorylation status per site were observed. Conclusion: Analysis of our candidate proteins suggests that multiple signaling pathways are impaired in FD.
Renal Rhbg is localized to the basolateral membrane of intercalated cells and is involved in NH3/NH4+ transport. The structure of Rhbg is not yet resolved however a high-resolution crystal structure of AmtB, a bacterial homologue of Rh, has been determined. We aligned the sequence of Rhbg to AmtB and identified important sites of Rhbg that may affect transport. Our analysis positioned three conserved amino acids, Histidine-183 (H183), Histidine-342 (H342) and Tryptophan-230 (W230), within the hydrophobic pore where they presumably serve to control NH3 transport. A fourth residue, Phenylalanine-128 (F128) was positioned at the upper vestibule presumably contributing to recruitment of NH4+. We generated three mutations each of H183, H342, W230 and F128 and expressed them in frog oocytes. Immunolabeling showed that W230 and F128 mutants were localized to the cell membrane whereas H183 and H342 staining was diffuse and mostly intracellular. To determine function, we compared measurements of NH3/NH4+ and methyl amine/ammonium (MA/MA+)-induced currents, intracellular pH and surface pH (pHs) among oocytes expressing the mutants, Rhbg or injected with H2O. In H183 and W230 mutants, NH4+-induced current and intracellular acidification were inhibited compared to Rhbg and MA-induced intracellular alkalinization was completely absent. Expressing H183A or W230A mutants inhibited NH3/NH4+- and MA/MA+-induced decrease in pHs to the level observed in H2O-injected oocytes. Mutations of F128 did not affect transport of NH3 or NH4+ significantly. These data demonstrated that mutating H183 or W230 caused loss of function but not F128. H183 and H342 may affect membrane expression of the transporter.
Renal ischemia/reperfusion injury is the state of which a tissue experience injury after a phase of restrictive blood supply and recirculation. Ischemia/reperfusion injury (I/R-I) is a leading cause of acute kidney injury (AKI) in several disease states, including kidney transplantation, sepsis and hypovolemic shock. The most common methods to evaluate AKI are creatinine clearance, plasma creatinine, blood urea nitrogen (BUN) or renal histology. However, there is currently a lack of precise methods to directly assess renal injury state non-invasively. Hyperpolarized 13C-pyruvate magnetic resonance imaging (MRI) enables non-invasive accurate quantification of the in vivo conversion of pyruvate to lactate, alanine and bicarbonate. In the present study, we investigated the in situ alterations of metabolic conversion of pyruvate to lactate, alanine and bicarbonate in a unilateral I/R-I rat model with 30 min and 60 min of ischemia followed by 24 hours of reperfusion. The pyruvate conversion was unaltered compared to sham in the 30 min I/R-I group while a significant reduced metabolic conversion was found in the post-ischemic kidney after 60 min of ischemia. This indicates that after 30 min of ischemia the kidney maintains normal metabolic function in spite of decreased kidney function, whereas the post-ischemic kidney after 60 min of ischemia show a generally reduced metabolic enzyme activity concomitant with a reduced kidney function. We have confidence in that these findings can have a high prognostic value in prediction of kidney injury and the outcome of renal injury.
Protein mimotopes or blocking peptides are small therapeutic peptides that prevent protein-protein interactions by selectively mimicking a native binding domain. Inexpensive technology facilitates straightforward design and production of blocking peptides in sufficient quantities to undertake preventive and therapeutic trials in both in vitro and in vivo experimental disease models. The kidney is an ideal peptide target since small molecules undergo rapid filtration and efficient bulk absorption by tubular epithelial cells. Compared to the blood stream, the half-life of peptides in the kidney is markedly prolonged, making blocking peptides an attractive tool for treating diverse renal diseases including ischemia, proteinuric states such as membranous glomerulonephritis (MGN) and focal and segmental glomerulosclerosis (FSGS) or renal cell carcinoma. Therapeutic peptides represent one of the fastest growing reagents classes for novel drug development in human disease partly due their ease of administration, high binding affinity and minimal off target effects. This review introduces the concepts of blocking peptide design, production, and administration and highlights the potential use of therapeutic peptides to prevent or treat specific renal diseases.
Transient Receptor Potential Vanilloid Type 1 (TRPV1) is a major nociceptive ion channel implicated in bladder physiology and/or pathophysiology. However, the precise expression of TRPV1 in neuronal versus non-neuronal bladder cells is uncertain. Here we used reporter mouse lines (TRPV1-Cre:tdTomato and TRPV1PLAP-nLacZ) to map the expression of TRPV1 in post-natal bladder. TRPV1 was not detected in the urothelium, however, we found marked expression of TRPV1 lineage in sensory nerves, and surprisingly, in arterial/arteriolar smooth muscle (ASM) cells. Tomato fluorescence was prominent in the vesical arteries and in small diameter (15 to 40 μm) arterioles located in the sub-urothelial layer with a near equal distribution in bladder dome and base. Notably, arteriolar TRPV1 expression was greater in females compared with males and increased in both sexes after 90 days of age suggesting a sex-hormone and age dependence. Analysis of whole bladder and vesical artery TRPV1 mRNA revealed a similar sex and developmental dependence. Pharmacological experiments confirmed functional TRPV1 protein expression; capsaicin increased intracellular Ca2+ in approximately 15% of ASM cells from wild-type female bladders but we observed no responses to capsaicin in bladder arterioles isolated from TRPV1-null mice. Further, capsaicin triggered arteriole constriction that was rapidly reversed by the TRPV1 antagonist, BCTC. These data show that bladder ASM cells, predominantly in post-pubertal female mice, express functional TRPV1 channels that may act to constrict arterioles. TRPV1 may therefore play an important role in regulating the microcirculation of the female bladder and this effect may be of significance during inflammatory conditions.
MRI can provide excellent detail of renal structure and function. Recently, novel MR contrast mechanisms and imaging tools have been developed to evaluate microscopic kidney structures including the tubules and glomeruli. Quantitative MRI can assess local tubular function and is able to determine the kidney's concentrating mechanism non-invasively in real time. Measuring single nephron function is now a near possibility. In parallel to advancing imaging techniques for kidney microstructure is a need to carefully understand the relationship between the local source of MRI contrast and the underlying physiological change. The development of these imaging markers can impact the accurate diagnosis and treatment of kidney disease. This article reviews the novel tools to examine kidney microstructure and local function, and demonstrates the application of these methods in renal pathophysiology.
Kidney cell injury may be associated with protein misfolding and induction of endoplasmic reticulum (ER) stress. Examples include complement-induced glomerular epithelial cell (GEC)/podocyte injury in membranous nephropathy, and ischemia-reperfusion injury. Renal cell injury can also result from mutations in integral proteins, which lead to their misfolding and accumulation. Certain nephrin missense mutants misfold, accumulate in the ER, and induce ER stress. We examined if enhancement of ubiquitin-proteasome system (UPS) function may facilitate proteostasis and confer protection against injury. Ubiquitin-specific protease 14 (Usp14) is reported to retard proteasomal protein degradation. Thus, inhibition of Usp14 may enhance degradation of misfolded proteins, and attenuate cell injury. In GEC, the reporter proteins, GFPu (a "misfolded" protein) and CD3delta (an ER-associated degradation substrate) undergo time-dependent proteasomal degradation. Complement did not affect degradation of CD3delta-YFP, but accelerated degradation of GFPu, and the Usp14-directed inhibitor, IU1, further accelerated this degradation. Conversely, overexpression of Usp14 reduced degradation of GFPu and CD3delta-YFP. In 293T cells, IU1 did not enhance degradation of disease-associated nephrin missense mutants, I171N and S724C, whereas overexpression of Usp14 reduced degradation. IU1 was cytoprotective after injury induced by the ER stressor, tunicamycin, and in vitro ischemia-reperfusion, but did not affect complement-induced cytotoxicity. In conclusion, Usp14 controls proteasomal degradation of some misfolded proteins. In addition, a Usp14-directed inhibitor reduces cytotoxicity in the context of global protein misfolding during certain types of renal cell injury.
Nephrocalcinosis describes the ectopic deposition of calcium salts in the kidney parenchyma. Nephrocalcinosis can result from a number of acquired causes, but also an even greater number of genetic diseases, predominantly renal, but also extra-renal. Here we provide a review of the genetic causes of nephrocalcinosis, along with putative mechanisms, illustrated by human and animal data.
In addition to improving sexual function, testosterone has been reported to have beneficial effects in ameliorating lower urinary tract symptoms by increasing bladder capacity and compliance, while decreasing bladder pressure. However, the cellular mechanisms by which testosterone regulates detrusor smooth muscle (DSM) excitability have not been elucidated. Here, we used amphotericin-B perforated whole cell patch-clamp and single channel recordings on inside-out excised membrane patches to investigate the regulatory role of testosterone in guinea pig DSM excitability. Testosterone (100 nM) significantly increased the depolarization-induced whole cell outward currents in DSM cells. The selective pharmacological inhibition of the large conductance voltage- and Ca2+-activated K+ (BK) channels with paxilline (1 µM) completely abolished this stimulatory effect of testosterone, suggesting a mechanism involving BK channels. At a holding potential of -20 mV, DSM cells exhibited transient BK currents (TBKCs). Testosterone (100 nM) significantly increased TBKC activity in DSM cells. In current-clamp mode, testosterone (100 nM) significantly hyperpolarized the DSM cell resting membrane potential and increased spontaneous transient hyperpolarizations. Testosterone (100 nM) rapidly increased the single BK channel open probability in inside-out excised membrane patches from DSM cells, clearly suggesting a direct BK channel activation via a non-genomic mechanism. Live-cell Ca2+ imaging showed that testosterone (100 nM) caused a decrease in global intracellular Ca2+ concentration, consistent with testosterone-induced membrane hyperpolarization. In conclusion, the data provide compelling mechanistic evidence that under physiological conditions, testosterone at nanomolar concentrations directly activates BK channels in DSM cells, independent fromgenomic testosterone receptors, and thus regulates DSM excitability.
The regulation of Na/K-ATPase in various tissues is under the control of a number of hormones and peptides that exert both short- and long-term control over its activity. Present study was performed to investigate the effect of chronic insulin treatment on Na/K-ATPase in renal proximal tubular cells. Incubation of opossum kidney (OK) cells, transfected with rat Na/K-ATPase α1- subunit, with 1 nmol/L insulin for 48hr decreased Na/K-ATPase activity. Insulin decreased α1 protein content and increased α1 serine phosphorylation and α1-adaptor protein (AP) 2 interaction. Removal of 26 N-terminal (-NT) amino acid from α1-subunit containing serine/threonine sites abolished the insulin-mediated serine-phosphorylation and inhibition of Na/K-ATPase. Substitution of serine 16 and 23 with alanine showed comparable effect to -NT. Insulin increased the activity of protein kinase (PK) C which was blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. Both PI3K and PKC inhibitors abolished the insulin mediated inhibition of Na/K-ATPase. Insulin increased the expression of PKC β1, , and , however, only PKC / specific inhibitors blocked insulin-induced phosphorylation and inhibition of Na/K-ATPase. Our data demonstrate that insulin activates atypical PKC isoforms / via PI3K pathway. PKC /-induced phosphorylation of α1-subunit at serine 16 and 23 leads to AP-2 recruitment, degradation and a decrease in Na/K-ATPase activity.
Diabetic kidney disease (DKD) remains the leading cause of end-stage renal disease. A major challenge in preventing DKD is the difficulty in identifying high-risk patients at an early, pre-clinical stage. Albuminuria and eGFR as measures of renal function in DKD research and clinical practice are limited by regression of one-third of patients with microalbuminuria to normoalbuminuria and eGFR is biased and imprecise in the normal-elevated range. Moreover, existing methods that are used to assess renal function do not give detailed insight into the location of the renal hemodynamic effects of pharmacological agents at the segmental level. To gain additional information about the intrarenal circulation in-vivo in humans, mathematical equations were developed by Gomez et al in the 1950s. These equations used measurements of GFR, renal blood flow (RBF), effective renal plasma flow (ERPF), renal vascular resistance (RVR), hematocrit and serum protein to calculate afferent and efferent arteriolar resistances, glomerular hydrostatic pressure and filtration pressure. Although indirect and based on physiological assumptions, these techniques have the potential to improve researchers' ability to identify early pre-clinical changes in renal hemodynamic function in patients with a variety of conditions including DKD, thereby offering tremendous potential in mechanistic human research studies. In this review, we focus on the application of Gomez' equations and summarize the potential and limitations of this technique in DKD research. We also summarize illustrative data derived from Gomez' equations in patients with type 1 (T1D) and type 2 diabetes (T2D) and hypertension.
Aquaporin 11 (AQP11) is a channel protein with unknown biological function that is expressed in multiple tissues, including the kidney proximal tubule (PT) epithelium. Constitutive deletion of Aqp11 in mice (Aqp11-/-) results in early postnatal vacuolization in the PT and development of apparent cysts at 2 weeks of age. Electron microscopy of adult Aqp11-/- mouse PT cells revealed a dilated rough endoplasmic reticulum. These changes may cause renal failure and premature death. This study examined 1) whether postnatal deletion of Aqp11 affects PT injury and cyst formation, 2) the temporal role of Aqp11 deletion on cyst development, and 3) the nature of apparent cysts. Tamoxifen-inducible Aqp11-/- mice were generated (Ti-Aqp11-/-). Deletion of Aqp11 at post-natal (P) days 2, 4, 6, 8 and 12 was investigated. Deranged renal development, especially in kidney cortex, PT cell vacuolization and apparent tubular cysts developed only in mice where Aqp11 gene disruption was induced until P8. Aqp11 gene deletion from P12 onwards did not result in a clear deficiency in renal development, PT injury or cyst formation. Intra-peritoneal injection of biotinylated-dextran (10 kDa) into adult mice resulted in extensive endocytic dextran uptake in both cystic Aqp11-/- and control PT epithelium, respectively. This suggests that apparent cysts are not membrane enclosed structures but represent PT dilations. We conclude that Aqp11-/- mice develop cyst-like dilated proximal tubules without documented cysts at time of death.
Recent studies suggested a direct link between circadian rhythms and regulation of sodium excretion. Endothelin-1 (ET-1) regulates sodium balance by promoting natriuresis through the endothelin B receptor (ETB) in response to increased salt in the diet, but the effect that the time of day has on this natriuretic response is not known. Therefore, this study was designed to test the hypothesis that ETB receptor activation contributes to the diurnal control of sodium excretion, and that sex differences contribute to this control as well. 12-hour urine collections were used to measure sodium excretion. On day 3 of the experiment, a NaCl load (900 µEq) was given by oral gavage either at Zeitgeber [ZT] time 0 (inactive period) or ZT12 (active period) to examine the natriuretic response to the acute salt load. Male and female ETB deficient (ETB def) rats showed an impaired natriuretic response to a salt load at ZT0 compared to their respective transgenic controls (Tg cont). Male ETB def rats showed a delayed natriuretic response to a salt load given at ZT12 compared to male Tg cont, a contrast to the prompt response shown by female ETB def rats. Treatment with ABT-627, an ETA receptor antagonist, improved the natriuretic response seen within the first 12 hours of a ZT0 salt load in both sexes. These findings demonstrate that diurnal excretion of an acute salt load 1) requires ET-1 and the ETB receptor, 2) is more evident in male versus female rats, and 3) is opposed by the ETA receptor.
Gitelman syndrome (GS) is an autosomal recessive salt-wasting tubular disorder resulting from loss-of-function mutations in the thiazide-sensitive NaCl cotransporter (NCC). Functional analysis of these mutations has been limited to the use of Xenopus laevis oocytes. The aim of the present study was, therefore, to analyze the functional consequences of NCC mutations in a mammalian cell-based assay, followed by analysis of mutated NCC protein expression as well as glycosylation and phosphorylation profiles using human embryonic kidney (HEK) 293 cells. NCC activity was assessed with a novel assay based on thiazide-sensitive iodide uptake in HEK293 cells expressing wild-type or mutant NCC (N59I, R83W, I360T, C421Y, G463R, G731R, L859P, or R861C). All mutations caused a significantly lower NCC activity. Immunoblot analysis of the HEK293 cells revealed that (i) all NCC mutants have decreased NCC protein expression; (ii) mutant N59I, R83W, I360T, C421Y, G463R, and L859P have decreased NCC abundance at the plasma membrane; (iii) mutants C421Y and L859P display impaired NCC glycosylation, and (iv) mutants N59I, R83W, C421Y, C731R, and L859P show affected NCC phosphorylation. In conclusion, we developed a mammalian cell-based assay in which NCC activity assessment together with a profiling of the mutated protein processing aids to our understanding of the pathogenic mechanism of the NCC mutations.
Proteinuria is a major risk factor for chronic kidney disease progression. Furthermore, exposure of proximal tubular epithelial cells to excess albumin promotes tubular atrophy and fibrosis, key predictors of progressive organ dysfunction. However, the link between proteinuria and tubular damage is unclear. We propose that pathologic albumin exposure impairs proximal tubular autophagy, an essential process for recycling damaged organelles and toxic intracellular macromolecules. In both mouse primary proximal tubule and immortalized human kidney cells, albumin exposure decreased the number of autophagosomes, visualized by the autophagosome-specific fluorescent markers monodansylcadaverine and GFP-LC3, respectively. Similarly, renal cortical tissue harvested from proteinuric mice contained reduced numbers of autophagosomes on electron micrographs and immunoblot showed reduced steady state LC3-II content. Albumin exposure decreased autophagic flux in vitro in a concentration-dependent manner as assessed by LC3-II accumulation rate in the presence of bafilomycin, an H+-ATPase inhibitor that prevents lysosomal LC3-II degradation. In addition, albumin treatment significantly increased the half-life of radiolabeled long-lived proteins, indicating that the primary mechanism of degradation, autophagy, is dysfunctional. In vitro, mTOR activation, a potent autophagy inhibitor, suppressed autophagy as a result of intracellular amino acid accumulation from lysosomal albumin degradation. mTOR activation was demonstrated by the increased phosphorylation of its downstream target S6K with free amino acid or albumin exposure. We propose that excess albumin uptake and degradation inhibits proximal tubule autophagy via an mTOR-mediated mechanism and contributes to progressive tubular injury.
Previous studies have shown that increased parathyroid hormone (PTH) due to secondary hyperparathyroidism in chronic kidney disease accelerates the arteriosclerotic fibrosis and calcification. Although the underlying mechanisms remain largely unknown, endothelial cells (ECs) have recently been demonstrated to participate in calcification in part by providing chondrogenic cells via the endothelial-to-mesenchymal transition (EndMT). Therefore, this study aimed to investigate whether elevated PTH could induce endothelial to chondrogenic transition in aortic ECs and to determine the possible underlying signaling pathway. We found that treatment of ECs with PTH significantly upregulated the expression of EndMT-related markers. Accordingly, ECs treated with PTH exhibited chondrogenic potential. In vivo, a lineage tracing model subjected mice with endothelial-specific GFP fluorescence to chronic PTH infusion showed a marked increase in the aortic expression of chondrocyte markers, and confocal microscopy revealed the endothelial origin of cells expressing chondrocyte markers in the aorta after PTH infusion. Furthermore, this in vitro study showed that PTH enhanced the nuclear localization of β-catenin in ECs, whereas, β-catenin siRNA or DKK1, an inhibitor of β-catenin nuclear translocation, attenuated the upregulation of EndMT-associated and chondrogenic markers induced by PTH. In summary, our study demonstrated that elevated PTH could induce the transition of ECs to chondrogenic cells via EndMT, possibly mediated by the nuclear translocation of β-catenin.
Acute kidney injury (AKI) is associated with poor patient outcome and a global burden for end-stage renal disease. Ischemia-reperfusion injury (IRI) is one of the major causes of AKI, and experimental work has revealed many details of the inflammatory response in the kidney. Here, we investigated whether deletion of the NF-B kinases IKK2 or NEMO in lymphocytes or systemic inhibition of IKK2 would cause different kidney inflammatory responses after IRI induction. Serum creatinine, BUN level and renal tubular injury score were significantly increased in CD4creIKK2f/f (CD4xIKK2) and CD4creNEMOf/f (CD4xNEMO) mice compared with CD4cre mice after IRI induction. The frequency of Th17 cells infiltrating the kidneys of CD4xIKK2 or CD4xNEMO mice was also significantly increased at all time points. CCL20, an important chemokine in Th17 cell recruitment, was significantly increased at early time points after the induction of IRI. IL1β, TNFα and CCL2 were also significantly increased in different patterns. A specific IKK2 inhibitor, KINK-1, reduced BUN and serum creatinine compared with non-treated mice after IRI induction, but the frequency of kidney Th17 cells was also significantly increased. In conclusion, although systemic IKK2 inhibition improved kidney function, lymphocyte-specific deletion of IKK2 or NEMO aggravated kidney injury after IRI, and in both conditions, the percentage of Th17 cells was increased. Our findings demonstrate the critical role of the NF-B pathway in Th17 activation, which advises caution when using systemic IKK2 inhibitors in patients with kidney injury as they might impair the T cell response and aggravate renal disease.
The distal nephron is a heterogeneous part of the nephron composed by six different cell types forming the epithelium of the distal convolute (DCT), connecting (CNT) and collecting duct (CD). To dissect the function of these cells, knock out models specific for their unique cell marker have been created. However, since this part of the nephron develops at the border between the ureteric bud and the metanephric mesenchyme, the specificity of the single cell-markers has been recently questioned. Here, by mapping the fate of the AQP2, NCC-positive cells using transgenic mouse lines expressing the YFP fluorescent marker, we showed that the origin of the distal nephron is extremely composite. Indeed, AQP2 expressing precursors results to give rise, not only, to the principal cells, but also, to some of the A- and B-type intercalated cells and even to cells of the DCT. On the other side, some principal cells and B-type intercalated cells can develop from NCC expressing precursors. In conclusion, these results demonstrate that the origin of different cell types in the distal nephron is not as clearly defined as originally thought. Importantly, they highlight the fact that knocking out a gene encoding for a selective functional marker in the adult life does not guarantee cell specificity during the overall kidney development. Tools allowing not only cell-specific but also time-controlled recombination will be useful in this sense.
Diabetic nephropathy (DN) is the leading cause of chronic kidney disease in the United States and is a major cause of cardiovascular disease and death. DN develops insidiously over a span of years before clinical manifestations, including microalbuminuria and declining glomerular filtration rate (GFR), are evident. During the clinically silent period, structural lesions develop, including glomerular basement membrane (GBM) thickening, mesangial expansion, and glomerulosclerosis. Once microalbuminuria is clinically apparent, structural lesions are often considerably advanced and GFR decline may then proceed rapidly toward end-stage kidney disease. Given the current lack of sensitive biomarkers for detecting early DN, a shift in focus towards examining the cellular and molecular basis for the earliest structural change in DN, i.e., GBM thickening, may be warranted. Observed within 1-2 years following the onset of diabetes, GBM thickening precedes clinically evident albuminuria. In the mature glomerulus, the podocyte is likely key in modifying the GBM, synthesizing and assembling matrix components, both in physiological as well as pathological states. Podocytes also secrete matrix metalloproteinases, crucial mediators in extracellular matrix turnover. Studies have shown that the critical podocyte-GBM interface is disrupted in the diabetic milieu. Just as healthy podocytes are essential for maintaining the normal GBM structure and function, injured podocytes likely have a fundamental role in upsetting the balance between the GBM's synthetic and degradative pathways. This article will explore the biological significance of GBM thickening in DN by reviewing what is known about the GBM's formation, its maintenance during health, and its disruption in DN.
The chloride intracellular channel (CLIC) 5A is expressed at very high levels in renal glomeruli, in both endothelial cells (EC) and podocytes. CLIC5A stimulates Rac1- and PI[4,5]P2-dependent ERM (ezrin, radixin, moesin) activation. ERM proteins, in turn, function in lumen formation and in the development of actin-based cellular projections. In mice lacking CLIC5A, ERM phosphorylation is profoundly reduced in podocytes, but preserved in glomerular EC. Since glomerular EC also express CLIC4, we reasoned if CLIC4 activates ERM proteins like CLIC5A, then CLIC4 could compensate for the CLIC5A loss in glomerular EC. In glomeruli of CLIC5 deficient mice, CLIC4 expression was up-regulated and co-localized with moesin and ezrin in glomerular EC, but not in podocytes. In cultured glomerular EC, CLIC4 silencing reduced ERM phosphorylation and cytoskeletal association, and expression of exogenous CLIC4 or CLIC5A rescued ERM de-phosphorylation due to CLIC4 silencing. In mice lacking either CLIC4 or CLIC5, ERM phosphorylation was retained in glomerular EC, but in mice lacking both CLIC4 and CLIC5, glomerular EC ERM phosphorylation was profoundly reduced. Although glomerular EC fenestrae developed normally in dual CLIC4/CLIC5 deficient mice, the density of fenestrae declined substantially by 8 months of age, along with the deposition of subendothelial electron-lucent material. The dual CLIC4/CLIC5 deficient mice developed spontaneous proteinuria, glomerular cell proliferation and matrix deposition. Thus, CLIC4 stimulates ERM activation, and can compensate for CLIC5A in glomerular EC. The findings indicate that CLIC4/CLIC5A-mediated ERM activation is required for maintenance of the glomerular capillary architecture.
The bone-secreted hormone fibroblast growth factor 23 (FGF23) has an essential role in phosphate homeostasis by regulating expression of the kidney proximal tubule sodium-phosphate co-transporters as well as parathyroid hormone levels. Induction of FGF23 early in chronic kidney disease (CKD) helps to maintain normal phosphorous levels. However, high FGF23 levels become pathologic as kidney disease progresses and are associated with an increased risk of CKD progression, cardiovascular events, and death. The factors responsible for increasing FGF23 levels early in CKD are unknown, but recent work has proposed a role for inflammation and disordered iron homeostasis. Notably, FGF23 has recently been shown to elicit and inflammatory response and to display immunomodulatory properties. Here, we will review emerging evidence on the crosstalk between inflammation, iron, FGF23, and bone and mineral metabolism, and discuss the relevance for CKD patients.
Ischemia-reperfusion (IR)-induced kidney injury is a major clinical problem, but the underlying mechanisms remain unclear. The transcription factor Nuclear Factor, Erythroid 2-like 2 (NFE2L2 or Nrf2) is crucial for protection against oxidative stress generated by pro-oxidant insults. We have previously shown that Nrf2 deficiency enhances susceptibility to IR-induced kidney injury in mice and its upregulation is protective. Here, we examined Nrf2 target antioxidant gene expression and the mechanisms of its activation in both human and murine kidney epithelia following acute (2 h) and chronic (12 h) hypoxia and re-oxygenation conditions. We found that acute hypoxia modestly and chronic hypoxia strongly stimulates Nrf2 putative target HMOX1 expression, but not other antioxidant genes. Inhibition of AKT1/2 or ERK1/2 signaling blocked this induction; AKT1/2 but not ERK1/2 inhibition affected Nrf2 levels in basal state and acute hypoxia-reoxygenation. Unexpectedly, chromatin immunoprecipitation assays revealed reduced levels of Nrf2 binding at the distal AB1 and SX2 enhancers and proximal promoter of HMOX1 in acute hypoxia, accompanied by diminished levels of nuclear Nrf2. In contrast, Nrf2 binding at the AB1 and SX2 enhancers differentially increased during chronic hypoxia and reoxygenation, with re-accumulation of nuclear Nrf2 levels. Si-RNA mediated Nrf2 depletion attenuated acute and chronic hypoxia-inducible HMOX1 expression, and primary Nrf2-null kidney epithelia showed reduced levels of HMOX1 induction in response to acute and chronic hypoxia. Collectively, our data demonstrate that Nrf2 upregulates HMOX1 expression in kidney epithelia through a distinct mechanism during acute and chronic hypoxia reoxygenation, and AKT1/2 and ERK1/2 signaling are required for this process.
Mutations in genes encoding subunits of the epithelial Na+ channel (ENaC) can cause early-onset familial hypertension, demonstrating the importance of this channel in modulating blood pressure. It remains unclear whether other genetic variants resulting in subtler alterations of channel function result in hypertension or altered sensitivity of blood pressure to dietary salt. This study sought to identify functional human ENaC variants, to examine how these variants alter channel activity, and to explore whether these variants are associated with altered sensitivity of blood pressure to dietary salt. Six-hundred participants of the Genetic Epidemiology Network of Salt Sensitivity (GenSalt) study with salt-sensitive or salt-resistant blood pressure underwent sequencing of the genes encoding ENaC subunits. Functional effects of identified variants were examined in a Xenopus oocyte expression system. Variants that increased channel activity included three in the gene encoding the α subunit (αS115N, αR476W and αV481M), one in the β subunit (βS635N), and one in the subunit (L438Q). One α subunit variant (αA334T) and one β subunit variant (βD31N) decreased channel activity. Several α subunit extracellular domain variants altered channel inhibition by extracellular Na+ (Na+ self-inhibition). One variant (αA334T) decreased and one (αV481M) increased cell-surface expression. Association between these variants and salt-sensitivity did not reach statistical significance. This study identifies novel, functional human ENaC variants and demonstrates that some variants alter channel cell surface expression and/or Na+ self-inhibition.
Extracellular vesicles (EV) are endogenously produced, membrane-bound vesicles that contain various molecules. Depending on their size and origins, EVs are classified into apoptotic bodies, microvesicles, and exosomes. A fundamental function of EVs is to mediate intercellular communication. In kidneys, recent research has begun to suggest a role of EVs, especially exosomes, in cell-cell communication by transferring proteins, mRNAs, and microRNAs to recipient cells as nanovectors. EVs may mediate the cross-talk between various cell types within kidneys for the maintenance of tissue homeostasis. They may also mediate the cross-talk between kidneys and other organs under physiological and pathological conditions. EVs have been implicated in the pathogenesis of both acute kidney injury and chronic kidney diseases, including renal fibrosis, end-stage renal disease, glomerular diseases, and diabetic nephropathy. The release of EVs with specific molecular contents into urine and plasma may be useful biomarkers for kidney disease. In addition, EVs produced by cultured cells may have therapeutic effects for these diseases. However, the role of EVs in kidney diseases is largely unclear and the mechanism underlying EV production and secretion remains elusive. In this review, we introduce the basics of EVs and then analyze the current information about the involvement, diagnostic value, and therapeutic potential of EVs in major kidney diseases.
Age is associated with an increased prevalence of chronic kidney disease (CKD), which, through progressive tissue damage and fibrosis, ultimately leads to loss of kidney function. Although much effort is put into studying CKD development experimentally, age has rarely been taken into account. Therefore, we investigated the effect of age on the development of renal tissue damage and fibrosis in a mouse model of obstructive nephropathy (i.e. unilateral ureter obstruction; UUO). We observed that after 14 days, obstructed kidneys of old mice had more tubulointerstitial atrophic damage but less fibrosis than those of young mice. This was associated with reduced connective tissue growth factor (CTGF), and higher BMP6 expression and pSMAD1/5/8 signaling, while TGF-β expression and transcriptional activity were no different in obstructed kidneys of old and young mice. In vitro, CTGF bound to and inhibited BMP6 activity. In summary, our data suggest that in obstructive nephropathy atrophy increases and fibrosis decreases with age, and that this relates to increased BMP signaling, most likely due to higher BMP6 and lower CTGF expression.
Apical membrane targeting of the collecting duct water channel aquaporin-2 (AQP2) is essential for body water balance. As this event is regulated by Gs coupled 7-transmembrane receptors such as the vasopressin type 2 receptor (V2R) and the prostanoid receptors EP2 and EP4, it is believed to be cAMP-dependent. However, on the basis of recent reports, it was hypothesized in the current study that increased cAMP levels are not necessary for AQP2 membrane targeting. The role and dynamics of cAMP signaling on AQP2 membrane targeting in Madin-Darby Canine Kidney and mouse cortical collecting duct (mpkCCD14) cells was examined using selective agonists against the V2R (dDAVP), EP2 (butaprost) and EP4 (CAY10580). During EP2 stimulation, AQP2 membrane targeting continually increased during 80 min of stimulation; whereas cAMP levels reached a plateau after 10 min. EP4 stimulation caused a rapid and transient increase in AQP2 membrane targeting, but did not significantly increase cAMP levels. After washout of EP2 agonist or dDAVP, AQP2 membrane abundance remained elevated for at least 80 min, whereas cAMP levels rapidly decreased. Similar effects of the EP2 agonist were also observed for AQP2 constitutively non-phosphorylated at ser-269. The adenylyl cyclase inhibitor SQ22536 did not prevent AQP2 targeting during stimulation of each receptor, nor after dDAVP washout. In conclusion, this study demonstrates that although direct stimulation with cAMP causes AQP2 membrane targeting, cAMP is not necessary for receptor-mediated AQP2 membrane targeting and Gs coupled receptors can also signal through an alternative pathway that increase AQP2 membrane targeting.
Increasing incidences of obesity and diabetes have made diabetic kidney disease (DKD) the leading cause of chronic kidney disease and end-stage renal disease worldwide. Despite current pharmacological treatments, including strategies for optimizing glycemic control and inhibitors of the renin-angiotensin system, DKD still makes up almost half of all cases of end-stage renal disease in the United States. Compelling and mounting evidence has clearly demonstrated that immunity and inflammation play a paramount role in the pathogenesis of DKD. This article reviews the involvement of the immune system in DKD and identifies important roles of key immune and inflammatory mediators. One of the most recently identified biomarkers is serum amyloid A (SAA), which appears to be relatively specific for DKD. Novel and evolving treatment approaches target protein kinases, transcription factors, chemokines, adhesion molecules, growth factors, advanced glycation end-products and other inflammatory molecules. This is the beginning of a new era in the understanding and treatment of DKD and we may have finally reached a tipping point in our fight against the growing burden of DKD.
Lower urinary tract symptoms (LUTS), including frequency, urgency, incomplete voiding, slow stream are common in both men and women with advancing age. The most common cause for LUTS in aging men is benign prostatic hyperplasia (BPH). Some studies have also revealed an inverse association of serum testosterone levels with LUTS; however the underlying mechanisms by which gonadal hormones affect the LUT have not been clarified. In the present study, we examined the effect of orchiectomy and testosterone replacement on lower urinary tract function in adult male Sprague-Dawley rats. Six weeks after bilateral orchiectomy or sham operations and 3 weeks after injection of long acting testosterone undecanoate (100 mg/kg i.m, transvesical cystometry and external urethral sphincter electromyogram (EUS EMG) recordings were performed under urethane anesthesia. The micturition reflex was elicited in both sham and orchiectomized animals. In orchiectomized rats volume threshold for inducing micturition decreased by 47.6%; however contraction amplitude, duration, and voiding efficiency were similar in sham and orchiectomized rats. The active period during EUS EMG bursting was lengthened during micturition in orchiectomized animals. Testosterone treatment which normalized plasma testosterone levels reversed these changes but also increased the duration of EUS EMG bursting (BD). Orchiectomy also reduced mean voiding flow rate estimated from the duration of BD, an effect that was not reversed by testosterone. The results indicate that orchiectomy affects both the active and passive properties of the bladder and urethra and that many but not all of the changes can be reversed by testosterone.
Aquaporin-2 (AQP2) is essential to maintain body water homeostasis. AQP2 traffics from intracellular vesicles to the apical membrane of kidney collecting duct principal cells in response to vasopressin (AVP), a hormone released with low intravascular volume, which causes decreased kidney perfusion. Decreased kidney perfusion activates AMP-activated kinase (AMPK), a metabolic sensor that inhibits the activity of several transport proteins. We hypothesized that AMPK activation also inhibits AQP2 function. These putative AMPK effects could protect interstitial ionic gradients required for urinary concentration during metabolic stress when low intravascular volume induces AVP release. Here we found that short-term AMPK activation by treatment with 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR; 75 min) in kidney tissue prevented baseline AQP2 apical accumulation in principal cells but did not prevent AQP2 apical accumulation in response to the AVP analog desmopressin (dDAVP). Prolonged AMPK activation prevented AQP2 cell membrane accumulation in response to forskolin in mouse collecting duct mpkCCDc14 cells. Moreover, AMPK inhibition accelerated hypotonic lysis of Xenopus oocytes expressing AQP2. We performed phosphorylation assays to elucidate the mechanism by which AMPK regulates AQP2. Although AMPK weakly phosphorylated immunoprecipitated AQP2 in vitro, no direct AMPK phosphorylation of the AQP2 C-terminus was detected by mass spectrometry. AMPK promoted Ser-261 phosphorylation and antagonized dDAVP-dependent phosphorylation of other AQP2 COOH-terminal sites in cells. Our findings suggest an increasing, time-dependent antagonism of AMPK on AQP2 regulation with AICAR-dependent inhibition of cAMP-dependent apical accumulation and AVP-dependent phosphorylation of AQP2. This inhibition likely occurs via a mechanism that does not involve direct AQP2 phosphorylation by AMPK.
The pleiotropic actions of the renin-angiotensin system (RAS) depend on the availability of angiotensinogen (AGT) which generates angiotensin I (Ang I) when cleaved by renin. Thus, quantification of the intact AGT (iAGT) concentrations is important to evaluate the actual renin substrate available. The iAGT conformation exists as oxidized AGT (oxi-AGT) and reduced AGT (red-AGT) in a disulfide bond, and oxi-AGT has a higher affinity for renin, which may exacerbate RAS associated diseases. Accordingly, we determined iAGT, oxi-AGT and red-AGT levels in plasma from rats and mice. Blood samples were obtained by cardiac puncture, then immediately mixed with an inhibitor solution containing a renin inhibitor. Total AGT (tAGT) levels were measured by tAGT ELISA which detects both cleaved and iAGT. iAGT levels were determined by iAGT ELISA which was found to only detect red-AGT. Thus, it was necessary to treat samples with dithiothreitol, a reducing agent, to quantify total iAGT concentration. tAGT levels in rat and mouse plasma were 1839±139 and 1082±77 ng/ml, respectively. iAGT levels were 53% of tAGT in rat plasma but only 22% in mouse plasma, probaby reflecting the greater plasma renin activity in mice. The ratios of oxi-AGT and red-AGT were approximately 4:1 (rat) and 16:1 (mouse). Plasma iAGT consists of oxi-AGT and red-AGT suggesting that oxidative stress can influence Ang I generation by the AGT conformation switch. Furthermore, the lower availability of plasma iAGT in mice suggests that it may serve as a limiting factor in Ang I formation in this species.
While angiotensin II blockade slows the progression of diabetic nephropathy, current data suggest that it alone cannot stop disease process. New therapies or drug combinations will be required to further slow or halt disease progression. Inhibition of PAI-1 aimed at enhancing ECM degradation has been shown therapeutic potential in diabetic nephropathy. Here using a mouse model of type II diabetes, the maximally therapeutic dose of the PAI-1 neutralizing mouse monoclonal antibody (MEDI-579) was determined and compared with the maximally effective dose of enalapril. We then examined whether addition of the MEDI-579 to enalapril would enhance the efficacy in slowing the progression of diabetic nephropathy. Untreated uninephrectomized diabetic db/db mice developed progressive albuminuria and glomerulosclerosis associated with increased expression of TGF-ß1, PAI-1, type IV collagen and fibronectin from weeks 18 to 22, which were reduced by MEDI-579 at 3mg/kg BW, similar to enalapril given alone from weeks 12 to 22. Adding MEDI-579 to enalapril from weeks 18 to 22 resulted in further reduction in albuminuria and markers of renal fibrosis. Renal plasmin generation was dramatically reduced by 57% in diabetic mice, a decrease that was partially reversed by MEDI-579 or enalapril given alone but was further restored by these two treatments given in combination. Our results suggest that MEDI-579 is effective in slowing the progression of diabetic nephropathy in db/db mice and that the effect is additive to an ACEI. While enalapril is renal protective, add-on PAI-1 antibody may offer additional renoprotection in progressive diabetic nephropathy via enhancing ECM turnover.
Vasopressin triggers the phosphorylation and apical plasma membrane accumulation of aquaporin 2 (AQP2), and plays an essential role in urine concentration. Vasopressin, acting through protein kinase A, phosphorylates AQP2. However, the phosphorylation state of AQP2 could also be affected by the action of protein phosphatases (PPs). Rat inner medullas (IM) were incubated with calyculin (PP1 and PP2A inhibitor, 50 nM) or tacrolimus (PP2B inhibitor, 100 nM). Calyculin did not affect total AQP2 protein abundance (by Western blot) but did significantly increase the abundances of pS256-AQP2 and pS264-AQP2. It did not change pS261-AQP2 or pS269-AQP2. Calyculin significantly enhanced the membrane accumulation (by biotinylation) of total AQP2, pS256-AQP2, and pS264-AQP2. Likewise, immunohistochemistry showed an increase in the apical plasma membrane association of pS256-AQP2 and pS264-AQP2 in calyculin-treated rat IM. Tacrolimus also did not change total AQP2 abundance but significantly increased the abundances of pS261-AQP2 and pS264-AQP2. In contrast to calyculin, tacrolimus did not change the amount of total AQP2 in the plasma membrane (by biotinylation and immunohistochemistry). Tacrolimus did increase the expression of pS264-AQP2 in the apical plasma membrane (by immunohistochemistry). In conclusion, PP1/PP2A regulates the phosphorylation and apical plasma membrane accumulation of AQP2 differently than PP2B. Serine-264 of AQP2 is a phosphorylation site that is regulated by both PP1/PP2A and PP2B. This dual regulatory pathway may suggest a previously unappreciated role for multiple phosphatases in the regulation of urine concentration.
Acute kidney injury (AKI) and autosomal dominant polycystic kidney disease (ADPKD) are considered separate entities that both frequently cause renal failure. Since ADPKD appears to depend on Pc1- or Pc2-dosage mechanism, we investigated whether slow progression of cystogenesis in two Pkd1 transgenic mouse models can be accelerated with moderate ischemia-reperfusion injury (IRI). Transient unilateral left ischemic kidney in both non-transgenic and transgenic mice reproducibly develop tubular dilatations, cysts and typical PKD cellular defects within 3 months post-IRI. Similar onset and severity of IRI induced-cystogenesis independently of genotype revealed that IRI is sufficient to promote renal cyst formation, however this response was not further amplified by the transgene in Pkd1 mouse models. The IRI non-transgenic and transgenic kidneys showed from 16 days post-IRI, strikingly increased and sustained Pkd1/Pc1 (>3-fold) and Pc2 (>8-fold) expression that can individually be cystogenic in mice. In parallel, long-term and important stimulation of Hif1α expression was induced as in polycystic kidney disease. While mTOR signaling is activated, stimulation of the Wnt pathway, with markedly increased active β-catenin and c-Myc expression in IRI renal epithelium, uncovered a similar regulatory cystogenic response shared by IRI and ADPKD. Our study demonstrates that AKI on long-term, induces cystogenesis and a crosstalk with ADPKD Pc1/Pc2 pathogenic signaling.
The renal phenotype in Bardet-Biedl syndrome (BBS) is highly variable. The present study describes renal findings in 41 BBS patients and analyzes the pathogenesis of hyposthenuria, the most common renal dysfunction. 5/41 patients (12%) showed an estimated glomerular filtration rate <60ml/min/1.73m2. Urine protein and urine albumin-to-creatinine ratio were over 200 and 30 mg/g in 9/24 and 7/23 patients respectively. 4/41 patients showed no renal anomalies on ultrasound. 20/34 patients had hyposthenuria in the absence of renal insufficiency. In all 8 of the hyposthenuric patients studied, dDAVP failed to elevate urine osmolality (Uosm), suggesting a nephrogenic origin. Interestingly, water loading (WL) did not result in a significant reduction of Uosm, indicating combined concentrating and diluting defects. DDAVP infusion induced a significant increase of plasma Factor VIII and von Willebrand Factor levels, supporting normal function of the type 2 vasopressin receptor at least in endothelial cells. While urinary aquaporin 2 (u-AQP2) abundance was not different between patients and controls at baseline, the dDAVP-induced increased u-AQP2 and the WL-induced reduction of u-AQP2 were blunted in patients with a combined concentrating and diluting defect, suggesting a potential role of AQP2 in the defective regulation of water absorption. Urine Uromodulin excretion was reduced in all hyposthenuric patients suggesting a thick ascending limb defect. Interestingly, renal Na, Cl, Ca but not K handling was impaired after acute WL but not at basal. In summary, BBS patients show combined urinary concentration and dilution defects; a thick ascending limb and collecting duct tubulopathy may underlie impaired water handling
Aging-associated kidney disease is usually considered a degenerative process associated with aging. Recently, it has been shown that animals can produce fructose endogenously, and that this can be a mechanism for causing kidney damage in diabetic nephropathy and in association with recurrent dehydration. We therefore hypothesized that low level metabolism of endogenous fructose might play a role in aging-associated kidney disease. Wild-type and fructokinase knockout mice were fed a normal diet for 2 years that had minimal (<5%) fructose content. At the end of two years, wild-type mice showed elevations in systolic blood pressure, mild albuminuria, and glomerular changes with mesangial matrix expansion, variable mesangiolysis, and segmental thrombi. The renal injury was amplified by provision of high salt diet for 3 weeks, as noted by the presence of glomerular hypertrophy, mesangial matrix expansion and alpha smooth muscle actin expression, and with segmental thrombi. Fructokinase knockout mice were protected from renal injury both at baseline and after high salt intake (3 week) compared with wild-type mice. This was associated with higher levels of active (phosphorylated serine 1177) endothelial nitric oxide synthase in their kidneys. These studies suggest that aging-associated renal disease might be due to activation of specific metabolic pathways that could theoretically be targeted therapeutically, and raise the hypothesis that aging-associated renal injury may represent a disease process as opposed to normal age-related degeneration.
During urinary bladder filling the bladder urothelium releases chemical mediators which in turn transmit information to the nervous and muscular systems to regulate sensory sensation and detrusor muscle activity. Defects in release of urothelial mediators may cause bladder dysfunctions that are characterized with aberrant bladder sensation during bladder filling. Previous studies have demonstrated release of adenosine 5'-triphosphate (ATP) from the bladder urothelium during bladder filling and ATP remains the most studied purine mediator that is released from the urothelium. However, the micturition cycle is likely regulated by multiple purine mediators as various purine receptors are found present in many cell types in the bladder wall, including urothelial cells, afferent nerves, interstitial cells in lamina propria and detrusor smooth muscle cells. Information about the release of other biologically-active purines during bladder filling is still lacking. Decentralized bladders from C57BL/6 mice and Cynomolgus monkeys (Macaca fascicularis) were filled with physiological solution at different rates. Intraluminal fluid was analyzed by high performance liquid chromatography with fluorescence detection (HPLC-FLD) for simultaneous evaluation of ATP, ADP, AMP, adenosine, nicotinamide adenine dinucleotide (NAD+), ADP-ribose and cyclic ADP-ribose content. We also measured ex vivo bladder filling pressures and performed cystometry in conscious unrestrained mice at different filling rates. ATP, ADP, AMP, NAD+, ADPR, cADPR and adenosine were detected released intravesically at different ratios during bladder filling. Purine release increased with increased volumes and rates of filling. Our results support the concept that multiple urothelium-derived purines likely contribute to the complex regulation of bladder sensation during bladder filling.
The mechanisms by which prostanoids contribute to the maintenance of whole body water homeostasis are complex and not fully understood. The present study demonstrates that an EP3-dependent feedback mechanism contributes to the regulation of water homeostasis under high salt conditions. Rats on a normal diet and tap water were placed in metabolic cages and given either sulprostone (20 µg/kg/day) or vehicle for 3 days to activate EP3 receptors in thick ascending limb (TAL). Treatment was continued for another 3 days in rats given either 1% NaCl in the drinking water or tap water. Sulprostone decreased expression of COX-2 expression by approximately 75% in TAL tubules from rats given 1% NaCl concomitant with a ~60% inhibition of COX-2-dependent PGE2 levels in the kidney. Urine volume increased after ingestion of 1% NaCl, but was reduced ~40% by sulprostone. In contrast, the highly selective EP3 receptor antagonist (L-798106, 100 µg/kg/day), which increased COX-2 expression and renal PGE2 production, increased urine volume in rats given 1% NaCl. Sulprostone increased expression of AQP2 in inner medullary collecting duct plasma membrane in association with an increase in phosphorylation at Ser269 and decrease in Ser261 phosphorylation; antagonism of EP3 with L798106 reduced AQP2 expression. Thus, although acute activation of EP3 by PGE2 in the TAL and collecting duct inhibits NKCC2 and AQP2 activity, respectively, chronic activation of EP3 in vivo limits the extent of COX-2-derived PGE2 synthesis thereby mitigating the inhibitory effects of PGE2 on these transporters and decreasing urine volume.
The current study examined the effect of obesity on the development of renal injury within the genetic background of the Dahl salt-sensitive rat with a dysfunctional leptin receptor derived from Zinc-finger nucleases (SSLepRmutant strain). At 6 weeks of age, body weight was 35% higher in the SSLepRmutant strain compared to SSWT rats and remained elevated throughout the entire study. The SSLepRmutant strain exhibited impaired glucose tolerance and increased plasma insulin levels at 6 weeks of age suggesting insulin resistance while SSWT rats did not. However, blood glucose levels were normal throughout the course of the study. Systolic arterial pressure (SAP) was similar between the two strains from 6-10 weeks of age. However, by 18 weeks of age, the development of hypertension was more severe in the SSLepRmutant strain compared to SSWT rats (201±10 vs. 155±3 mmHg, respectively). Interestingly, proteinuria was substantially higher at 6 weeks of age in the SSLepRmutant strain versus SSWT rats (241±27 vs. 24±2 mg/day, respectively) and remained elevated until the end of the study. The kidneys from the SSLepRmutant strain displayed significant glomerular injury including podocyte foot process effacement and lipid droplets when compared to SSWT rats as early as 6 weeks of age. By 18 weeks of age, plasma creatinine levels were 2-fold higher in the SSLepRmutant strain versus SSWT rats suggesting the presence of chronic kidney disease (CKD). Overall, these results indicate that the SSLepRmutant strain develops podocyte injury and proteinuria independent of hyperglycemia and elevated arterial pressure that later progresses to CKD.
Fenofibrate activates not only peroxisome proliferator-activated receptor α (PPARα) but also adenosine monophosphate-activated protein kinase (AMPK). AMPK-mediated cellular responses protect kidney from high-fat diet (HFD)-induced injury, and autophagy resulting from AMPK activation has been regarded as a stress-response mechanism. Thus, the present study examined the role of AMPK and autophagy in the renotherapeutic effects of fenofibrate. C57BL/6J mice were divided into 3 groups: normal diet (ND), HFD, and HFD+fenofibrate (HFD+FF). Fenofibrate was administered 4 weeks after the initiation of the HFD when renal injury was initiated. Mouse proximal tubule cells (mProx24) were used to clarify the role of AMPK. Feeding mice with HFD for 12 weeks induced insulin resistance and kidney injury such as albuminuria, glomerulosclerosis, tubular injury, and inflammation, which were effectively inhibited by fenofibrate. In addition, fenofibrate treatment resulted in the activation of renal AMPK, upregulation of fatty acid oxidation (FAO) enzymes and antioxidants, and induction of autophagy in the HFD mice. In mProx24 cells, fenofibrate activated AMPK in a concentration-dependent manner, upregulated FAO enzymes and antioxidants, and induced autophagy, all of which were inhibited by treatment of compound C, an AMPK inhibitor. Fenofibrate-induced autophagy was also significantly blocked by AMPKα1 siRNA but not by PPARα siRNA. Collectively, these results demonstrate that delayed treatment with fenofibrate has a therapeutic effect on HFD-induced kidney injury, at least in part, through the activation of AMPK and induction of subsequent downstream effectors; autophagy, FAO enzymes, and antioxidants.
T helper 17 (Th17) lymphocytes promote renal inflammation in anti-glomerular basement membrane glomerulonephritis (anti-GBM GN), and signal transducer and activator of transcription 3 (STAT3) mediates activation of Th17 lymphocytes by interleukin 6 (IL-6) and transforming growth factor beta (TGFβ). Cln three requiring 9 (Ctr9), a subunit of RNA polymerase-associated factor complex (PAFc), regulates the transcription of IL-6/STAT3-dependent genes. Here, we investigated the role of Ctr9 in regulating Th17-driven inflammation in anti-GBM GN. In mice, STAT3β or IL-17 knockout ameliorated anti-GBM autoantibody-induced renal injury. This phenomenon was associated with decreases in retinoic acid receptor-related orphan receptor t (RORt), IL-17, phosphorylated STAT3, and pro-inflammatory cytokines. Compared with wild-type mice, Ctr9 increased in both STAT3β-/- and IL-17-/- mice injected with anti-GBM IgG, showing a negative correlation with Th17-related transcripts. Small interfering RNA (siRNA)-mediated knockdown of Ctr9 in intrarenal lymphocytes further upregulated Th17-related transcripts, consistent with repression of Th17 differentiation by Ctr9. Interestingly, Ctr9 was also expressed in human and mouse mesangial cells and downregulated in response to anti-GBM IgG or to TGFβ plus IL-17. Ctr9 in mesangial cells was even more repressed in the presence of both anti-GBM IgG and Th17-activating cytokines. Consistent with these findings, renal biopsies obtained from patients with anti-GBM GN showed consistent downregulation of Ctr9 and upregulation of phosphorylated STAT3 and IL-17 in the glomerulus. We conclude that Ctr9 is a negative regulator of Th17 differentiation in anti-GBM GN and repressed by anti-GBM IgG and IL-17 in mesangial cells.
Hydronephrosis is a commonly found disease state characterized by the dilation of renal calices and pelvis, resulting in the loss of kidney function in the severest cases. A generally accepted etiology of hydronephrosis involves the obstruction of urine flow along the urinary tract. In the recent years, we have developed a mouse model of hydronephrosis induced by lactational exposure to dioxin and demonstrated a lack of anatomical obstruction in this model. We also showed that prostaglandin E2 synthesis system plays a critical role in the onset of hydronephrosis. In the present study, we found that neonatal hydronephrosis was not likely to be associated with functional obstruction (impaired peristalsis), but was found to be associated with polyuria and low urine osmolality with the downregulation of proteins involved in the urine concentrating process. The administration of an antidiuretic hormone analogue to the dioxin-exposed pups not only suppressed the increased urine output but also decreased the incidence and severity of hydronephrosis. In contrast to the case in pups, administration of dioxin to adult mice failed to induce polyuria and upregulation of prostaglandin E2 synthesis system, and the adult mice were resistant to develop hydronephrosis. These findings suggest the possibility that polyuria could induce hydronephrosis in the absence of anatomical or functional obstruction of the ureter. It is concluded that the present animal model provides a unique example of polyuria-associated type of hydronephrosis, suggesting a need to redefine this disease state.
(Pro)renin receptor (PRR) is highly expressed in the distal nephron with unclear functional implication. The present study was conducted to explore a potential role of renal PRR during high K+ (HK) loading. In normal Sprague-Dawley rats, a 1-week HK intake increased renal expression of full-length PRR and urinary excretion of soluble PRR (sPRR). Administration of PRO20, a decoy peptide antagonist of PRR, to K+-loaded animals elevated plasma K+ level and decreased urinary K+ excretion, accompanied with suppressed aldosterone excretion. HK downregulated Na+-Cl- cotransporter (NCC) expression but upregulated CYP11B2 (cytochrome P450, family 11, subfamily B, polypeptide 2), renal outer medullary K+ channel (ROMK), calcium-activated potassium channel subunit alpha-1 (α-BK), α-Na+-K+-ATPase (α-NKA), and epithelial Na+ channel subunit beta (β-ENaC), all of which were blunted by PRO20. Following HK, urinary but not plasma renin was upregulated, which was blunted by PRO20. The same experiments performed using adrenalectomized (ADX) rats yielded similar results. Interestingly, spironolactone treatment in HK-loaded ADX rats attenuated kaliuresis but promoted natriuresis associated with the suppressed responses of β-ENaC, α-NKA, ROMK, and α-BK protein expression. Taken together, we discovered a novel role of renal PRR in regulation of K+ homeostasis that is likely through a local mechanism involving intrarenal renin-angiotensin-aldosterone system and coordinated regulation of membrane Na+ and K+ transporting proteins.
Growth delay is common in children with chronic kidney disease (CKD), often associated with poor quality of life. The role of anemia in uremic growth delay is poorly understood. Here we describe an induction of uremic growth retardation by 0.2% adenine diet in wild type (WT) and hepcidin gene (Hamp) knock-out (KO) mice, compared with their respective littermates fed a regular diet. Experiments were started at weaning (3 weeks). After 8 weeks, blood was collected and mice were euthanized. Adenine-fed WT mice developed CKD (BUN 82.8±11.6 mg/dL, creatinine 0.57±0.07 mg/dL) and were 2.1 cm shorter compared to WT controls. WT adenine-fed mice were anemic, had low serum iron, elevated Hamp, elevated IL6 and TNF-alpha. WT adenine fed mice had advanced mineral bone disease (serum phosphorus 16.9±3.1 mg/dL, FGF23 204.0±115.0 ng/mL) with loss of cortical and trabecular bone volume seen on micro-CT. Hamp disruption rescued the anemia phenotype resulting in improved growth rate in mice with CKD, thus providing direct experimental evidence of the relationship between Hamp pathway and growth impairment in CKD. Hamp disruption ameliorated CKD-induced growth hormone - insulin-like growth factor 1 axis derangements and growth plate alterations. Disruption of Hamp did not mitigate the development of uremia, inflammation, mineral and bone disease in this model. Taken together, these results indicate that adenine diet can be successfully used to study growth in mice with CKD. Hepcidin appears to be related to pathways of growth retardation in CKD suggesting that investigation of hepcidin lowering therapies in juvenile CKD is warranted.
Diabetic Nephropathy (DN) remains an unmet medical challenge as its prevalence is projected to continue to increase and specific medicines for treatment remain undeveloped. Activation of the immune system, in particular T-cells, is emerging as a possible mechanism underlying DN disease progression in humans and animal models. We hypothesized that inhibition of T-cell activation will ameliorate DN. Interaction of B7-1 (CD80) on the surface of antigen presenting cells with its binding partners, CTLA4 (CD152) and CD28 on T-cells, is essential for T-cell activation. In this study we used the soluble CTLA4-Fc fusion protein Abatacept to block cell surface B7-1, preventing the cellular interaction and inhibiting T-cell activation. When Abatacept was dosed in an animal model of diabetes-induced albuminuria, it reduced albuminuria in both prevention and intervention modes. The number of T-cells infiltrating the kidneys of DN animals correlated with the degree of albuminuria and treatment with Abatacept reduced the number of renal T-cells. As B7-1 induction has been recently proposed to underlie podocyte damage in DN, Abatacept could be efficacious in DN by protecting podocytes. However, this does not appear to be the case as B7-1 was not expressed in: 1) kidneys of DN animals; 2) stimulated human podocytes in culture; or 3) glomeruli of DN patients. We conclude that Abatacept ameliorates DN by blocking systemic T-cell activation and not by interacting with podocytes.
Parietal epithelial cells (PEC) response to glomerular injury may underlie a common pathway driving fibrogenesis following podocyte loss that typifies several glomerular disorders. Although the mammalian target of rapamycin (mTOR) pathway is important in cell homeostasis, little is known of the biological role or impact of reducing mTOR activity on PECs response following podocyte depletion, nor in the aging kidney. The purpose of these studies was to determine the impact on PECs of reducing mTOR activity following abrupt experimental depletion in podocyte number, as well as in a model of chronic podocyte loss and sclerosis associated with aging. Podocyte depletion was induced by an anti-podocyte antibody and rapamycin started at D5 until sacrifice at D14. Reducing mTOR did not lead to a greater reduction in podocyte density despite greater glomerulosclerosis. However, mTOR inhibition lead to an increase in PEC density and PEC derived crescent formation. Additionally, markers of epithelial to mesenchymal transition (PDGFβR, α-SMA, Notch 3) and PEC activation (CD44, Collagen IV) were further increased by mTOR reduction. Aged mice treated with rapamycin for 1, 2 and 10-week before sacrifice at 26.5 months (75 year old human age) had increased number of glomeruli with a crescentic appearance. mTOR inhibition either at a high or low levels lead to changes in PEC phenotype indicating PEC morphology is sensitive to changes mediated by global mTOR inhibition.
Cyclic nucleotide signal transduction pathways are an emerging research field in kidney disease. Activated cell surface receptors transduce their signals via intracellular second messengers such as cyclic AMP and cyclic GMP (cGMP). There is increasing evidence that regulation of the cGMP-cGMP-dependent protein kinase1-phosphodiesterase (cGMP-cGK1-PDE) signaling pathway may be renoprotective. Selective PDE5 inhibitors have shown potential in treating kidney fibrosis in patients with chronic kidney disease, via their downstream signaling, and these inhibitors also have known activity as anti-thrombotic and anti-cancer agents. This review gives an outline of the cGMP-cGK1-PDE signaling pathways and details the downstream signaling and regulatory functions that are modulated by cGK1 and PDE inhibitors with regards to anti-fibrotic, anti-thrombotic and anti-tumor activity. Current evidence that supports the renoprotective effects of regulating cGMP-cGK1-PDE signaling is also summarized. Finally, the effects of icariin, a natural plant extract with PDE5 inhibitory function, are discussed. We conclude that regulation of cGMP-cGK1-PDE signaling might provide novel, therapeutic strategies for the worsening global public health problem of chronic kidney disease.
Connecting tubule glomerular feedback (CTGF) is a mechanism where an increase in sodium (Na) concentration in the connecting tubule (CNT) causes the afferent arteriole (Af-Art) to dilate. We recently reported that aldosterone within the CNT lumen enhances CTGF via a nongenomic effect involving GPR30 receptors and sodium/hydrogen exchanger (NHE), but the signaling pathways of this mechanism are unknown. We hypothesize that aldosterone enhances CTGF via cAMP/protein kinase A (PKA) pathway that activates protein kinase C (PKC) and stimulates superoxide (O-2) production. Rabbit Af-Arts and their adherent CNTs were microdissected and simultaneously perfused. Two consecutive CTGF curves were elicited by increasing the CNT luminal NaCl. We found that the main effect of aldosterone was to sensitize CTGF and we analyzed data by comparing NaCl concentration in the CNT perfusate needed to achieve half of the maximal response (EC50). During the control period, the NaCl concentration that elicited a half-maximal response (EC50) was 37.0±2.0 mmol/L; addition of aldosterone (10-8 mol/L) to the CNT lumen decreased EC50 to 19.3±1.3 mmol/L (p≤0.001 vs. Control). The specific adenylyl cyclase inhibitor 2',3'-Dideoxyadenosine (ddA) (2x10-4 mol/L) and the PKA inhibitor H-89 dihydrochloride hydrate (H-89) (2x10-6 mol/L) prevented the aldosterone effect. Selective PKC inhibitor GF109203X (10-8 mol/L) also prevented EC50 reduction caused by aldosterone. CNT intraluminal addition of O-2 scavenger tempol (10-4 mol/L) blocked the aldosterone effect. We conclude that aldosterone inside the CNT lumen enhances CTGF via a cAMP/PKA/PKC pathway and stimulates O2 generation and this process may contribute to renal damage by increasing glomerular capillary pressure.
Obstructive kidney disease is a common complication in the clinic. Downregulation of aquaporins (AQPs) in obstructed kidneys has been thought as a key factor leading to the polyuria and impairment of urine-concentrating capability after the release of kidney obstruction. The present study was to investigate the role of mitochondrial complex-1 in modulating AQPs in obstructive nephropathy. Following 7-day unilateral ureteral obstruction (UUO), AQP1, AQP2, AQP3, and V2 receptor were remarkably reduced as determined by qRT-PCR and/or Western blotting. Notably, inhibition of mitochondrial complex-1 by rotenone markedly reversed the downregulation of AQP1, AQP2, AQP3, and V2. In contrast, AQP4 was not affected by kidney obstruction or rotenone treatment. In a separate study, rotenone also attenuated AQPs' downregulation after 48h UUO. To study the potential mechanisms in mediating the rotenone effects on AQPs, we examined the regulation of COX-2/mPGES-1/PGE2/EP pathway and found that COX-2, mPGES-1,and renal PGE2 content were all significantly elevated in obstructive kidneys, which was not affected by rotenone treatment. For EP receptors, EP2 and EP4 but not EP1 and EP3 were upregulated in obstructive kidneys. Importantly, rotenone strikingly suppressed EP1 and EP4 but not EP2 and EP3 receptors. However, treatment of EP1 antagonist SC-51322 could not affect AQPs' reduction in obstructed kidneys. Collectively, these findings suggested an important role of mitochondrial dysfunction in modulating AQPs and V2 receptor in obstructive nephropathy possibly via prostaglandin-independent mechanisms.
Postnatal inhibition or deletion of angiotensin II (ANG II) AT1 receptors impairs renal medullary mircrovascular development through a mechanism that may include vascular endothelial growth factor (VEGF). The present study was designed to test if VEGF/VEGF receptor signaling is necessary for development of the renal medullary microcirculation. Endothelial cell-specific immunolabeling of kidney sections from rats showed immature vascular bundles at postnatal (P) day 10 with subsequent expansion of bundles until P21. Medullary VEGF protein abundance coincided with vasa recta bundle formation. In human fetal kidney tissue, immature vascular bundles appeared early in third trimester (GA27-28) and expanded in size until term. Rat pups treated with the VEGF receptor-2 (VEGFR2) inhibitor vandetanib (100 mg/kg/day) from P7-P12 or P10-P16 displayed growth retardation and proteinuria. Stereological quantification showed a significant reduction in total length (386±13 vs 219±16 meters), surface area and volume of medullary microvessels. Vascular bundle architecture was unaffected. ANGII-AT1A/1B-/- mice kidneys displayed poorly defined vasa recta bundles whereas mice with collecting duct principal cell-specific AT1A deletion displayed no medullary microvascular phenotype. In conclusion, VEGFR2 signaling during postnatal development is necessary for expansion of the renal medullary microcirculation, but not structural patterning of the vasa recta bundles, which occurs through an AT1-mediated mechanism.
We examined renal Na and K transporters in mice with deletions in the gene encoding the aldosterone-induced protein SGK1. The knockout mice were hyperkalemic, and had altered expression of the subunits of the epithelial Na channel (ENaC). The kidneys showed decreased expression of the cleaved forms of the ENaC subunit, and the fully glycosylated form of the βENaC subunits when animals were fed a high-K diet. Knockout animals treated with exogenous aldosterone also had reduced subunit processing and diminished surface expression of βENaC and ENaC. Expression of the three upstream Na transporters NHE3, NKCC2 and NCC was reduced in both wild-type and knockout mice in response to K loading. The activity of ENaC measured as whole-cell amiloride-sensitive current (INa) in principal cells of the cortical collecting duct (CCD) was minimal under control conditions but was increased by a high-K diet to a similar extent in knockout and wild-type animals. INa in the connecting tubule also increased similarly in the two genotypes in response to exogenous aldosterone administration. The activities of both ROMK channels in principal cells and BK channels in intercalated cells of the CCD were unaffected by the deletion of SGK1. Acute treatment of animals with amiloride produced similar increases in Na excretion and decreases in K excretion in the two genotypes. The absence of changes in ENaC activity suggests compensation for decreased surface expression. Altered K balance in animals lacking SGK1 may reflect defects in ENaC-independent K excretion.
Tubulointerstitial fibrosis is a major feature associated with declining kidney function in chronic kidney disease of diverse etiology. No effective means as yet exists to prevent the progression of fibrosis. We have shown that the transcription factor SREBP-1 is an important mediator of the profibrotic response to TGFβ and Angiotensin II, both key cytokines in the fibrotic process. Here we examined the role of SREBP in renal interstitial fibrosis in the unilateral ureteral obstruction (UUO) model. The two isoforms of SREBP (-1 and -2) were activated by 3 days after UUO, with SREBP-1 showing a more sustained activation to 21 days. We then examined whether SREBP1/2 inhibition with the small molecule inhibitor fatostatin could attenuate fibrosis after 14 days of UUO. SREBP activation was confirmed to be inhibited by fatostatin. Treatment decreased interstitial fibrosis, TGFβ signaling, and upregulation of αSMA, a marker of fibroblast activation. Fatostatin also attenuated inflammatory cell infiltrate and apoptosis. Associated with this, fatostatin preserved proximal tubular mass. The significant increase in atubular glomeruli observed after UUO, known to correlate with irreversible renal functional decline, was also decreased by treatment. In cultured primary fibroblasts, TGFβ1 induced the activation of SREBP-1 and -2. Fatostatin blocked TGFβ1-induced αSMA and matrix protein upregulation. The inhibition of SREBP is thus a potential novel therapeutic target in the treatment of fibrosis in chronic kidney disease.
Endoplasmic reticulum (ER) stress has been implicated in some types of glomerular and tubular disorders. The objectives of this study were to elucidate the role of ER stress in lithium-induced nephrogenic diabetes insipidus (NDI) and to investigate whether attenuation of ER stress by 4-phenybutyric acid (4-PBA) improves urinary concentrating defect in a lithium-treated rats. Wistar rats have received lithium (40 mmol/kg food), 4-PBA (320 mg/kg BW by gavage every day), or no treatment (control) for two weeks and some of them were dehydrated for 24 hours. Lithium treatment resulted in increased urine output and decreased urinary osmolality, which was significantly improved by 4-PBA. 4-PBA also prevented reduced protein expression of AQP2, pS256-AQP2, and pS261-AQP2 in inner medullar of kidneys from lithium-treated rats after 24 hour dehydration. Lithium treatment resulted in increased expression of ER stress markers in inner medulla, which was associated with dilated cisternae and expansion of ER in the inner medullary collecting duct (IMCD) principal cells. Confocal immunofluorescence studies showed colocalization of a molecular chaperone BiP with AQP2 in principal cells. Immunohistochemistry demonstrated increased intracellular expression of BiP and decreased AQP2 expression in IMCD principal cells of kidneys from lithium-treated rats. 4-PBA attenuated expression of ER stress markers and recovered ER morphology. In IMCD suspensions isolated from lithium-treated rats, 4-PBA incubation was associated with increased AQP2 expression and ameliorated ER stress. In conclusion, in experimental lithium-induced NDI, 4-PBA improved urinary concentrating defect and increased AQP2 expression, likely via attenuating ER stress in inner medullary collecting duct principal cells.
High mobility group box 1 (HMGB1) is a nuclear protein released extracellularly in response to infection or injury, where it activates immune responses and contributes to the pathogenesis of kidney dysfunction in sepsis and sterile inflammatory disorders. Recently we demonstrated that HMGB1 inhibits HCO3- absorption in perfused rat medullary thick ascending limbs (MTAL) through a basolateral RAGE-dependent pathway that is additive to TLR4-ERK-mediated inhibition by LPS. Here, we examined signaling and transport mechanisms that mediate inhibition by HMGB1. Inhibition of HCO3- absorption by HMGB1 was eliminated by the Rho-associated kinase (ROCK) inhibitor Y27632 and by a specific inhibitor of Rho, the major upstream activator of ROCK. HMGB1 increased RhoA and ROCK1 activity. HMGB1-induced ROCK1 activation was eliminated by the RAGE antagonist FPS-ZM1 and by inhibition of Rho. The Rho and ROCK inhibitors had no effect on inhibition of HCO3- absorption by bath LPS. Inhibition of HCO3- absorption by HMGB1 was eliminated by bath amiloride, 0 Na+ bath, and the F-actin stabilizer jasplakinolide, three conditions that selectively prevent inhibition of MTAL HCO3- absorption mediated through NHE1. HMGB1 decreased basolateral Na+/H+ exchange activity through activation of ROCK. We conclude that HMGB1 inhibits HCO3- absorption in the MTAL through a RAGE-RhoA-ROCK1 signaling pathway coupled to inhibition of NHE1. The HMGB1-RAGE-RhoA-ROCK1 pathway thus represents a potential target to attenuate MTAL dysfunction during sepsis and other inflammatory disorders. HMGB1 and LPS inhibit HCO3- absorption through different receptor signaling and transport mechanisms, which enables these pathogenic mediators to act directly and independently to impair MTAL function.
We developed a model of calcium homeostasis in the rat in order to better understand the impact of dysfunctions such as primary hyperparathyroidism and vitamin D deficiency on calcium balance. The model accounts for the regulation of calcium intestinal uptake, bone resorption, and renal reabsorption by parathyroid hormone (PTH), vitamin D3, and Ca2+ itself. It is the first such model to incorporate recent findings regarding the role of the calcium sensing receptor (CaSR) in the kidney, the presence of a rapidly exchangeable pool in bone, and the delayed response of vitamin D3 synthesis. Accounting for two (fast and slow) calcium storage compartments in bone allows the model to properly predict the effects of bisphophonates on the plasma levels of Ca2+ ([Ca2+]p), PTH, and vitamin D3. Our model also suggests that Ca2+ exchange rates between plasma and the fast pool vary with both sex and age, allowing [Ca2+]p to remain constant in spite of sex- and age-based hormonal and other differences. Our results suggest that the inconstant hypercalciuria that is observed in primary hyperparathyroidism can be attributed in part to counterbalancing effects of PTH and CaSR in the kidney. Our model also correctly predicts that calcimimetic agents such as Cinacalcet bring down [Ca2+]p to within its normal range in primary hyperparathyroidism. In addition, the model provides a simulation of CYP24A1 inactivation that leads to a situation reminiscent of infantile hypercalcemia. In summary, our model of calcium handling can be used to decipher the complex regulation of calcium homeostasis.
The introduction of calcineurin inhibitors (CNI) into clinical practice in the late 1970s transformed organ transplantation and led to significant improvement in acute rejection episodes. However, despite their significant clinical utility, the use of these agents is hampered by the development of hypertension and nephrotoxicity, which ultimately lead to end-stage kidney disease and overt cardiovascular outcomes. There are currently no effective agents to treat or prevent these complications. Importantly, CNI-free immunosuppressive regimens lack the overall efficacy of CNI-based treatments and put patients at risk of allograft rejection. Cytochrome P-450 epoxygenase metabolites of arachidonic acid, epoxyeicosatrienoic acids (EETs) have potent vasodilator and anti-hypertensive properties in addition to many cytoprotective effects, but their effects on CNI-induced nephrotoxicity have not been explored. Here, we show that PVPA, a novel orally active analog of 14,15-epoxyeicosatrienoic acid, effectively prevents the development of hypertension and ameliorates kidney injury in cyclosporine-treated rats. PVPA treatment reduced proteinuria and renal dysfunction induced by cyclosporine. PVPA inhibited inflammatory cell infiltration into the kidney and decreased renal fibrosis. PVPA also reduced tubular epithelial cell apoptosis, attenuated the generation of reactive oxygen species and modulated the unfolded protein response that is associated with endoplasmic reticulum stress. Consistent with the in vivo data, PVPA attenuated cyclosporine-induced apoptosis of NRK-52E cells in vitro. These data indicate that the cytochrome P-450/ EET system offers a novel therapeutic strategy to treat or prevent CNI-induced nephrotoxicity.
Rationale: Overactive Bladder (OAB) is an idiopathic condition, characterized by urgency, urinary frequency and urgency incontinence, in the absence of routinely traceable urinary infection. We have described microscopic pyuria (≥10 wbc μl-1) in patients suffering from the worst symptoms. It is established that inflammation is associated with increased ATP release from epithelial cells, and extracellular ATP originating from the urothelium following increased hydrostatic pressure, is a mediator of bladder sensation. Objectives: Here, using bladder-biopsy samples, we have investigated urothelial ATP signaling in OAB patients with microscopic pyuria. Findings: Basal, but not stretch-evoked, release of ATP was significantly greater from urothelium of OAB patients with pyuria than from non-OAB patients or OAB patients without pyuria (<10 wbc μl-1). Basal ATP release from urothelium of OAB patients with pyuria was inhibited by the P2 receptor antagonist suramin and abolished by the hemichannel blocker carbenoxolone, which differed from stretch-activated ATP release. Altered P2 receptor expression was evident in urothelium from pyuric OAB patients. Furthermore, intracellular bacteria were visualized in shed urothelial cells from ~80% of OAB patients with pyuria. Conclusions: These data suggest that increased ATP release from the urothelium, involving bacterial colonization, may play a role in the heightened symptoms associated with pyuric OAB patients.
Percutaneous nephrolithotomy (PCNL) causes a rapid increase in renal pelvic pressure in the kidney, which induces an inflammatory response. High-mobility group box-1 (HMGB1) is known to trigger the recruitment of inflammatory cells and the release of pro-inflammatory cytokines following ischemia reperfusion injury in the kidney, but the contribution of HMGB1 to the inflammatory response following high-pressure renal pelvic perfusion has not been investigated. In this study, high-pressure renal pelvic perfusion was induced in anesthetized pigs to examine the effect of HMGB1 on the inflammatory response. HMGB1 levels in the kidney increased following high pressure renal pelvic perfusion, together with elevated levels of inflammatory cytokines in the plasma and kidney and an accumulation of neutrophils and macrophages. Inhibition of HMGB1 alleviated this inflammatory response, while perfusion with recombinant HMGB1 had an augmentative effect, confirming the involvement of HMGB1 in the inflammatory response to high pressure renal pelvic perfusion. HMGB1 regulated the inflammatory response by activating Toll-like receptor 4 (TLR4) signaling. In conclusion, this study has demonstrated that HMGB1/TLR4 signaling contributes to the inflammatory response following high-pressure renal pelvic perfusion in a porcine model and has implications for the management of inflammation after PCNL.
Pericytes are tissue-resident mesenchymal progenitor cells anatomically associated with the vasculature, that have been shown to participate in tissue regeneration. Here we tested the hypothesis that kidney pericytes derived from FoxD1+ mesodermal progenitors during embryogenesis, are necessary for postnatal kidney homeostasis. Diphtheria toxin delivery to FoxD1Cre::RsDTR transgenic mice resulted in selective ablation of >90% of kidney pericytes but not other cell lineages. Abrupt increases in plasma creatinine, BUN and albuminuria within 96h indicated acute kidney injury in pericyte-ablated mice. Loss of pericytes led to rapid accumulation of neutral lipid vacuoles, swollen mitochondria and apoptosis in tubular epithelial cells. Pericyte ablation led to endothelial cell swelling, reduced expression of vascular homeostasis markers and peritubular capillary loss. Despite the observed injury, no signs of acute inflammatory response were observed. Pathway enrichment analysis of genes expressed in kidney pericytes in vivo identified basement membrane proteins, angiogenic factors, and factors regulating vascular tone as major regulators of vascular function. Using novel microphysiological devices we recapitulated human kidney peritubular capillaries coated with pericytes and show that pericytes regulate permeability, basement membrane deposition, and microvascular tone. These findings suggest that through the active support of the microvasculature, pericytes are essential to adult kidney homeostasis.
Podocytes are the key target for injury in proteinuric glomerular diseases that result in podocyte loss, progressive focal segmental glomerular sclerosis (FSGS) and renal failure. Current evidence suggests that the initiation of podocyte injury and associated proteinuria can be separated from factors that drive and maintain these pathogenic processes leading to FSGS. In nephrotic urine aberrant glomerular filtration of Plasminogen (Plg) is activated to the biologically active serine protease Plasmin by urokinase type plasminogen activator (uPA). In-vivo inhibition of uPA mitigates Plg activation and development of FSGS in several proteinuric models of renal disease including 5/6 nephrectomy. Here we show that Plasminogen (Plg) is markedly increased in the urine in two murine models of proteinuric kidney disease associated with podocyte injury: Tg26 HIV-associated nephropathy and the Cd2ap-/- model of FSGS. We show that human podocytes express uPA and three Plg receptors: uPAR, tPA and Plg-RKT. We demonstrate that Plg treatment of podocytes specifically upregulates NADPH oxidase isoforms NOX2/NOX4 and increases production of mitochondrial-dependent superoxide anion (O2- ) that promotes endothelin-1 synthesis. Plg via O2- also promotes expression of the B scavenger receptor CD36 and subsequent increased intracellular cholesterol uptake resulting in podocyte apoptosis. Taken together our findings suggest that following disruption of the glomerular filtration barrier at the onset of proteinuric disease, podocytes are exposed to Plg resulting in further injury mediated by oxidative stress. We suggest that chronic exposure to Plg could serve as a "second hit" in glomerular disease and that Plg is potentially an attractive target for therapeutic intervention.
In chronic kidney disease (CKD), simultaneous mineral and skelecton changes are prevalent, known as CKD-mineral bone disorder (CKD-MBD). Arterial calcification (AC) is a clinically important complication of CKD-MBD. It can increase arterial stiffness, which leads to severe cardiovascular events. However, current treatments have little effects on regression of AC, as its mechanisms are still unclear. There are multiple risk factors of AC, among which Malnutrition-Inflammation Complex Syndrome (MICS) is a new and crucial one. MICS, a combined syndrome of malnutrition and inflammation, generally begins at the early stage of CKD and becomes obvious in end-stage renal disease (ESRD). It was linked to reverse epidemiology and associated with increased cardiovascular mortality in ESRD patients. Recent data suggests that MICS can trigger CKD-MBD and accelerate the course of AC. In this present review, we summarized the recent understandings about the aggravating effects of MICS on AC and discussed the possible underlying mechanisms. A series of findings indicate that targeting MICS will provide a potential strategy for treating AC in CKD.
Overlapping symptoms of overactive bladder (OAB) and urinary tract infection UTI often complicate the diagnosis and contribute to over- prescription of antibiotics. Inflammatory response is a shared characteristic of both UTI and OAB and here we hypothesized that molecular differences in inflammatory response seen in urine can help discriminate OAB from UTI. Methods: Subjects in the age range of (20-88 years) of either sex were recruited for this urine analysis study. Urine specimens were available from 62 UTI patients with positive dipstick test before antibiotic treatment. Six of these patients also provided urine after completion of antibiotic treatment. Subjects in cohorts of OAB (n= 59) and asymptomatic controls (n=26) were negative for dipstick test. Urinary chemokines were measured by MILLIPLEX MAP Human Cytokine/Chemokine immunoassay and their association with UTI and OAB was determined by univariate and multivariate statistics. RESULTS: Significant elevation of CXCL-1, CXCL-8(IL-8) and CXCL-10 together with reduced levels for receptor antagonist of IL-1a (sIL-1RA) were seen in UTI relative to OAB and asymptomatic controls. Elevated CXCL-1 urine levels predicted UTI with odds ratio of 1.018 and showed a specificity of 80.77% and sensitivity of 59.68%. Post-antibiotic treatment, reduction was seen in all CXC chemokines with significant reduction for CXCL-10. CONCLUSIONS: Strong association of CXCL-1 and CXCL-10 for UTI over OAB indicates mechanistic differences in signaling pathways driving inflammation secondary of infection in UTI compared to a lack of infection in OAB. Urinary chemokines highlight molecular differences in the paracrine signaling driving the overlapping symptoms of UTI and OAB.
We previously demonstrated that renal peptidyl arginine deiminase-4 (PAD4) is induced after renal ischemia and reperfusion (IR) injury and exacerbates acute kidney injury (AKI) by increasing the renal tubular inflammatory response. Here, we tested whether genetic ablation of PAD4 attenuates renal injury and inflammation after IR in mice. After 30 min renal IR, PAD4 wild type mice develop severe AKI with large increases in plasma creatinine, neutrophil infiltration as well as significant renal tubular necrosis, apoptosis and pro-inflammatory cytokine generation. In contrast, PAD4 deficient mice are protected against ischemic AKI with reduced real tubular neutrophil infiltration, renal tubular necrosis and apoptosis. In addition, hepatic injury and inflammation observed in PAD4 wild type mice after renal IR is significantly attenuated in PAD4 deficient mice. We also show that increased renal tubular PAD4 expression after renal IR is associated with translocation of PAD4 from the nucleus to the cytosol. Consistent with PAD4 cytosolic translocation, we show increased renal tubular cytosolic peptidyl-citrullination after ischemic AKI. Mechanistically, recombinant PAD4 treatment increased nuclear translocation of NFkB in cultured human as well as murine proximal tubule cells that is inhibited by a PAD4 inhibitor (2-chloroamidine). Taken together, our studies further support the hypothesis that renal tubular PAD4 plays a critical role in renal IR injury by increasing the renal tubular inflammatory response and neutrophil infiltration after renal IR perhaps by interacting with the pro-inflammatory transcription factor NFkB in the cytosol and promoting its nuclear translocation.
Animal models are indispensable for the study of glomerulonephritis, a group of diseases that destroy kidneys but for which specific therapies do not yet exist. Novel interventions are urgently needed, but their rational design requires suitable in vivo platforms to identify and test new candidates. Animal models can recreate the complex immunologic microenvironments that foster human autoimmunity and nephritis, and provide access to tissue compartments not readily examined in patients. Study of rat Heymann nephritis identified fundamental disease mechanisms that ultimately revolutionized our understanding of human membranous nephropathy. Significant species differences in expression of a major target antigen, however, and lack of spontaneous autoimmunity in animals remain roadblocks to full exploitation of preclinical models in this disease. For several glomerulonephritides, humanized models have been developed to circumvent cross-species barriers and to study the effects of human genetic risk variants. Herein we review humanized mouse prototypes that provide fresh insight into mediators of IgA nephropathy and origins of anti-glomerular basement membrane nephritis and Goodpasture's disease, as well as a means to test novel therapies for ANCA vasculitis. Additional and refined model systems are needed to mirror the full spectrum of human disease in a genetically diverse population, to facilitate development of patient-specific interventions, to determine the origin of nephritogenic autoimmunity, and to define the role of environmental exposures in disease initiation and relapse
The antidiuretic hormone vasopressin (AVP) regulates renal salt and water reabsorption along the distal nephron and collecting duct system. These effects are mediated by V2 receptors (V2R) and release of intracellular Gs-mediated cAMP to activate epithelial transport proteins. Inactivating mutations in the V2R gene lead to the X-linked form of nephrogenic diabetes insipidus (NDI) which has chiefly been related with impaired aquaporin 2-mediated water reabsorption in the collecting ducts. Previous work also suggested the AVP-V2R-mediated activation of Na+-K+-2Cl--cotransporters (NKCC2) along the thick ascending limb (TAL) in the context of urine concentration, but its individual contribution to NDI or, more generally, to overall renal function was unclear. We hypothesized that V2R-mediated effects in TAL essentially determine its reabsorptive function. To test this, we reevaluated V2R expression. Basolateral membranes of medullary and cortical TAL were clearly stained, whereas cells of the macula densa cells were unreactive. A dominant-negative, NDI-causing truncated V2R mutant (Ni3-Glu242 stop) was then introduced into the rat genome under control of the Tamm-Horsfall protein promoter to cause a tissue-specific AVP-signalling defect exclusively in TAL. Resulting Ni3-V2R transgenic rats revealed decreased basolateral but increased intracellular V2R signal in TAL epithelia, suggesting impaired trafficking of the receptor. Rats displayed significant baseline polyuria, failure to concentrate the urine in response to water deprivation, and hypercalciuria. NKCC2 abundance, phosphorylation, and surface expression were markedly decreased. In summary, these data indicate that suppression of AVP-V2R signaling in TAL causes major impairment in renal fluid and electrolyte handling. Our results may have clinical implications.
Despite increasing numbers of patients on dialysis, the numbers of renal transplants performed yearly have remained relatively static. During the last 50 years, there have been many advances in the pharmacology of prevention of organ rejection. However, most patients will suffer from a slow but steady decline in renal function leading to graft loss. The most common cause of long-term graft loss is chronic allograft nephropathy (CAN). Therefore, elucidating and understanding the mechanisms involved in CAN is crucial for achieving better post-transplant outcomes. It is thought that the development of epithelial to mesenchymal transition (EMT) in proximal tubules is one of the first steps towards CAN, and has been shown to be a result of cellular senescence. Cells undergoing senescence acquire a senescence associated secretory phenotype (SASP) leading to the production of interleukin-1 alpha (IL-1α), which has been implicated in several degenerative and inflammatory processes including renal disease. A central mediator in SASP activation is the production of reactive oxygen species (ROS), which are produced in response to numerous physiologic and pathologic stimuli. This review explores the connection between SASP and the development of EMT/CAN in an effort to suggest future directions for research leading to improved long-term graft outcomes.
In recent years, genome-wide RNA expression analysis has become a routine tool that offers a great opportunity to study and understand the key role of genes that contribute to carcinogenesis. Various microarray platforms and statistical approaches can be used to identify genes that might serve as prognostic biomarkers and be developed as antitumor therapies in the future. Metastatic renal cell carcinoma (mRCC) is a serious, life-threatening disease, and there are few treatment options for patients. In this study, we performed one-color microarray gene expression (4x44K) analysis of the mRCC cell line Caki-1 and the healthy kidney cell line ASE-5063. A total of 1921 genes were differentially expressed in the Caki-1 cell line (1023 upregulated and 898 downregulated). Gene set enrichment analysis (GSEA) and Ingenuity Pathway Analysis (IPA) approaches were used to analyze the differential-expression data. The objective of this research was to identify complex biological changes that occur during metastatic development using Caki-1 as a model mRCC cell line. Our data suggest that there are multiple deregulated pathways associated with metastatic clear cell renal cell carcinoma (mccRCC), including integrin-linked kinase (ILK) signaling, leukocyte extravasation signaling, IGF-I signaling, CXCR4 signaling, and PI3K/AKT/mTOR signaling. The IPA upstream analysis predicted top transcriptional regulators that are either activated or inhibited, such as estrogen receptors, TP53, KDM5B, SPDEF, and CDKN1A. The GSEA approach was used to further confirm enriched pathway data following IPA.
Nox4 and Nox2 are the most abundant NADPH oxidases (Nox) in the kidney and have been shown to contribute to hypertension, renal oxidative stress and injury in Dahl salt-sensitive (SS) hypertensive rats. The present study focused on the role of Nox4 and p67phox/Nox2 on the generation of H2O2 and O2•- in mTAL of SS rats in response to increasing luminal flow (5 to 20 nl/min). Nox4 and p67phox/Nox2 genes were found to be expressed in the renal medullary thick ascending limb of Henle (mTAL) of SS rats. Responses of SS rats were compared to SS rats with knockout of Nox4 (SSNox4-/-) or functional mutation of p67phox (SSp67phox-/-). Results show that Nox4 was the dominant source of increased intracellular H2O2 production in response to increased luminal flow as determined with the fluorescent dye PF6-AM. The rate of mitochondrial H2O2 production (mito-PY1) was also significantly reduced in SSNox4-/- compared to SS rats but not in SSp67phox-/- rats. In contrast, intracellular O2•- production (Eth/DHE) in mTAL of SSNox4-/- rats was nearly identical to SS rats in response to luminal flow indicating Nox4 made no measurable contribution. SSp67phox-/- rats exhibited reduced mTAL O2•- production compared to SS rats at the lower luminal flow of 5 nl/min and progressively increased when perfusion was changed to 20 nl/min. We conclude that increased mTAL luminal flow results in increases of both intracellular and mitochondrial H2O2 which are dependent upon the presence of Nox4 and that p67phox/Nox2 accounts solely for increases of O2•- production.
Water and salt metabolism are tightly regulated processes. Maintaining this milieu intérieur within narrow limits is critical for normal physiological processes to take place. Disturbances to this balance can result in disease and even death. Some of the better characterized regulators of water and salt homeostasis include angiotensin II (ANGII), aldosterone, arginine vasopressin (AVP) and oxytocin. Although secretin (SCT) was first described more than hundred years ago, little is known about the role of this classic gastrointestinal hormone in the maintenance of water-salt homeostasis. In recent years, increasing body of evidence suggested that SCT and its receptor play important roles in the central nervous system (CNS) and kidney to ensure that the mammalian extracellular fluid (ECF) osmolarity is kept within healthy range. In this review, we focus on recent advances in our understanding of the molecular, cellular and network mechanisms by which SCT and its receptor mediates the control of osmotic homeostasis. Implications of hormonal cross-talk and receptor-receptor interaction are highlighted.
Since changes in the plasma water sodium concentration ([Na+]pw) are clinically due to changes in the mass balance of Na+, K+ and H2O, the analysis and treatment of the dysnatremias are dependent on the validity of the Edelman equation in defining the quantitative interrelationship between the [Na+]pw and the total exchangeable sodium (Nae), total exchangeable potassium (Ke), and total body water (TBW) (5):[Na+]pw = 1.11(Nae+ Ke)/TBW - 25.6 (Eq. 1). The interrelationship between [Na+]pw and Nae, Ke and TBW in the Edelman equation is empirically determined by accounting for measurement errors in all these variables. In contrast, linear regression analysis of the same data set using [Na+]pw as the dependent variable yields the following equation:[Na+]pw = 0.93(Nae + Ke)/TBW + 1.37 (Eq. 2). Moreover, based on the study by Boling et al. (1), the [Na+]pw is related to the Nae, Ke and TBW by the following linear regression equation:[Na+]pw = 0.487(Nae + Ke)/TBW + 71.54 (Eq. 3). In this mathematical analysis, we demonstrate that the disparities between the slope and y-intercept in these three equations can be explained by how the osmotically inactive Na+ and K+ storage pool is quantitatively accounted for. Our analysis also indicates that the osmotically inactive Na+ and K+ storage pool is dynamically regulated and that changes in the [Na+]pw can be predicted based on changes in the Nae, Ke and TBW despite dynamic changes in the osmotically inactive Na+ and K+ storage pool.
Na+/H+ exchanger 3 (NHE3), a major Na+ transporter in the luminal membrane of proximal tubule, is subject to angiotensin II (ANG II) regulation in renal Na+/fluid absorption and blood pressure control. We have previously shown that inositol 1,4,5-triphosphate receptor-binding protein released with inositol 1,4,5-triphosphate (IRBIT) mediates ANG II-induced exocytosis of NHE3 in cultured proximal tubule epithelial cells. In searching for scaffold protein(s) that coordinates with IRBIT in NHE3 trafficking, we found that Na+/H+ exchanger regulatory factor 1 (NHERF1), NHE3 and IRBIT proteins were co-expressed in the same macrocomplexes and loss of type 1 receptor of angiotensin II (AT1R) decreased their expression in the renal brush border membrane. We found that NHERF1 was required for ANG II-mediated forward trafficking and activation of NHE3 in cultured cells. ANG II induced a concomitant increase of NHERF1 interaction with NHE3 and IRBIT, which was abolished when NHERF1 PDZ1 domain was removed. Overexpression of an YFP-NHERF1 construct that lacks PDZ1, but not PDZ2, failed to exaggerate ANG II-dependent increase of NHE3 expression in the apical membrane. Moreover, exogenous expression of PDZ1 exerted a dominant-negative effect on NHE3 activation by ANG II. We further demonstrated that IRBIT was indispensable for ANG II-provoked increase in NHERF1-NHE3 interaction, and that phosphorylation of IRBIT at Ser68 was necessary for the assembly of the NHEF1-IRBIT-NHE3 complex. Taken together, our findings suggest that NHERF1 mediates ANG II-induced activation of renal NHE3, which requires coordination between IRBIT and NHERF1 PDZ1 domain in binding and transporting NHE3.
An analytical Population Balance Equation model is developed and used to assess the risk of critical renal stone formation for astronauts during future space missions. The model uses the renal biochemical profile of the subject as input and predicts the steady state size distribution of the nucleating, growing, and agglomerating calcium oxalate crystals during their transit through the kidney. The model is verified through comparison with published results of several crystallization experiments. Numerical results indicate that the model is successful in clearly distinguishing between 1g normal and 1g recurrent stone-former subjects based solely on their published 24 hr urine biochemical profiles. Numerical case studies further show that the predicted renal calculi size distribution for a microgravity astronaut are closer to those of a recurrent stone-former on earth rather than to a normal subject in 1g. This interestingly implies that the increase in renal stone risk level in microgravity is relatively more significant for a normal person than a stone former. However, numerical predictions still underscore that the stone-former subject carries by far the highest absolute risk of critical stone formation during space travel.
An analytical Population Balance Equation model is used to assess the efficacy of citrate, pyrophosphate, and augmented fluid in-take as dietary countermeasures aimed at reducing the risk of renal stone formation for the astronauts. The model uses the measured biochemical profile of the astronauts as input and predicts the steady state size distribution of the nucleating, growing, and agglomerating renal calculi subject to biochemical changes brought about by administration of these dietary countermeasures. Numerical predictions indicate that an increase in citrate levels beyond its average normal ground-based urinary values is beneficial but only to a limited extent. Unfortunately, results also indicate that any decline in the citrate levels during space travel below its normal urinary values on Earth can easily move the astronaut into the stone-forming risk category. Pyrophosphate is found to be an effective inhibitor since numerical predictions indicate that even at quite small urinary concentrations, it has the potential of shifting the maximum crystal aggregate size to a much smaller and plausibly safer range. Finally, our numerical results predict a decline in urinary volume below 1.5 liters/day can act as a dangerous promoter of renal stone development in microgravity while urinary volume levels of 2.5 - 3 liters/day can serve as effective space countermeasures.
Immune cells in the kidney are implicated in the development of hypertension and renal damage in the Dahl Salt-Sensitive (SS) rat. Interestingly, interleukin 6 (IL-6) mRNA is 54-fold higher in T lymphocytes isolated from the kidney in comparison to circulating T lymphocytes. The present experiments assessed the role of IL-6 in the development of SS hypertension by treating rats (n=13-14/group) with an IL-6 neutralizing antibody or normal IgG during an 11 day period of high salt (4.0% NaCl chow) intake. The mean arterial pressure (MAP) and urine albumin excretion rates (Ualb) were not different between the groups fed low salt (0.4% NaCl). Following 11 days of drug-treatment and high salt, however, the rats receiving anti-IL-6 demonstrated a 47% reduction of IL-6 in the renal medulla compared to control SS. Moreover, the increase in MAP following 11 days of high NaCl intake was significantly attenuated in SS administered anti-IL-6 compared with the control group (138±3 vs. 149±3 mmHg) as was the salt-induced increase in Ualb and glomerular and tubular damage. To investigate potential mechanisms of action, a flow cytometric analysis of immune cells in the kidney (n=8-9/group) demonstrated that the total number of monocytes and macrophages was significantly lower in the treatment vs the control group. The total number of T and B lymphocytes in the kidneys was not different between groups. These studies indicate that IL-6 production may participate in the development of SS hypertension and end-organ damage by mediating increased infiltration or proliferation of macrophages into the kidney.
Sepsis is an uncontrolled systemic inflammatory response against an infection and a major public health issue worldwide. This condition affects several organs, and kidneys are particularly damaged when it is caused by Gram-negative bacteria. Due to the importance of renin-angiotensin system (RAS) in regulating renal function, in the present study, we aimed to investigate the effects of endotoxemia over the renal RAS. Wistar rats were injected with E. coli lipopolysaccharide (LPS) (4 mg/kg), mimicking the endotoxemia induced by Gram-negative bacteria. Three days after treatment, body mass, blood pressure and plasma nitric oxide (NO) were reduced, indicating that endotoxemia triggered cardiovascular and metabolic consequences and that hypotension was maintained by NO-independent mechanisms. Regarding the effects in renal tissue, inducible NO synthase (iNOS) was diminished, but no changes in the renal level of NO was detected. RAS was also highly affected by endotoxemia, since renin, Angiotensin-Converting Enzyme (ACE) and ACE2 presented decreased activity in renal tissue. Although these enzymes were modulated, only Angiotensin (Ang) II was augmented in kidneys, Ang I and Ang 1-7 levels were not influenced by LPS. Cathepsin G and chymase activities were increased in the endotoxemia group, suggesting alternative pathways for Ang II formation. Taken together, our data suggests the activation of non-canonical pathways for Ang II production and the presence of renal vasoconstriction and tissue damage in our animal model. In summary, the systemic administration of LPS affects renal RAS, what may contribute for several deleterious effects of endotoxemia over kidneys.
Renal ischemia/reperfusion (I/R) in male rats causes reductions in plasma testosterone, and infusion of testosterone 3 hours post-reperfusion is protective. We tested the hypotheses that acute high doses of testosterone promote renal injury after I/R; and that acute low dose testosterone is protective by: 1) increasing renal IL-10 and reducing TNF-α; 2) it's effects on nitric oxide (NO); and 3) reducing intrarenal T cell infiltration. Rats were subjected to renal I/R followed by intravenous infusion of vehicle or testosterone (20, 50 or 100 μg/kg) 3 hrs post-reperfusion. Low dose testosterone (20 μg/kg) reduced plasma creatinine, increased nitrate/nitrite excretion, increased intrarenal IL-10 and reduced intrarenal TNF-a, whereas 50 μg/kg testosterone failed to reduce plasma creatinine, increased IL-10, but failed to reduce TNF-α; a higher dose of testosterone (100 mg/kg) not only failed to reduce plasma creatinine, but significantly increased both IL-10 and TNF-α compared to other groups. Low dose nitro-L-arginine methyl ester (1 mg/kg/d), given 2 days prior to I/R, prevented low dose testosterone (20 μg/kg) from protecting against I/R injury, and was associated with lack of increase in intrarenal IL-10. Intrarenal CD4+ and CD8+ T cells were significantly increased with I/R but were attenuated with low dose testosterone as were effector T helper 17 cells. The present studies suggest that acute, low dose testosterone is protective against I/R AKI in males due to its effects on inflammation by reducing renal T cell infiltration and by shifting the balance to favor anti-inflammatory cytokine production rather than pro-inflammatory cytokines.
Ischemia-reperfusion-mediated acute kidney injury can necessitate renal replacement therapy and is a major cause of morbidity and mortality. We have identified BB3, a small molecule, which when first administered at 24 hr following renal ischemia in rats, improved survival, augmented urine output and reduced the increase in serum creatinine and blood urea nitrogen. Compared with control kidneys, the kidneys of BB3-treated animals exhibited reduced levels of kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, reduced tubular apoptosis and acute tubular necrosis but enhanced tubular regeneration. Consistent with its hepatocyte growth factor-like mode of action, BB3 treatment promoted phosphorylation of renal cMet and Akt and upregulated renal expression of the survival protein Bcl-2. These data suggest that the kidney is amenable to pharmacotherapy even 24 hr after ischemia-reperfusion, and that activation of the hepatocyte growth factor signaling pathway with the small molecule BB3 confers interventional benefit late into ischemia-reperfusion injury. These data, formed, in part, the basis for use of BB3 in a clinical trial in kidney recipients presenting with delayed graft function
This study had two objectives: 1) to determine whether preconditioning cultured proximal tubular cells (PTCs) with pharmacologic activators of AMPK protects these cells from apoptosis induced by metabolic stress in vitro and 2) to assess the effects of preconditioning mice with these agents on the severity of ischemic acute renal kidney injury (AKI) in vivo. We demonstrate that preconditioning PTCs with AICAR or A-769662 reduces apoptosis of PTCs induced by subsequent stress. We also show that the reduction in cell death during metabolic stress associated with pre-treatment AMPK activators, is associated with an increase in the cytosolic level of ATP, which is mediated by an increase in the rate of glycolysis. In addition, we provide evidence that the effect of AMPK activators on glycolysis is mediated, at least in part, by an increased uptake of glucose, and by the induction of hexokinase II (HK II) expression. Our data also show that the increased in HK II expression associated with preconditioning with AMPK activators is mediated by the activation (phosphorylation) of the cAMP-response element binding protein (CREB). We also provide entirely novel evidence that that A-79662 is substantially more effective than AICAR in mediating these alterations in PTCs in vitro. Finally, we demonstrate that preconditioning mice with AICAR or A-769662 substantially reduces the severity of renal dysfunction and tubular injury in a model of ischemic AKI in vivo, and that the efficacy of AICAR and A-768662 in ameliorating ischemic AKI in vivo is comparable.
Renal endothelin-1 (ET-1) and purinergic signaling systems regulate Na+ reabsorption in the renal medulla. A link between the renal (ET-1) and purinergic systems was demonstrated in-vitro, however, the in-vivo interaction between these systems has not been defined. To test whether renal medullary activation of purinergic (P2) receptors promotes ET-dependent natriuresis, we determined the effect of increased medullary NaCl loading on Na+ excretion and inner medullary ET-1 mRNA expression in anesthetized adult male Sprague Dawley rats in the presence and absence of purinergic receptor antagonism. Isosmotic saline (NaCl; 284 mOsmol/kg H2O) was infused into the medullary interstitium (500 μl/h) during a 30 min baseline urine collection period, followed by isosmotic or hyperosmotic saline (1800 mOsmol/kg H2O) for two further 30 min urine collection periods. Na+ excretion was significantly increased during intramedullary infusion of hyperosmotic saline. Compared with isosmotic saline, hyperosmotic saline infused into the renal medulla caused significant increases in inner medullary ET-1 mRNA expression. Renal intramedullary infusion of the purinergic (P2) receptor antagonist, suramin, inhibited the increase in Na+ excretion and inner medullary ET-1 mRNA expression induced by NaCl loading in the renal medulla. Activation of medullary purinergic (P2Y2/4) receptors by infusion of UTP increased urinary Na+ excretion. Combined ETA and ETB receptor blockade abolished the natriuretic response to intramedullary infusion of UTP. These data demonstrate that activation of medullary purinergic (P2) receptors promotes ET-dependent natriuresis in male rats, suggesting that the renal ET-1 and purinergic signaling systems interact to efficiently facilitate excretion of a NaCl load.
Human organic anion transporter 1 (hOAT1) expressed at the basolateral membrane of the kidney proximal tubule cells mediates the active renal secretion of a diverse array of clinically important drugs, including anti-HIV therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. We previously demonstrated that post-translational modification of hOAT1 by ubiquitination is an important mechanism for the regulation of this transporter. The current study aimed at identifying the ubiquitin ligase for hOAT1 and its mechanism of action. We showed that overexpression of Nedd4-1 (neural precursor cell expressed, developmentally down-regulated 4-1), an E3 ubiquitin ligase, enhanced hOAT1 ubiquitination, decreased hOAT1 expression at the cell surface, and inhibited hOAT1 transport activity. In contrast, overexpression of the ubiquitin ligase-dead mutant Nedd4-1/C867S was without effects on hOAT1. Furthermore, knockdown of endogenously expressed Nedd4-1 by Nedd4-1-specific siRNA reduced hOAT1 ubiquitination. Immunoprecipitation experiments in cultured cells and in rat kidney slices and immunofluorescence study in rat kidney slices showed that there was a physical interaction between OAT1 and Nedd4-1. Nedd4-1 contains four protein-protein interacting WW domains. When these WW domains were inactivated by mutating two amino acid residues in each of the four WW domains respectively (Mut-WW1: V210W/H212G; Mut-WW2: V367W/H369G; Mut-WW3: I440W/H442G; Mut-WW4: I492W/H494G), only Mut-WW2 and Mut-WW3 significantly lost their ability to bind and to ubiquitinate hOAT1. As a result, Mut-WW2 and Mut-WW3 were unable to suppress hOAT1-mediated transport as effectively as wild type Nedd4-1. In conclusion, this is the first demonstration that Nedd4-1 regulates hOAT1 ubiquitination, expression, and transport activity through its WW2 and WW3 domains.
Stress-induced activation of p38 MAPK and JNK signaling is a feature of both acute and chronic kidney disease and is associated with disease progression. Inhibitors of p38 MAPK or JNK activation provide protection against inflammation and fibrosis in animal models of kidney disease; however, clinical trials of p38 MAPK and JNK inhibitors in other diseases (rheumatoid arthritis and pulmonary fibrosis) have been disappointing. Apoptosis signal-regulating kinase 1 (ASK1) acts as an upstream regulator for the activation of p38 MAPK and JNK in kidney disease. Mice lacking the Ask1 gene are healthy with normal homeostatic functions and they are protected from acute kidney injury induced by ischemia-reperfusion and from renal interstitial fibrosis induced by ureteric obstruction. Recent studies have shown that a selective ASK1 inhibitor substantially reduced renal p38 MAPK activation and halted the progression of nephropathy in diabetic mice and this has led to a current clinical trial of an ASK1 inhibitor in patients with stage 3 or 4 diabetic kidney disease. This review explores the rationale for targeting ASK1 in kidney disease and the therapeutic potential of ASK1 inhibitors based on current experimental evidence.
Transactivation of EGF receptor (EGFR) by angiotensin II (Ang II) plays important roles in the initiation and progression of chronic kidney diseases. Studies suggest that heparin-binding EGF-like factor (HB-EGF) may be a critical mediator in this process, but its role in vivo has not been investigated. In the current study, we found that in response to Ang II-infusion, kidneys from endothelial HB-EGF deletion mice had significantly reduced EGFR activation compared to controls. Meanwhile, it decreased Ang II-infusion related renal injury, as demonstrated by 1) less albuminuria; 2) less glomerulosclerosis; 3) preserved endothelial integrity and decreased podocyte injury, as shown by greater glomerular tuft area and WT1 positive cells, and fewer apoptotic cells measured by cleaved caspase 3 staining; and 4), reduced Ang II-infusion induced renal inflammation as indicated by less infiltration of F4/80 positive macrophage and CD3 positive T lymphocytes and lower levels of proinflammatory cytokines, such as IL-6 and MCP-1 in both serum and kidney. In conclusion, our results suggest that shedding of HB-EGF from endothelium plays an important role in Ang II-induced renal injury by linking Ang II-AT1R with EGFR transactivation. Inhibition of HB-EGF shedding could be a potential therapeutic strategy for chronic kidney disease.
Acute kidney injury (AKI) is a common and independent risk factor for death and chronic kidney disease (CKD). Despite promising preclinical data, there is no evidence that anti-oxidants reduce the severity of injury, increase recovery, or prevent CKD in patients with AKI. Pyridoxamine (PM) is a structural analog of vitamin B6 that interferes with oxidative macromolecular damage via a number of different mechanisms, and is in a phase 3 clinical efficacy trial to delay CKD progression in patients with diabetic kidney disease. Since oxidative stress is implicated as one of the main drivers of renal injury after AKI, the ability of PM to interfere with multiple aspects of oxidative damage may be favorable for AKI treatment. In these studies we therefore evaluated PM treatment in a mouse model of AKI. Pretreatment with PM caused a dose-dependent reduction in acute tubular injury, long-term post-injury fibrosis, as well as improved functional recovery after ischemia-reperfusion AKI (IR-AKI). This was associated with a dose-dependent reduction in oxidative stress marker isofuran/F2-isoprostane ratio, indicating that PM reduces renal oxidative damage post-AKI. PM also reduced post-injury fibrosis when administered 24 hours after the initiating injury, but this was not associated with improvement in functional recovery after IR-AKI. This is the first report showing that treatment with PM reduces short and long-term injury, fibrosis and renal functional recovery after IR-AKI., These pre-clinical findings suggest that PM, which has favorable clinical safety profile, holds therapeutic promise for AKI and, most importantly, for prevention of adverse long-term outcomes after AKI.
There is an alarming global increase in the incidence of end-stage kidney disease, for which early biomarkers and effective treatment options are lacking. Largely based on the histology of the end-stage kidney and on the model of unilateral ureteral obstruction, current investigation is focused on the pathogenesis of renal interstitial fibrosis as a central mechanism in the progression of chronic kidney disease (CKD). It is now recognized that cumulative episodes of acute kidney injury (AKI) can lead to CKD and conversely, CKD is a risk factor for AKI. Based on recent and historic studies, this review shifts attention from the glomerulus and interstitium to the proximal tubule as the primary sensor and effector in the progression of CKD as well as AKI. Packed with mitochondria and dependent on oxidative phosphorylation, the proximal tubule is particularly vulnerable to injury (obstructive, ischemic, hypoxic, oxidative, metabolic), resulting in cell death and ultimately in the formation of atubular glomeruli. Animal models of human glomerular and tubular disorders have provided evidence for a broad repertoire of morphologic and functional responses of the proximal tubule, revealing processes of degeneration and repair that may lead to new therapeutic strategies. Most promising are studies that encompass the entire life cycle from fetus to senescence, recognizing epigenetic factors. The application of techniques in molecular characterization of tubule segments, and the development of human kidney organoids may provide new insights into the mammalian kidney subjected to stress or injury, leading to biomarkers of early CKD and new therapies.
Cisplatin, a wildly used chemotherapy drug, induces nephrotoxicity that is characterized by renal tubular cell apoptosis. In response to toxicity, tubular cells can activate cytoprotective mechanisms, such as heat shock response. However, the role and regulation of heat shock response in cisplatin-induced nephrotoxicity remain largely unclear. Here we demonstrate the induction of Heat shock factor 1 (Hsf1) and the small heat shock protein Crystallin-αB (CryAB) during cisplatin nephrotoxicity in mice. Consistently, cisplatin induced Hsf1 and CryAB in cultured renal tubular RPTC cells. RPTC cells underwent apoptosis during cisplatin treatment, which was increased when Hsf1 was knocked down. Transfection or restoration of Hsf1 into Hsf1-knockdown cells suppressed cisplatin-induced apoptosis, further supporting a cytoprotective role of Hsf1 and its associated heat shock response. Moreover, Hsf1 knockdown increased Bax translocation to mitochondria and cytochrome c release into cytosol. In RPTC cells, Hsf1-knockdown led to a specific down-regulation of CryAB. Transfection of CryAB into Hsf1-knockdown cells diminished their sensitivity to cisplatin-induced apoptosis, suggesting that CryAB may be a key mediator of the cytoprotective effect of Hsf1. Together, these results demonstrate a heat shock response in cisplatin nephrotoxicity that is mediated by Hsf1 and CryAB to protect tubular cells against apoptosis.
Acute kidney injury (AKI) dramatically increases mortality of hospitalized patients. Incidences have been increased in recent years. The most frequent cause is transient renal hypoperfusion or ischemia which induces significant tubular cell dyfunction / damage. In addition, two further events take place: interstitial inflammation and microvasculopathy (MV). The latter evolves within minutes to hours post-ischemia and may results in permanent deterioration of the peritubular capillary network, ultimately increasing the risk for chronic kidney disease (CKD) in the long-term. In recent years, our understanding of the molecular / cellular processes responsible for acute and sustained microvasculopathy has increasingly been expanded. The methodical approaches for visualizing impaired peritubular blood-flow and increased vascular permeability have been optimized, even allowing to depict tissue abnormalities in a three-dimensional manner. In addition, endothelial dysfunction, a hallmark of MV is increasingly been recognized as inductor of both, vascular malfunction and interstitial inflammation. In this regard, so-called regulated necrosis of the endothelium could potentially play a role in postischemic inflammation. Endothelial Progenitor Cells, represented by at least two major subpopulations have been shown to promote vascular repair in experimental AKI, not only in the short- but also in the long-term. The discussion about the true biology of the cells continues. It has been proposed that early EPCs are most likely myelomonocytic in nature, thus they may simply termed as proangiogenic cells (PACs). Nevertheless, the reliably protect certain types of tissues / organs from ischemia-induced damage, mostly by modulating the perivascular microenvironment in a direct manner. Aim of the current article is to summarize the current knowledge on postischemic MV and EPC-mediated renal repair.
Background: Postoperative renal failure is a common complication after open repair of an abdominal aneurysm. The amino-acid arginine is formed in the kidneys from its precursor citrulline, citrulline from glutamine in the intestines. Arginine enhances the function of the immune and cardiovascular system, which is important for recovery after and during surgery. We hypothesized that renal arginine production is diminished after ischemia-reperfusion injury by clamping of the aorta during open abdominal aortic surgery and that parenteral glutamine supplementation may compensate this impaired arginine synthesis. Methods: two groups of patients who underwent clamping of the aorta during open abdominal aortic surgery were randomized to this open label randomized clinical trial, to receive a perioperative supplement of intravenous alanyl-glutamine (0,5 g/kg/day) (group A, n=5) or no supplement (group B, n=5). Metabolism and conversion of glutamine, citrulline and arginine were analyzed using stable isotopes and tracer methodology. Results: Whole body metabolism of glutamine, citrulline and arginine was significantly higher in group A compared to B (p<0.01). Synthesis of citrulline from glutamine (p<0,01, 4,8±0,7 versus 1,6±0,3 μmol/kg/h) and citrulline from arginine (p<0,01, 2,3±0,3 versus 0,96±0,1 μmol/kg/h) and arginine from glutamine (p<0,001, 7,7±0,4 versus 2,8±0,2 μmol/kg/h) was significantly higher in group A compared to B. Conclusion: Production of citrulline and arginine is severely reduced after clamping during aortic surgery. This study shows that an intravenous supplement of glutamine increases the production of citrulline and arginine, and compensates for the inhibitory effect of ischemia-reperfusion injury.
In angiotensin II (Ang II) dependent hypertension, there is an AT1 receptor dependent amplification mechanism enhancing intrarenal angiotensinogen (AGT) formation and secretion into the tubular fluid. To evaluate the role of increased arterial pressure, AGT mRNA, protein expression, and urinary AGT (uAGT) excretion and tissue injury were assessed in both kidneys of 2-kidney, 1-clip (2K1C) Sprague Dawley hypertensive rats subjected to left renal arterial clipping (0.25 mm gap). By 18-21 days, systolic arterial pressure increased to 180±3 mmHg, and uAGT increased. Water intake, body weights, 24 hr urine volumes and sodium excretion were similar. In separate measurements of renal function in anesthetized rats, renal plasma flow and glomerular filtration rate (GFR) were similar in clipped and non-clipped kidneys and not different from those in sham rats indicating that the perfusion pressure to the clipped kidneys remained within the autoregulatory range. The non-clipped kidneys exhibited increased urine flow and sodium excretion. The uAGT excretion was significantly greater in non-clipped kidneys compared to clipped and sham kidneys. AGT mRNA was 2.15 fold greater in the non-clipped kidneys compared to sham (1.0±0.1) or clipped kidneys (0.98±0.15). AGT protein levels were also greater in the non-clipped kidneys. The non-clipped kidneys exhibited greater glomerular expansion and immune cell infiltration, medullary fibrosis, and cellular proliferation than the clipped kidneys. Because both kidneys have elevated Ang II levels, the greater tissue injury in the non-clipped kidneys indicates that an increased arterial pressure synergizes with increased intrarenal Ang II to stimulate AGT production and exert greater renal injury.
In male rats androgen supplements increase 20-hydroxyeicosatetraenoic acid (20-HETE) via cytochrome P450 (CYP)4A -hydroxylase and cause an increase in blood pressure (BP). In this study we determined the roles of 20-HETE and CYP4A2 on the elevated BP in hyperandrogenemic female rats. Chronic dihydrotestosterone (DHT) increased mean arterial pressure (MAP) in female Sprague Dawley rats (96±2 vs 108±2 mmHg; p<0.05), and was associated with increased renal microvascular CYP4A2 mRNA expression (15 fold), endogenous renal 20-HETE (5 fold), and -hydroxylase activity (3 fold). Chronic DHT also increased MAP in low salt fed Dahl R females (81±4 vs 95±1 mmHg, p<0.05), but had no effect on MAP Dahl S (154±3 vs 153±3 mmHg) that are known to be 20-HETE deficient. To test the role of CYP4A2, female CYP4A2-/- and SS.5Bn (WT) rats were treated with DHT. DHT increased MAP in SS.5Bn (104±1 vs 128±1 mmHg; p<0.05), but had no effect in CYP4A2-/- females (118±1 vs 120±1 mmHg). Renal microvascular 20-HETE was reduced in CYP4A2-/- control females, and was increased with DHT in SS.5Bn females (6 fold), but not CYP4A2-/- females. -Hydroxylase activity was 40% lower in control CYP4A2-/- females than SS.5Bn, and DHT decreased -hydroxylase activity in SS.5Bn (by 50%), but significantly increased -hydroxylase activity in CYP4A2-/- females (3 fold). These data suggest that 20-HETE via CYP4A2 contributes to the elevation in BP in hyperandrogenemic females. The data also suggest that 20-HETE synthesis inhibition may be effective in treating the elevated BP in women with hyperandrogenemia, such as women with polycystic ovary syndrome.
The Hippo signaling pathway is an evolutionarily conserved kinase cascade, playing multiple roles in embryonic development that controls organ size, cell proliferation and apoptosis. At the center of this network lie the Hippo kinase target and downstream pathway effector Yes-associated protein (YAP) and its paralog TAZ. In its phosphorylated form, cytoplasmic YAP is sequestered in an inactive state. When it is dephosphorylated, YAP a potent oncogene, is activated and relocates to the nucleus to interact with a number of transcription factors and signaling regulators that promote cell growth, differentiation and survival. The identification of YAP activation in human cancers has made it an attractive target for chemotherapeutic drug development. Little is known to date about the function of the Hippo pathway in the kidney but that is rapidly changing. Recent studies have shed light on the role of Hippo-YAP signaling in glomerular and lower urinary tract embryonic development, maintenance of podocyte homeostasis, the integrity of the glomerular filtration barrier, regulation of renal tubular cyst growth, renal epithelial injury in diabetes and renal fibrogenesis. This review summarizes the current knowledge of the Hippo-YAP signaling axis in the kidney under normal and disease conditions.
Estrogen treatment causes renal phosphate (Pi) wasting and hypophosphatemia in rats and humans; however the signaling mechanisms mediating this effect are still poorly understood. To determine the specific roles of estrogen receptor isoforms (ERα and ERβ) and Klotho pathway in mediating these effects, we studied the effects of estrogen on renal Pi handling in female mice with null mutations of ERα or ERβ or Klotho and their wild-type (WT) using balance studies in metabolic cages. Estrogen treatment of WT and ERβ KO mice caused a significant reduction in food intake along with increased renal phosphate wasting. The later resulted from a significant downregulation of NaPi-IIa and NaPi-IIc protein abundance. The mRNA expression levels of both transporters were unchanged in estrogen-treated mice. These effects on both food intake and renal Pi handling were abolished in ER KO mice. Estrogen treatment of Klotho KO mice or PTH-depleted thyro-parathyroidectomized mice exhibited a significant downregulation of NaPi-IIa with no change in the abundance of NaPi-IIc. Estrogen treatment of a cell line (U20S) stably co-expressing both ERα and ERβ caused a significant downregulation of NaPi-IIa protein, when transiently transfected with a plasmid containing full-length or ORF-3'UTR but not 5'UTR-ORF of mouse NaPi-IIa transcript. Conclusions: estrogen causes phosphaturia and hypophosphatemia in mice. These effects result from downregulation of NaPi-IIa and NaPi-IIc proteins in the proximal tubule through the activation of ERα. The downregulation of NaPi-IIa by estrogen involves 3'UTR of its mRNA and is independent of Klotho/fgf23 and PTH signaling pathways.
It is now recognized that the metabolic disorders observed in diabetes are not, or not only due to the lack of insulin or insulin resistance, but also to elevated glucagon secretion. Accordingly, selective glucagon receptor antagonists are now proposed as a novel strategy for the treatment of diabetes. However, besides its metabolic actions, glucagon also influences kidney function. The glucagon receptor is expressed in the thick ascending limb, distal tubule and collecting duct, and glucagon regulates the transepithelial transport of several solutes in these nephron segments. Moreover, it also influences solute transport in the proximal tubule, possibly by an indirect mechanism. This review summarizes the knowledge accumulated over the last 30 years about the influence of glucagon on the renal handling of electrolytes and urea. It also describes a possible novel role of glucagon in the short term regulation of potassium homeostasis. Several original findings suggest that pancreatic alpha-cells may express a "potassium sensor" sensitive to changes in plasma K concentration, and could respond by adapting glucagon secretion that, in turn, would regulate urinary K excretion. By their combined actions, glucagon and insulin, working in a combinatory mode, could ensure an independent regulation of both plasma glucose and plasma K concentrations. The results and hypotheses reviewed here suggest that the use of glucagon-receptor antagonists for the treatment of diabetes should take into account their potential consequences on electrolyte handling by the kidney.
This study investigated the role of the hypogastric nerve and β-adrenergic mechanisms in the inhibition of nociceptive and non-nociceptive reflex bladder activity induced by pudendal nerve stimulation (PNS). In α-chloralose anesthetized cats the non-nociceptive reflex bladder activity was induced by slowly infusing saline into the bladder, while the nociceptive reflex bladder activity was induced by replacing saline with 0.25% acetic acid (AA) to irritate the bladder. PNS was applied at multiple threshold (T) intensities for inducing anal sphincter twitching. During saline infusion, PNS at 2T and 4T significantly (p<0.01) increased bladder capacity to 184.7±12.6% and 214.5±10.4% of the control capacity. Propranolol (3 mg/kg, i.v.) had no effect on PNS inhibition, but MTEP (1-3 mg/kg, i.v.) significantly (p<0.05) reduced the inhibition. During AA irritation, the control bladder capacity was significantly (p<0.05) reduced to about 22% of saline control capacity. PNS at 2T and 4T significantly (p<0.01) increased bladder capacity to 406.8±47% and 415.8±46% of the AA control capacity. Propranolol significantly (p<0.05) reduced the bladder capacity to 276.3%±53.2% (at 2T PNS) and 266.5±72.4% (at 4T PNS) of AA control capacity, while MTEP removed the residual PNS inhibition. Bilateral transection of the hypogastric nerves produced an effect similar to that produced by propranolol. This study indicates that the hypogastric nerve and a β-adrenergic mechanism in the detrusor play an important role in PNS inhibition of nociceptive but not non-nociceptive reflex bladder activity. In addition to this peripheral mechanism, a CNS mechanism involving metabotropic glutamate 5 receptors also has a role in PNS inhibition.
Alpha-1-microglobulin (A1M) is a low molecular weight heme-binding, anti-oxidant protein that is readily filtered by the glomerulus and reabsorbed by proximal tubules. Given these properties, recombinant A1M (rA1M) has been proposed as a renal antioxidant and therapeutic agent. However, little direct evidence to support this hypothesis exists. Hence, we have sought 'proof of concept' in this regard. Cultured proximal tubule (HK-2) cells, or isolated mouse proximal tubule segments, were challenged with a variety of pro-oxidant insults: i) hemin; ii) myoglobin; iii) 'catalytic' iron; iv) H2O2/Fenton reagents; v) Ca2+ ionophore, vi) antimycin A; or vii) hypoxia (with/without rA1M treatment). HK-2 injury was gauged by %LDH release and MTT uptake. In vivo protection was sought in rA1M-treated mice subjected to: i) graded myohemoglobinura (2, 4, 8, or 9 ml/Kg glycerol injection); ii) purified myoglobinemia/uria; or iii) endotoxemia. In vivo injury was assessed by BUN, creatinine, and the expression of redox sensitive genes (HO-1, NGAL, MCP-1 mRNAs). Although rA1M totally blocked in vitro hemin toxicity, equimolar albumin (another heme binder), or 10% serum, induced equal protection. rA1M failed to mitigate any non-hemin forms of either in vitro or in vivo injury. A1M appeared to be rapidly degraded within proximal tubules (Western blotting). Surprisingly, rAIM exerted select injury-promoting effects (increased in vitro catalytic iron / antimycin toxicities; increased in vivo MCP-1/NGAL mRNA expression following glycerol or endotoxin injection). We conclude that rA1M has questionable utility as a renal antioxidant/cytoprotective agent, particularly in the presence of larger amounts of competitive free heme (e.g. albumin) binders.
Inflammasomes are supramolecular structures that sense molecular patterns from pathogenic organisms or damaged cells and trigger an innate immune response, most commonly through production of the pro-inflammatory cytokine IL-1β and IL-18, but also through less understood mechanisms independent of these cytokines. Great strides have been made in understanding these structures and their dysfunction in various inflammatory diseases, lending new insights into urological and renal problems. From a clinical perspective, benign urinary pathology almost universally involves the inflammatory process and understanding how inflammasomes translate etiological conditions (diabetes, obstruction, stones, UTIs, etc.) into acute and chronic inflammatory responses is critical to understanding these diseases at a molecular level. To date, inflammasome components have been found in the bladder, prostate, and kidney and have been shown to be activated in response to several infectious and non-infectious insults. In this review we summarize what is known regarding inflammasomes in both the upper and lower urinary tract and describe several common disease states where they potentially play critical roles.
Placental insufficiency programs an increase in blood pressure associated with a two-fold increase in serum testosterone in male growth-restricted offspring at 4 months of age. Population studies indicate that the inverse relationship between birth weight and blood pressure is amplified with age. Thus, we tested the hypothesis that intrauterine growth restriction programs an age-related increase in blood pressure in male offspring. Growth-restricted offspring retained a significantly higher blood pressure at 12 but not at 18 months of age compared to age-matched controls. Blood pressure was significantly increased in control offspring at 18 months of age relative to control counterparts at 12 months; however, blood pressure was not increased in growth-restricted at 18 months relative to growth-restricted counterparts at 12 months. Serum testosterone levels were not elevated in growth-restricted offspring relative to control at 12 months of age. Thus, male growth-restricted offspring no longer exhibited a positive association between blood pressure and testosterone at 12 months of age. Unlike hypertension in male growth-restricted offspring at 4 months of age, inhibition of the renin angiotensin system with enalapril (250 mg/L for 2 weeks) did not abolish the difference in blood pressure in growth-restricted offspring relative to control counterparts at 12 months of age. Therefore, these data suggest that intrauterine growth restriction programs an accelerated age-related increase in blood pressure in growth-restricted offspring. Furthermore, this study suggests that the etiology of increased blood pressure in male growth-restricted offspring at 12 months of age differs from that at 4 months of age
Systemic fibrosis from gadolinium-based magnetic resonance imaging contrast is a scourge for the afflicted. Although gadolinium-associated systemic fibrosis is a rare condition, the threat of litigation has vastly altered clinical practice. Most theories concerning the etiology of the fibrosis are grounded in case reports rather than experiment. This has led to the widely-accepted conjecture that the relative affinity of certain contrast agents for the gadolinium ion inversely correlates with the risk of succumbing to the disease. How gadolinium-containing contrast agents trigger widespread and site-specific systemic fibrosis and how chronicity is maintained is largely unknown. This review highlights experimentally-derived information from our laboratory and others that pertain to our understanding of the pathophysiology of gadolinium-associated systemic fibrosis.
The availability of oxygen in renal tissue is determined by the complex interactions among a host of processes, including renal blood flow, glomerular filtration, arterial-to-venous oxygen shunting, medullary architecture, Na$^+$ transport, and oxygen consumption. When this delicate balance is disrupted, the kidney may become susceptible to hypoxic injury. Indeed, renal hypoxia has been implicated as one of the major causes of acute kidney injury and chronic kidney diseases. This review highlights recent advances in our understanding of renal hypoxia; some of these studies were published in response to a recent Call for Papers of this journal: Renal Hypoxia.
Alport syndrome is a familial kidney disease caused by defects in the collagen IV network of the glomerular basement membrane. The lack of collagen α3α4α5(IV) changes the glomerular basement membrane morphologically and functionally, rendering it leaky to albumin and other plasma proteins. Filtered albumin has been suggested to be a cause of the glomerular and tubular injuries observed at advanced stages of Alport syndrome. To directly investigate the role albumin plays in progression of disease in Alport syndrome, we generated albumin knockout (Alb-/-) mice to use as a tool for removing albuminuria as a component of kidney disease. Mice lacking albumin were healthy and indistinguishable from control littermates, although they developed hypertriglyceridemia. Dyslipidemia was observed in Alb+/- mice, which displayed half the normal plasma albumin concentration. Alb mutant mice were bred to Col4a3-/- mice, which are a model for human Alport syndrome. The lack of circulating and filtered albumin in Col4a3-/-;Alb-/- mice resulted in dramatically improved kidney disease outcomes, as these mice lived 64% longer than did Col4a3-/-;Alb+/+ and Col4a3-/-;Alb+/- mice, despite similar blood pressures and serum triglyceride levels. Further investigations showed that the absence of albumin correlated with reduced TGFβ1 signaling as well as reduced tubulointerstitial, glomerular, and podocyte pathology. We conclude that filtered albumin is injurious to kidney cells in Alport syndrome and perhaps in other proteinuric kidney diseases, including diabetic nephropathy.
The role of 5-HT1A receptors in regulating voiding functions remains unclear, particularly regarding the urine flow rate (UFR) during voiding. This study examined the effects of 5-HT1A receptors on regulating urethral functions in female rats, and investigated underlying modulatory mechanisms. Intravesical pressure (IVP), external urethral sphincter-electromyography (EUS-EMG), and UFR were simultaneously recorded during continuous transvesical infusion to examine the effects of a 5-HT1A receptor agonist (8-OH-DPAT) and antagonist (WAY-100635) on bladder and urethral functions. In addition, this study evaluated the independent roles of urethral striated and smooth muscles in the UFR in rats after the neuromuscular blockade (NMB) treatment and the bilateral hypogastric nerve transection. Our results revealed that 8-OH-DPAT significantly increased the maximal UFR, but reduced the mean UFR. This discrepancy may be because 8-OH-DPAT markedly increased the maximal UFR during the initial segment of the flow duration and subsequently induced an approximately zero level of long oscillatory waves during the remaining flow duration. Thus, the mean UFR was reduced because of the prolonged approximately zero level of the UFR. However, after paralyzing the EUS with an NMB agent, 8-OH-DPAT significantly increased the maximal and mean UFRs because the prolonged zero level of the oscillatory UFR did not continue. These results support the hypothesis that the increased UFR in female rats during voiding is due to the induction of the urethral smooth muscle relaxation by 8-OH-DPAT. This paper provides a detailed understanding of the role of 5-HT1A receptors in controlling the UFR in female rats.
Activators of soluble guanylyl cyclase (sGC) interact directly with its prosthetic heme group, enhancing the enzyme responsiveness in pathological conditions. This study aimed to evaluate the effects of the sGC activator BAY 58-2667 on voiding dysfunction, protein expressions of α1 and β1 sGC subunits and cGMP levels in the bladder tissues after cyclophosphamide (CYP) exposure. Female C57BL/6 mice (20-25 g) were injected with CYP (300 mg/kg, i.p) to induce cystitis. Mice were pretreated or not with BAY 58-2667 (1 mg/kg, gavage), given 1 h prior to CYP injection. The micturition patterns and in vitro bladder contractions were evaluated at 24 h. In freely-moving mice, CYP injection produced reduced the micturition volume and increased the number of urine spots. Cystometric recordings in CYP-injected mice revealed significant increases of basal pressure, voiding frequency and non-voiding contractions (NVCs), along with decreases in bladder capacity, intercontraction interval and compliance. BAY 58-2667 significantly prevented the micturition alterations observed in both freely-moving mice and cystometry, and normalized the reduced in vitro carbachol-induced contractions in CYP group. Reduced protein expressions of α1 and β1 sGC subunits, and of cGMP levels were observed in CYP group, all of which were prevented by BAY 58-2667. CYP exposure significantly increased the reactive-oxygen species (ROS) generation in both detrusor and urothelium, and this was normalized by BAY 58-2667. The increased MPO and COX-2 activities in the bladders of CYP group remained unchanged by BAY 58-2667. Activators of sGC may constitute a novel and promising therapeutic approach for management of interstitial cystitis.
Kir.4.1 is an inwardly-rectifying potassium (K+) channel and is expressed in the brain, inner ear and kidney. In the kidney, Kir4.1 is expressed in the basolateral membrane of the late thick ascending limb (TAL), the distal convoluted tubule (DCT) and the connecting tubule (CNT)/ cortical collecting duct (CCD). It plays a role in K+ recycling across the basolateral membrane in corresponding nephron segments and in generating negative membrane potential. The renal phenotypes of the loss-function-mutations of Kir4.1 include mild salt wasting, hypomagnesemia, hypokalemia, and metabolic alkalosis, suggesting that the disruption of Kir.4.1 mainly impairs the transport in the DCT. Patch-clamp experiments and immunostaining demonstrate that Kir4.1 plays a predominant role in determining the basolateral K+ conductance in the DCT. However, the function of Kir4.1 in the TAL and CNT/CCD is not essential because K+ channels other than Kir4.1 are also expressed. The down-regulation of Kir.4.1 in the DCT reduced basolateral Cl- conductance, suppressed the expression of ste20 proline-alanine rich kinase (SPAK), and decreased NCC expression and activity. This suggests that Kir4.1 regulates NCC expression by the modulation of the Cl--sensitive with-No-Lysine Kinase (WNK)-SPAK pathway.
Diabetic nephropathy is the most common microvascular complication of diabetes mellitus, manifesting as mesangial expansion, glomerular basement membrane thickening, glomerular sclerosis and progressive tubulointerstitial fibrosis leading to end-stage renal disease. Here we describe the functional characterization of Wnt6, whose expression is progressively lost in diabetic nephropathy and animal models of acute tubular injury and renal fibrosis. We have shown prominent Wnt6 and FzD7 expression in the mesonephros of the developing mouse kidney, suggesting a role for Wnt6 in epithelialization. Importantly, TCF/Lef reporter activity is also prominent in the mesonephros. Analysis of Wnt family members in human renal biopsies identified differential expression of Wnt6, correlating with severity of the disease. In animal models of tubular injury and fibrosis, loss of Wnt6 was evident. Wnt6 signals through the canonical pathway in renal epithelial cells as evidenced by increased phosphorylation of GSK3β (Ser9), nuclear accumulation of β-catenin and increased TCF/Lef transcriptional activity. FzD7 was identified as a putative receptor of Wnt6. In vitro Wnt6 expression leads to de novo tubulogenesis in renal epithelial cells grown in 3D culture. Importantly, Wnt6 rescued epithelial cell dedifferentiation in response to TGF-β; Wnt6 reversed TGF-β-mediated increases in vimentin and loss of epithelial phenotype. Wnt6 inhibited TGF-β-mediated p65-NFB nuclear translocation, highlighting crosstalk between the two pathways. The critical role of NFB in the regulation of vimentin expression was confirmed in both p65-/- and IKKα/β-/- embryonic fibroblasts. We propose that Wnt6 is involved in epithelialization and loss of Wnt6 expression contributes to the pathogenesis of renal fibrosis.
Polarity signaling through the aPKC-Par polarity complex is essential for the development and maintenance of the podocyte architecture and the function of the glomerular filtration barrier of the kidney. To study the contribution of Par3A in this complex we generated a novel Pard3 podocyte-specific knockout mouse model by targeting exon six of the Pard3 gene. Genetic deletion of Par3A did not impair renal function neither at birth nor later in life. Even challenging the animals did not result in glomerular disease. Despite its well-established role in aPKC-mediated signaling Par3A appears to be dispensable for the function of the glomerular filtration barrier. Moreover, its homolog Par3B and not Par3A, is the dominant Par3 gene expressed in podocytes and found at the basis of the slit diaphragm where it partially colocalizes with podocin. In conclusion, Par3A function is either dispensable for slit diaphragm integrity or compensatory mechanisms and a high redundancy of the different polarity proteins including Par3B, Lgl or PALS1 maintain the function of the glomerular filtration barrier even in the absence of Par3A.
(Pro)renin receptor (PRR) is abundantly expressed in the collecting duct (CD) and the expression is further induced by angiotensin II (AngII). The present study was conducted to investigate the role of CD PRR during AngII-induced hypertension and to further explore the underlying mechanism. Radiotelemetry demonstrated that a 1-wk AngII infusion gradually and significantly induced hypertensive response in floxed mice and this response was significantly attenuated in mice lacking PRR in the CD (termed CD PRR KO). AngII infusion in floxed mice increased urinary renin activity and selectively induced renal medullary α-ENaC mRNA and protein expression, all of which were blunted in the null mice. In cultured mpkCCD cells grown in Transwells, transepithelial Na+ transport as measured by using volt-ohmmeter was transiently stimulated by acute AngII treatment, which was abolished by a PRR antagonist PRO20. In a chronic setting, AngII treatment induced α-ENaC mRNA expression in mpkCCD cells, which was similarly blocked by PRO20. Chronic intramedullary infusion of an ENaC inhibitor amiloride in rats significantly attenuated AngII-induced hypertension. Overall, the present study suggests that CD PRR contributes to AngII-induced hypertension at least partially via activation of renal medullary ENaC.
MAD2B, an anaphase-promoting complex/cyclosome (APC/C) inhibitor and a small subunit of DNA polymerase zeta, is indispensible for mitotic checkpoint control and DNA repair. Previously we established MAD2B is expressed in glomerular and tubulointerstitial compartments and participates in high glucose induced podocyte injury. But its role in other renal diseases remains elusive. In the present study we aim to illustrate the potential role of MAD2B in the pathogenesis of renal fibrosis. By immunofluorescence and western blot, we found MAD2B expression is obviously increased in tubulointerstitial fibrosis (TIF) patients and unilateral ureteral obstruction (UUO) mice. It is widely accepted that resident fibroblasts are the major source of collagen-producing myofibroblasts during TIF. Therefore, we evaluated the level of MAD2B in fibroblasts (NRK-49F) exposed to TGF-β1 by immunoblotting and revealed that MAD2B is upregulated in a time dependent manner. Intriguingly, SnoN, a transcriptional repressor of TGF-β1/Smad signaling pathway, is decreased in TGF-β1 treated fibroblasts as well as the kidney cortex from TIF patients and UUO mice. Either in vitro or in vivo locally genetic depletion of MAD2B by Lentiviral transfection could preserve SnoN abundance and suppress Smad3 phosphorylation which finally dampens the fibroblast activation, ECM accumulation and alleviates the severity of TIF. However ubiquitin ligase APC/C is not involved in MAD2B mediated SnoN decline, although this process is ubiquitination dependent. In conclusion, our observation proposes that besides cell cycle management MAD2B has a profibrotic role during fibroblast activation and TIF by suppressing SnoN expression. Targeting MAD2B-SnoN pathway is a promising intervention for TIF.
The solute carrier family 12, as numbered according to HUGO nomenclature, encodes the electroneutral cation-coupled chloride cotransporters that are expressed in many cells and tissues; they play key roles in important physiological events, such as cell volume regulation, modulation of the intracellular chloride concentration and transepithelial ion transport. Most of these family members are expressed in specific regions of the nephron. The Na+:K+:2Cl- cotransporter NKCC2 in the thick ascending limb and the Na+:Cl- cotransporter in the distal convoluted tubule play a key role in salt reabsorption and serve as the receptors for loop and thiazide diuretics, respectively, which are among the most commonly prescribed drugs in the world. The activity of these transporters correlates with blood pressure levels; thus, their regulation has been a subject of intense research for more than a decade. The K+:Cl- cotransporters KCC1, KCC3, and KCC4 are expressed in several nephron segments, and their role in renal physiology is less understood but nevertheless important. Evidence suggests that they are involved in modulating proximal tubule glucose reabsorption, thick ascending limb salt reabsorption and collecting duct proton secretion. In this work, we present an overview of the physiological roles of these transporters in the kidney, with particular emphasis on the knowledge gained in the last few years.
Purinoceptors (ARs and P2Rs) are expressed on the cellular components of the glomerular filtration barrier and their activation may affect glomerular permeability to albumin, which may ultimately lead to albuminuria, a well-established risk factor for progression of chronic kidney disease and the development of cardiovascular diseases. We have investigated mechanisms underlying the in vitro and in vivo purinergic action on glomerular filter permeability to albumin, measuring convectional albumin permeability (Palb) in a single isolated rat glomerulus based on the video-microscopy method. Primary cultured rat podocytes were used for analysis of albumin permeability, cGMP accumulation, PKG Iα dimerization and immunofluorescence. In vitro, natural nucleotides (ATP, ADP, UTP, UDP) and non-metabolized ATP analogues (2-meSATP, ATP--S) increased Palb in a time- and concentration-dependent manner. The effects were dependent on P2Rs activation, nitric oxide synthase and cytoplasmic guanylate cyclase. ATP analogues significantly increased the permeability to albumin, cGMP accumulation and sub-cortical actin reorganization in a PKG-dependent but non dimmer-mediated route in cultured podocytes. In vivo, 2-meSATP and ATP--S increased Palb but did not significantly affect urinary albumin excretion. Both agonists enhanced the clathrin-mediated endocytosis of albumin in podocytes. A product of adenine nucleotides hydrolysis, adenosine, increased the permeability of the glomerular barrier via ARs in a dependent and independent manner. Our results suggest that the extracellular nucleotides that stimulate an increase of glomerular permeability to albumin involve nitric oxide synthase and cytoplasmic guanylate cyclase with actin reorganization in podocytes.
The physiological significance of the renal tubular prorenin receptor (PRR) has been difficult to elucidate due to developmental abnormalities associated with global or renal-specific PRR knockout (KO). We recently developed an inducible renal tubule-wide PRR KO using the Pax8/LC1 transgenes and demonstrated that disruption of renal tubular PRR at 1 month of age caused no renal histological abnormalities. Here, we examined the role of renal tubular PRR in blood pressure (BP) regulation and Na+ excretion and investigated the signaling mechanisms by which PRR regulates Na+ balance. No detectable differences in BP were observed between control and PRR KO mice fed normal or low Na+ diets. However, compared to controls, PRR KO mice had elevated plasma renin concentration and lower cumulative Na+ balance with normal and low Na+ intake. PRR KO mice had an attenuated hypertensive response and reduced Na+ retention following angiotensin-II infusion. Further, PRR KO mice had significantly lower epithelial Na+ channel (ENaC-α) expression. Treatment with mouse prorenin increased, while PRR antagonism decreased, ENaC activity in isolated split-open collecting ducts (CD). The prorenin effect was prevented by protein kinase A and Akt inhibition, but unaffected by blockade of AT-1, ERK1/2 or p38 MAPK pathways. Taken together, these data indicate that renal tubular PRR, likely via direct prorenin/renin stimulation of PKA/Akt-dependent pathways, stimulates CD ENaC activity. Absence of renal tubular PRR promotes Na+ wasting and reduces the hypertensive response to Ang-II.
The initial step in renal secretion of organic anions (OA) is mediated by transporters in the basolateral membrane (BLM). Contributors to this process are the primary-active Na+/K+-ATPase (EC 3.6.3.9), the secondary-active sodium-dicarboxylate cotransporter 3 (NaDC3/SLC13A3), and the tertiary-active OA transporters OAT1/SLC22A6, OAT2/SLC22A7 and OAT3/SLC22A8. In human kidneys we analyzed the localization of these transporters by immunochemical methods in tissue cryosections and isolated membranes. The specificity of antibodies was validated with HEK293 cells stably transfected with functional OA transporters. The Na+/K+-ATPase was immunolocalized to the BLM along the entire human nephron. NaDC3-related immunostaining was detected in the BLM of proximal tubules and in the BLM and/or luminal membrane of principal cells in connecting segments and collecting ducts. The thin and thick ascending limbs, macula densa, and distal tubules exhibited no reactivity with the anti-NaDC3 antibody. In OAT1-3-related immunostaining in human kidneys was detected only in the BLM of cortical proximal tubules; all three OATs were stained more intensely in S1/S2 segments as compared to S3 segment in medullary rays, whereas the S3 segment in outer stripe remained unstained. The expression of NaDC3, OAT1, OAT2, and OAT3 proteins exhibited considerable interindividual variability in both male and female kidneys, and sex differences in their expression could not be detected. Our studies provide a side-by-side comparison of basolateral transporters co-operating in renal OA secretion in the human kidney.
The stimulation of postprandial K+ clearance involves aldosterone-independent and -dependent mechanisms. In this context, SGK1, an ubiquitously expressed kinase, is one of the primary aldosterone induced proteins in the aldosterone sensitive distal nephron (ASDN). Germline inactivation of SGK1 suggests that this kinase is fundamental for K+ excretion under conditions of K+ load, but the specific role of renal SGK1 remains elusive. To avoid compensatory mechanisms that may occur during nephrogenesis, we employed inducible, nephron-specific Sgk1Pax8/LC1 mice to assess the role of renal-tubular SGK1 in K+ regulation. Under standard diet, these animals exhibited normal K+ handling. When challenged by a high K+ diet (HKD), they developed severe hyperkalemia, accompanied by a defect in K+ excretion. Molecular analysis revealed reduced NEDD4-2 phosphorylation and total expression. ENaC expression and α/ENaC proteolytic processing were also decreased in the mutant mice. Moreover, WNK1, which displayed in control mice punctuate staining in the DCT and diffuse distribution in the CNT/CCD, was diffused in the DCT and less expressed in the CNT/CD of SgkPax8/LC1 mice. Besides, SPAK phosphorylation, and NCC phosphorylation/apical localization were reduced in the mutant. Consistent with the altered WNK1 expression, increased ROMK apical localization was observed. In conclusion, our data suggest that renal-tubular SGK1 is important in the regulation of K+ excretion via the control of NEDD4-2, WNK1 and ENaC.
Increased O-linked ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) is a known contributor to diabetes; however its relevance in diabetic nephropathy is poorly elucidated. Here we studied the process and enzymes of O-GlcNAcylation with a special emphasis on Akt-endothelial nitric oxide synthase (eNOS) and heat shock protein 72 (HSP72) signaling. Since tubular injury is the prominent site of diabetic nephropathy the effect of hyperglycemia was first measured in proximal tubular (HK-2) cells cultured in high glucose. The in vivo O-GlcNAcylation and protein levels of O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), pAkt/Akt, peNOS/eNOS and HSP72 were assessed in streptozotocin-induced diabetic rat model. The effects of various renin-angiotensin-aldosterone system (RAAS)-inhibitors were also evaluated. In proximal tubular cells hyperglycemia-induced OGT expression led to increased O-GlcNAcylation which was followed by a compensatory increase of OGA. In parallel peNOS and pAkt levels decreased, while HSP72 increased. In diabetic rats elevated O-GlcNAcylation was accompanied by decreased OGT and OGA. RAAS-inhibitors ameliorated diabetes-induced kidney damage; prevented the elevation of O-GlcNAcylation and the decrement of pAkt, peNOS and HSP72. In conclusion hyperglycemia-induced elevation of O-GlcNAcylation contributes to the progression of DN via inhibition of Akt/eNOS phosphorylation and HSP72 induction. RAAS-blockers successfully inhibit this process suggesting a novel pathomechanism of their renoprotective action in the treatment of DN.
Overactive bladder (OAB) is a common debilitating bladder condition with unknown etiology and limited diagnostic modalities. Here, we explored a novel high throughput, and unbiased multiplex approach with cellular and molecular components in a well characterized patient cohort to identify biomarkers that could be reliably used to distinguish OAB from controls or provide insights into underlying etiology. As a secondary analysis, we determined if this method could discriminate between OAB and other chronic bladder conditions. We analyzed plasma samples from healthy volunteers (n=19) and patients diagnosed with OAB, IC/BPS, or urinary tract infections (UTI) (n= 51) for pro-inflammatory, chemokine, cytokine, angiogenesis, and vascular injury factors using Meso Scale Discovery (MSD) analysis and urinary cytological analysis. Wilcoxon rank-sum tests were used to perform univariate and multivariate comparisons between patient groups (controls, OAB, IC/BPS, and UTI). Multivariate logistic regression models were fit for each MSD analyte on 1) OAB patients and controls, 2) OAB and IC/BPS patients, and 3) OAB and UTI patients. Age, race, and sex were included as independent variables in all multivariate analysis. Receiver operating characteristic (ROC) curves were generated to determine the diagnostic potential of a given analyte. Our findings demonstrate that five analytes, interleukin 4, tumor necrosis factor alpha, macrophage inflammatory protein-1β, serum amyloid A, and Tie-2 can reliably differentiate OAB relative to controls and can be used to distinguish OAB from the other conditions. Together, our pilot study suggests a molecular imbalance in inflammatory proteins may contributes to OAB pathogenesis.
The With no Lysine [K] (WNK) family of enzymes are central in regulation of blood pressure. WNKs have been implicated in hereditary hypertension disorders, mainly through control of the activity and levels of ion cotransporters and channels. Actions of WNKs in the kidney have been heavily investigated, and recent studies have provided insight not only into the regulation of these enzymes but also how mutations in WNKs and their interacting partners contribute to hypertensive disorders. Defining the roles of WNKs in in the cardiovascular system will provide clues into additional mechanisms by which WNKs can regulate blood pressure. This review summarizes recent developments in the regulation of the WNK signaling cascade and its role in regulation of blood pressure.
Injectable hydrogels can be used to deliver drugs in situ over a sustained period of time. We hypothesized that sustained delivery of interleukin-10 (IL-10) following acute kidney injury (AKI) would mitigate the local and systemic pro-inflammatory cascade induced by AKI and reduce subsequent fibrosis. Wild-type C57BL/6 mice underwent ischemia-reperfusion AKI with avertin anesthesia. Three days later, mice were treated with either hyaluronic acid injectable hydrogel with or without IL-10, or IL-10 suspended in saline, injected under the capsule of the left kidney, or hydrogel with IL-10 injected subcutaneously. Untreated AKI served as controls. Serial in vivo optical imaging tracked the location and degradation of the hydrogel over time. Kidney function was assessed serially. Animals were sacrificed 28 days following AKI and the following were evaluated: serum IL-6, lung inflammation, urine NGAL and renal histology for fibroblast activity, collagen type III deposition and fibrosis. Our model shows systemic inflammation, and renal inflammation and fibrosis 28 days following AKI. The hydrogels are biocompatible and reduced serum interleukin-6 and renal collagen type III 28 days following AKI even when delivered without IL-10. Treatment with IL-10 reduced renal and systemic inflammation, regardless of whether the IL-10 was delivered in a sustained manner via the injectable hydrogel under the left kidney capsule, as a bolus injection via saline under the left kidney capsule, or via the injectable hydrogel subcutaneously. Injectable hydrogels are suitable for local drug delivery following renal injury, are biocompatible, and help mitigate local and systemic inflammation.
BACKGROUND AND PURPOSE: Attributing to their anti-proliferative effect, both rapamycin and PPAR could halt the progression of ADPKD. Whether combined use can enhance effect is unknown. EXPERIMENTAL APPROACH: The present study used rapamycin and the PPAR agonist rosiglitazone concomitantly to observe their effects on the proliferation of ADPKD cyst-lining epithelial cells and the progression of ADPKD in Han:SPRD rats. KEY RESULTS: Concomitant use of the two drugs inhibited the proliferation of WT9-12 cells significantly through a superimposition effect. Rosiglitazone inhibited the phosphorylation of mTOR target p70S6K. Concomitant use of rosiglitazone and rapamycin further down-regulated the p-p70S6K level. Rosiglitazone also inhibited the phosphorylation of Akt and antagonize the activation of Akt induced by rapamycin. Concomitant use of rosiglitazone and rapamycin significantly retarded the deterioration of renal function, decreased cyst cell proliferation and interstitial fibrosis in Han: SPRD rats. Rapamycin significantly increased cholesterol levels in the blood, whereas rosiglitazone mitigated rapamycin-induced hyperlipidemia. CONCLUSION AND IMPLICATIONS: These results indicate that the effects of concomitant use of rosiglitazone and rapamycin in inhibiting the proliferation of WT9-12 cells and delaying the progression of ADPKD in Han:SPRD rats are stronger than those of either drug alone. The present study may provide a new strategy for the long-term treatment of ADPKD.
Systemic lupus erythematosus (SLE) is a serious disorder of immune regulation characterized by over-production of autoantibodies, lupus nephritis, CD4+ T cell abnormal activation, and immune complex-mediated inflammation. Chronic graft versus host disease (cGVHD) mouse model is a well-established model of SLE. Quercitrin is a natural compound found in Tartary buckwheat with a potential anti-inflammation effect that is used to treat heart and vascular conditions. In our previous study, we determined that quercitrin is an immunosuppressant with beneficial effects in mouse models of immune diseases. We hypothesized that quercitrin could prevent lupus nephritis in the cGVHD mouse model by decreasing the production of autoantibodies and inflammatory cytokines, and reducing immune cell activation. cGVHD was induced by injecting DBA/2 spleen cells into the tail vein of BDF1 mice. The cGVHD mice exhibited a significant proteinuria, which is a marker of nephritis. Quercitrin decreased the amount of serum antibodies, CD4+ T cell activation, as well as the expression levels of T-bet, GATA-3, and selected cytokines. Moreover, quercitrin treatment decreased the expression of inflammatory genes and cytokines in the kidney, as well as in peritoneal macrophages. In addition, quercitrin inhibited LPS-induced cytokines as well as the phosphorylation of extracellular-signal regulated kinase, p38 MAPK and c-Jun N-terminal kinase in Raw264.7 cells. Overall, quercitrin ameliorated the symptoms of lupus nephritis in the cGVHD mouse model, which may be due to the inhibition of CD4 T cell activation and anti-inflammatory effects on macrophages.
The intrarenal renin-angiotensin system (RAS) has an important role in generating and maintaining hypertension in 2-kidney 1-clip (2K1C) rats. This study evaluated how various intrarenal RAS components contributed to hypertension not only in maintenance time (5w; 5 week after operation) but also in early time (2w; 2week after operation). We inserted a 2.5mm-sized clip into the left renal artery of the Sprague-Dawley rats and sacrificed them at 2w and 5w following operation. Systolic blood pressure increased within one week after operation and left ventricular hypertrophy was occurred in 2K1C rats. At 2w, juxtaglomerular apparatus (JGA) and collecting duct (CD) renin increased in CK. The tubular angiotensin I-converting enzyme (ACE) was not changed, but peritubular ACE2 decreased in NCK and CK. At 5w, ACE and CD rein were enhanced, and ACE2 was still lessened in both kidneys of 2K1C rat. However, plasma renin activity (PRA) was not different with that of sham rat. In proximal tubule of CK, AngII type 1 receptor (AT1R) was not suppressed, but Mas receptor (MasR) was reduced; thus, the AT1R/MasR ratio was elevated. Although hypoxic change of CK could not be excluded, the JGA renin of CK and CD renin of both kidneys was highly expressed independent of time. Peritubular ACE2 changed in early time and uninhibited AT1R in proximal tubule of CK was presented in maintenance time. In 2K1C rat, attenuated ACE2 seems to contribute to initiating hypertension while up-regulated ACE in combination with un-suppressed AT1R may have a key role in maintaining hypertension.
Klotho, a protein counteracting aging, is a powerful inhibitor of 1,25(OH)2D3 formation and regulator of mineral metabolism. In klotho-hypomorphic mice (kl/kl) excessive 1,25(OH)2D3 formation leads to hypercalcemia, hyperphosphatemia and vascular calcification, severe growth deficit, accelerated aging and early death. The kl/kl mice further suffer from extracellular volume depletion and hypotension, leading to stimulation of ADH and aldosterone release. Vitamin-D-deficient diet, restriction of dietary phosphate, inhibition of mineralocorticoid receptors with spironolactone and dietary NaCl all extend the life span of kl/kl mice. Kl/kl mice suffer from acidosis. The present study explored whether replacement of tap drinking water by 150 mM NaHCO3 affects growth, tissue calcification and life span of kl/kl mice. As a result, NaHCO3 administration to kl/kl mice did not reverse the growth deficit but substantially decreased tissue calcification and significantly increased the average life span from 78 days to 127 days. NaHCO3 did not significantly affect plasma concentrations of 1,25(OH)2D3 and Ca2+, but significantly decreased plasma phosphate concentration and plasma aldosterone concentration. The present study reveals a novel effect of bicarbonate, i.e. a favorable influence on vascular calcification and early death of klotho-deficient mice.
Chronic kidney disease is associated with vasculitis and is also an independent risk factor for peripheral vascular and coronary artery disease in Diabetic patients. Despite optimal management, a significant number of patients progress towards end-stage renal disease (ESRD), a suggestion that the disease mechanism is far from clear. A reduction in hydrogen sulfide (H2S) has been suggested to play a vital role in diabetic vascular complications including diabetic nephropathy (DN). This mini review highlights the recent findings on the role of hydrogen sulfide (H2S) in mitigating abnormal extracellular matrix metabolism in DN. A discussion on the development of the newer slow releasing H2S compounds and its therapeutic potential is also included.
Secretion of organic cations (OCs) across renal proximal tubules (RPTs) involves basolateral OCT2-mediated uptake from the blood, followed by apical MATE1/2-mediated efflux into the tubule filtrate. Whereas OCT2 supports electrogenic OC uniport, MATE is an OC/H+ exchanger. As assessed by epifluorescence microscopy, cultured CHO cells that stably express hMATE1 accumulated the fluorescent OC, N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][l,2,5]oxadiazol-4-yl)amino]ethanaminium (NBD-MTMA) in the cytoplasm and in a smaller, punctate compartment; accumulation in hOCT2 expressing cells was largely restricted to the cytoplasm. A second intracellular compartment was also evident in the multicompartmental kinetics of efflux of the prototypic OC, [3H]MPP, from MATE1-expressing CHO cells. Punctate accumulation of NBD-MTMA was markedly reduced by coexposure of MATE1-expressing cells with 5 μM bafilomycin (BAF), an inhibitor of the V-Type H-ATPase, and accumulation of [3H]MPP and [3H]NBD-MTMA was reduced by >30% by coexposure with 5 μM BAF. BAF had no effect on the initial rate of MATE1-mediated uptake of NBD-MTMA suggesting that the influence of BAF was a secondary effect involving inhibition of the V-type H-ATPase. The accumulation of [3H]MPP by isolated single non-perfused rabbit RPTs was also reduced >30% by coexposure to 5 μM BAF, suggesting that the native expression in RPTs of MATE protein within endosomes can increase steady-state OC accumulation. However, the rate of [3H]MPP secretion by isolated single perfused rabbit RPTs was not affected by 5µM BAF, suggesting that vesicles loaded with OCs+ are not likely to recycle into the apical plasma membrane at a rate sufficient to provide a parallel pathway for OC secretion.
Renal artery stenosis is the main cause of renovascular hypertension and results in ischemic nephropathy characterized by inflammation, oxidative stress, microvascular loss and fibrosis with consequent functional failure. Considering the limited number of strategies that effectively control renovascular hypertension and restore renal function, we propose that cell therapy may be a promising option based on the regenerative and immunosuppressive properties of stem cells.. This review addresses the effects of mesenchymal stem cells (MSC) in an experimental animal model of renovascular hypertension known as 2 kidney-1 clip (2K-1C). Significant benefits of MSC treatment have been observed on blood pressure and renal structure of the stenotic kidney. The mechanisms involved are discussed.
Hydronephrosis is associated with development of salt-sensitive hypertension. Studies suggest that increased sympathetic nerve activity (SNA) and oxidative stress play important roles in renovascular hypertension. This study aimed to investigate the link between renal SNA and NADPH oxidase (NOX) regulation in the development of hypertension in rats with hydronephrosis. Hydronephrosis was induced by partial unilateral ureteral obstruction (PUUO) in young rats. Sham surgery or renal denervation was performed at the same time. Blood pressure was measured during normal, high and low salt diets. Renal excretion pattern, NOX activity and expression, as well as components of RAAS were characterized. On normal salt diet, PUUO rats had elevated blood pressure compared with controls (115±3 vs 87±1 mmHg), and displayed increased urine production and lower urine osmolality. Blood pressure change in response to salt loading (salt-sensitivity) was more pronounced in the PUUO group compared with controls (15±2 vs 5±1mmHg). Renal denervation in PUUO rats attenuated hypertension (97±3mmHg) and salt-sensitivity (5±1mmHg), and normalized renal excretion pattern, whereas the degree of renal fibrosis and inflammation was not changed. NOX activity and expression, as well as renin and AT1A receptor expression, were increased in renal cortex from PUUO rats, and normalized by denervation. Plasma sodium and potassium levels were elevated in PUUO rats and normalized after renal denervation. Denervation in PUUO rats was also associated with reduced NOX expression, superoxide production and fibrosis in the heart. This study emphasizes a link between renal nerves, NOX function, and development of hypertension.
MicroRNAs (miRNA) are endogenously produced short non-coding regulatory RNAs that can repress gene expression by posttranscriptional mechanisms. They can therefore influence both normal and pathological conditions in diverse biological systems. Several miRNAs have been detected in kidneys where they have been found to be crucial for renal development and normal physiological functions as well as significant contributors to the pathogenesis of renal disorders such as diabetic nephropathy, acute kidney injury, lupus nephritis, polycystic kidney disease, and others due to their effects on key genes involved in these disease processes. miRNAs have also emerged as novel biomarkers in these renal disorders. Due to increasing evidence of their actions in various kidney segments, in this mini-review, we discuss the functional significance of altered miRNA expression during the development of renal pathologies and highlight emerging miRNA-based therapeutics and diagnostic strategies for early detection and treatment of kidney diseases.
We showed previously that an association of losartan and hydrochlorothiazide, initiated one month after 5/6 nephrectomy (Nx), reversed hypertension and albuminuria and promoted lasting renoprotection. In this new study, we investigated whether equal or even better protection can be obtained by combining losartan and furosemide. Nx was performed in 58 Munich-Wistar rats. One month later, tail-cuff pressure and albuminuria were markedly elevated. At this time, Nx rats were distributed among four groups: Nx (untreated); NxL, receiving losartan; NxLH, receiving losartan and hydrochlorothiazide; and NxLF, given losartan and furosemide. Seven months later, Nx rats exhibited high mortality, severe hypertension, albuminuria, glomerulosclerosis and interstitial fibrosis. Losartan treatment limited mortality and attenuated the renal and hemodynamic abnormalities associated with Nx. As shown previously, the LH association normalized TCP and ALB, prevented renal injury, and reduced mortality to zero. The LF treatment failed to reduce TCP or ALB to normal, and prevented renal injury less efficiently than the LH regimen. The reasons for the differing efficacies of the LF and LH schemes are unclear, and may include beneficial nondiuretic actions of thiazides, such as vasorelaxation and antiproliferative activity. These results refute the established concept that thiazides and thiazide-like diuretics are ineffective at advanced CKD stages. Rather, they suggest that, in view of their renoprotective action, these compounds may even be preferable to loop diuretics in the management of hypertension in advanced CKD.
Pendrin is a Na+-independent Cl-/HCO3- exchanger found in the apical regions of type B and non-A, non-B intercalated cells within the aldosterone-sensitive region of the nephron, i.e. the distal convoluted tubule (DCT), the connecting tubule (CNT) and the cortical collecting duct (CCD). Type B intercalated cells mediate Cl- absorption and HCO3- secretion primarily through pendrin-mediated Cl-/HCO3- exchange. This exchanger is upregulated with angiotensin II administration and in models of metabolic alkalosis, such as following administration of aldosterone or NaHCO3. In the absence of pendrin-mediated HCO3- secretion, an enhanced alkalosis is observed following aldosterone or NaHCO3 administration. However, probably of more significance is the role of pendrin in the pressor response to aldosterone. Pendrin mediates Cl- absorption and modulates aldosterone-induced Na+ absorption mediated by ENaC. Pendrin changes ENaC channel activity by changing both channel open probability (Po) and surface density (N), at least partly by altering luminal HCO3- and ATP concentration. Thus, aldosterone and angiotensin II stimulate pendrin expression and function, which stimulates ENaC activity, thereby contributing to the pressor response of these hormones. However, pendrin may modulate blood pressure partly through its extra-renal effects. For example, pendrin is expressed in the adrenal medulla, where it modulates catecholamine release. The increase in catecholamine release observed with pendrin gene ablation, likely contributes to the increment in vascular contractile force observed in the pendrin null mouse. This review summarizes the signaling mechanisms that regulate pendrin abundance and function as well as the contribution of pendrin to distal nephron function.
Nephrolithiasis is one of the most common urinary tract disorders with the majority of kidney stones composed of calcium oxalate (CaOx). Given its prevalence (US occurrence 10%), it is still poorly understood, lacking progress in identifying new therapies because of its complex etiology. Drosophila melanogaster (fruitfly) is a recently developed model of CaOx nephrolithiasis. Effects of sulfate and thiosulfate on crystal formation were investigated using the Drosophila model, as well as electrophysiological effects on both Drosophila (Slc26a5/6; dprestin) and mouse (mSlc26a6) oxalate transporters utilizing the Xenopus oocyte heterologous expression system. Results indicate that both transport thiosulfate with a much higher affinity than sulfate Additionally, both compounds were effective at decreasing CaOx crystallization when added to the diet. However, these results were not observed when compounds were applied to MTs ex vivo. Neither compound affected CaOx crystallization in dPrestin knock down animals indicating a role for principal cell-specific dPrestin in luminal oxalate transport. Furthermore,thiosulfate has a higher affinity for dPrestin and mSlc26a6 compared to oxalate These data indicate that thiosulfate's ability to act as a competitive inhibitor of oxalate via dPrestin, can explain the decrease in CaOx crystallization seen in the presence of thiosulfate, but not sulfate. Overall, our findings predict that thiosulfate or oxalate-mimics may be effective as therapeutic competitive inhibitors of CaOx crystallization.
The renal thiazide-sensitive NaCl cotransporter, NCC, is the major pathway for salt reabsorption in the distal convoluted tubule. The activity of this cotransporter is critical for regulation of several physiological variables such as blood pressure, serum potassium, acid base metabolism, and urinary calcium excretion. Therefore, it is not surprising that numerous hormone signaling pathways regulate NCC activity to maintain homeostasis. In this review we will provide an overview of the most recent evidence on NCC modulation by aldosterone, angiotensin II, vasopressin, glucocorticoids, insulin, norepinephrine, estradiol, progesterone, prolactin and parathyroid hormone.
A variety of macromolecules accumulate in the glomerular mesangium in many different diseases, but the physics of the transport of these molecules within the mesangial matrix has not been extensively studied. We present a computational model of convection and diffusion within the porous mesangial matrix, and apply this model to the specific instance of immunoglobulin A (IgA) transport in IgA nephropathy. We examine the influence of physiological factors including glomerular basement membrane (GBM) thickness and mesangial matrix density on the total accumulation of IgA. Our results suggest that IgA accumulation can be understood by relating convection and diffusion, thus demonstrating the importance of intrinsic glomerular factors.
Flow-induced K+ secretion in the aldosterone sensitive distal nephron is mediated by high conductance Ca2+-activated K+ (BK) channels. Familial hyperkalemic hypertension (pseudohypoaldosteronism type 2) is an inherited form of hypertension with decreased K+ secretion and increased Na+ reabsorption. This disorder is linked to mutations in genes encoding with-no-lysine kinase 1 (WNK1), WNK4, and Kelch-like 3/Cullin 3, two components of an E3 ubiquitin ligase complex that degrades WNKs. We examined whether the full length (or "long") form of WNK1 (L-WNK1) affected the expression of BK α subunits in HEK cells. Overexpression of L-WNK1 promoted a significant increase in BK α subunit whole-cell abundance and functional channel expression. BK α subunit abundance also increased with co-expression of a kinase dead L-WNK1 mutant (K233M) and with kidney specific WNK-1 (KS-WNK1), suggesting that the catalytic activity of L-WNK1 was not required to increase BK expression. A high K+ diet increases BK expression in intercalated cells. We examined whether dietary K+ intake affected L-WNK1 expression in the ASDN. We found a paucity of L-WNK1 labeling in cortical collecting ducts (CCDs) from rabbits on a low K+ diet, but observed robust staining for L-WNK1 primarily in intercalated cells when rabbits were fed a high K+ diet. Our results and previous findings suggest that L-WNK1 exerts different effects on renal K+ secretory channels, inhibiting ROMK and activating BK channels. A high K+ diet induced an increase in L-WNK1 expression selectively in intercalated cells and may contribute to enhanced BK channel expression and K+ secretion in CCDs.
Renal oxidative stress is increased in response to ureteral obstruction. In vitro, cyclooxygenase-2 (COX-2) activity contributes to protection against oxidants. Here, we test the hypothesis that COX-2 activity counters oxidative stress and apoptosis in an in vivo model of obstructive nephropathy. Renal oxidative stress markers, antioxidant enzymes and markers of tubular injury, tubular dilation and apoptosis were investigated in COX-2 knockout (COX-2-/-) and wild type (WT) mice subjected to 3 or 7 days of unilateral ureteral obstruction (UUO). In a separate series, sham and UUO WT mice were treated with a selective COX-2 inhibitor, parecoxib. COX-2 increased in response to UUO; oxidative stress markers 4-hydroxynonenal (4-HNE) and nitrotyrosine protein residues increased in kidney tissue with no genotype difference after UUO while antioxidant enzymes Heme oxygenase-1 (HO-1) and superoxide dismutases (SOD2) displayed higher levels in COX-2-/-. Tubular injury was aggravated by COX-2 deletion as measured by tubular dilatation, KIM-1 increase, cortical caspase-3 content and apoptosis index. In conclusion, COX-2 is necessary to protect against tubular injury and apoptosis after UUO but not necessary to protect against oxidative stress. COX-2 is not likely to directly regulate antioxidant enzymes HO-1 and SOD in the kidney.
To investigate kidney injury in preeclampsia, we analyzed 14 biomarkers in urine specimen from 4 groups of pregnant women (normotensive, pregnancy-complicated with chronic hypertension, mild or severe preeclampsia). These biomarkers were podocyte glycoproteins nephrin and podocalyxin; matrix metallopeptidases MMP2 and MMP9, and their inhibitor TIMP2; inflammatory molecules and cytokines sVCAM-1, TNFα, sTNFR-1, IL-6, IL-8, IL-10, and IL-18; and kidney injury biomarkers NGAL and KIM-1. Postpartum urine specimens (6-8 weeks) from normotensive and severe preeclampsia were also evaluated. We found 1) urine levels of nephrin, MMP2, MMP9, and KIM-1 were significantly higher before delivery in severe preeclampsia than in normotensive controls. The increased levels were all reduced to the levels similar to that of normotensive controls in postpartum specimens from severe preeclampsia; 2) sVCAM-1, sTNFR-1, and NGAL levels were significantly increased in severe preeclampsia compared to normotensive controls before delivery, but levels of these molecules were significantly reduced in postpartum specimens in both groups; 3) IL-6 and IL-8 levels were not different between preeclampsia and normotensive groups, but significantly increased in pregnancy-complicated with chronic hypertension; 4) TIMP2 and IL-18 levels were not different among the study groups before delivery, but significantly reduced in postpartum specimens from normotensive controls. Our results indicate that kidney experiences an increased inflammatory response during pregnancy. Most interestingly, tubular epithelial cell injury may also occur in severe preeclampsia. These biomarkers could be used to assess podocyte or tubular injury, and kidney inflammatory responses during pregnancy and to evaluate postpartum kidney injury recovery in pregnancy-complicated disorders.
Renal fibrosis is an inevitable outcome of chronic kidney disease (CKD). Erythropoietin (EPO) has been recently reported to be able to mitigate renal fibrosis. The mechanism underlying the protective effect of EPO, however, remains elusive. In the present study, employing mouse model of renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO), we demonstrated that EPO markedly reduced the disruption of tubular basement membrane (TBM) through attenuating the activation of tissue plasminogen activator (tPA) and matrix metalloproteinase 9 (MMP9), the major matrix proteolytic network in obstructed kidney. Instead of acting directly on tPA in kidney, EPO strongly increased the level of circulating miR-144, which was delivered to the injured renal fibroblasts via extracellular vesicles (EVs) to target tPA 3'-UTR and suppressed tPA expression. The protective effect of EPO on mouse TBM was inhibited by miR-144 antagomir. Furthermore, in vitro results confirmed that EPO could stimulate bone marrow-derived Sca-1+CD44+CD11b-CD19- cells to secret miR-144-containing EVs, which markedly suppressed tPA expression, as well as MMP9 level and activity, in cultured renal fibroblasts. In conclusion, our study provides the first evidence that EPO protects mouse renal TBM through promoting bone marrow cells to generate and secret miR-144, which in turn, is efficiently delivered into the mouse kidney via EVs to inhibit the activation of tPA/MMP9-mediated proteolytic network. This finding thus suggests that EPO, a hormone widely used to treat anemia in chronic kidney disease, is a potential therapeutic strategy for renal fibrosis.
The goal of this study was to investigate the effects of nitric oxide (NO)-mediated vasodilation in preventing medullary hypoxia, as well as the likely pathways by which superoxide (O2) conversely enhances medullary hypoxia. To do so, we expanded a previously-developed mathematical model of solute transport in the renal medulla that accounts for the reciprocal interactions among oxygen (O2), NO, and O2, to include the vasoactive effects of NO on medullary descending vasa recta. The model represents the radial organization of the vessels and tubules, centered around vascular bundles in the outer medulla and collecting ducts in the inner medulla. Model simulations suggest that NO helps to prevent medullary hypoxia both by inducing vasodilation of the descending vasa recta (thus increasing O2 supply) and by reducing the active sodium transport rate (thus reducing O2 consumption). That is, the vasodilative properties of NO significantly contribute to maintaining sufficient medullary oxygenation. The model further predicts that a reduction in tubular transport efficiency (i.e., the ratio of active sodium transport per O2 consumption) is the main factor by which increased O2 levels lead to hypoxia. It also predicts that hyperfiltration is not a likely pathway to medullary hypoxia due to oxidative stress. Finally, our results suggest that further increasing the radial separation between vessels and tubules would reduce the diffusion of NO towards descending vasa recta in the inner medulla, thereby diminishing its vasoactive effects therein and reducing O2 delivery to the papillary tip.
All cells in the body experience external mechanical forces such as shear stress and stretch. These forces are sensed by specialized structures in the cell known as mechanosensors. Cells lining the proximal tubule (PT) of the kidney are continuously exposed to variations in flow rates of the glomerular ultrafiltrate which manifest as changes in axial shear stress and radial stretch. Studies suggest that these cells respond acutely to variations in flow by modulating their ion transport and endocytic functions to maintain glomerulotubular balance. Conceptually, changes in the axial shear stress in the PT could be sensed by three known structures, namely, the microvilli, the glycocalyx, and primary cilia. The orthogonal component of the force produced by flow exhibits as radial stretch and can cause expansion of both the inner and outer diameters of the tubule. Forces of stretch are transduced by integrins, by stretch activated channels and by cell-cell contacts. This review summarizes our current understanding of flow sensing in PT epithelia, discusses challenges in dissecting the role of individual flow sensors in the mechanosensitive responses, and identifies potential areas of opportunity for new study.
Diabetic Nephropathy (DN) is the main cause of morbidity and mortality in diabetes, characterized by mesangial matrix deposition and podocytopathy including podocyte loss. The risk factors and mechanisms involved in the pathogenesis of DN are still not completely defined. In the current study we aim to understand the cellular mechanisms through which activation of B2 kinin receptors contribute to the initiation and progression of DN. Stimulation of cultured rat podocytes with bradykinin (BK) resulted in a significant increase in reactive oxygen species generation (ROS) and this was associated with a significant increase in NOX1 and NOX4 protein and mRNA levels. BK stimulation also resulted in a signicant increase in the phosphorylation of ERK1/2 and Akt and this effect was inhibited in the presence of NOX1 and Nox4 siRNA. Furthermore podocytes stimulated with BK resulted in a significant increase in the protein and mRNA levels of connective tissue growth factor (CTGF) and at the same time a significant decrease in the protein and mRNA levels of nephrin. siRNA targeted against NOX1 and NOX4 significantly inhibited the BK-induced increase in CTGF. Nephrin expression was increased in response to BK in the presence of NOX1 and NOX4 siRNA, thus implicating a role for NOXs in modulating the BK response in podocytes. Moreover, nephrin expression in response to BK was also significantly increased in the presence of siRNA targeted against CTGF. These findings provide novel aspects of BK signal transduction pathways in pathogenesis of DN and identify novel targets for interventional strategies.
Obesity and nonalcoholic fatty liver disease (NAFLD) are associated with the development and progression of chronic kidney disease. We recently showed that NAFLD induces liver specific CYP2E1-mediated metabolic oxidative stress following administration of a CYP2E1 substrate bromodichloromethane (BDCM). The present study examined the effects of CYP2E1 mediated oxidative stress in NAFLD leading to kidney toxicity. Mice were fed a high fat diet for 12 weeks to induce NAFLD. NAFLD mice were exposed to BDCM, a CYP2E1 substrate for 4 weeks. NAFLD+BDCM increased CYP2E1-mediated lipid peroxidation in proximal tubular cells compared to mice with NAFLD alone or BDCM -treated lean mice thus ruling out the exclusive role of BDCM. The lipid peroxidation increased IL1β, TNFα and IFN-. In parallel mesangial cell activation was observed by increased α-SMA and TGF-β, which was blocked by the CYP2E1 inhibitor Diallyl sulphide (DAS) both in vivo and in vitro Mice lacking NKT cells (CD1d KO) showed elevated (>4-fold) pro-inflammatory mediator release, increased TLR4 and PDGF2 mRNA and mesangial cell activation in the kidney. Finally, NAFLD-CD1D KO mice treated with BDCM exhibited increased HMGB1, FasL levels, and TUNEL positive nuclei indicating higher cell death that was attenuated in TLR4 KO mice. Tubular cells showed increased cell death and cytokine release when incubated with activated mesangial cells. In summary, an underlying condition of progressive NAFLD causes renal immunotoxicity and aberrant glomerular function possibly through HMGB1-dependent TLR4 signaling and mesangial cell activation which in turn is modulated by intrinsic CD1D dependent NKT cells.
This study describes a high-throughput fluorescence dilution technique to measure the albumin reflection coefficient (Alb) of isolated glomeruli. Rats were injected with fluorescein isothiocyanate-dextran 250 (75 mg/kg) and the glomeruli were isolated in a 6% bovine serum albumin solution. Changes in the fluorescence of the glomerulus due to water influx in response to an imposed oncotic gradient was used to determine Alb. Adjustment of the albumin concentration of the bath from 6% to 5%, 4%, 3%, and 2 % produced a 10%, 25%, 35%, and 50% decrease in the fluorescence of the glomeruli. Pretreatment of glomeruli with protamine sulfate (2 mg/ml) or TGF-β1 (10 ng/ml) decreased Alb from 1 to 0.54 and 0.48, respectively. Water and solute movement were modeled using the Kedem-Katchalsky equations and the measured responses closely fit the predicted behavior, indicating that loss of albumin by solvent drag or diffusion is negligible in comparison to the movement of water. We also found that Alb was reduced by 17% in Fawn Hooded Hypertensive rats, 33% in hypertensive Dahl Salt-sensitive (SS) rats, 26% in streptozotocin-treated diabetic Dahl SS rats and 21% in 6 month old Type II diabetic nephropathy rats relative to control SD rats. The changes in glomerular permeability to albumin were correlated with the degree of proteinuria in these strains. These findings indicate that the fluorescence dilution technique can be used to measure Alb in populations of isolated glomeruli and provides a means to assess the development of glomerular injury in hypertensive and diabetic models.
Endothelial dysfunction has been shown to be predictive of subsequent cardiovascular events and death. Through a mechanism that is incompletely understood, increased dietary salt intake promotes endothelial dysfunction in healthy, salt-resistant humans. The present study tested the hypothesis that dietary salt-induced TGF-β promoted endothelial dysfunction and salt-dependent changes in blood pressure (BP). Sprague-Dawley rats receiving diets containing 0.3% NaCl (LS) or 8.0% NaCl (HS) were treated with vehicle or SB525334 (SB), a specific inhibitor of TGF-β receptor I/activin receptor-like kinase 5 (ALK5), beginning on day 5. BP was monitored using radio-telemetry in the four groups of rats (LS, LS SB, HS, and HS SB) for up to 14 days. By day 14 of the study, mean daytime systolic blood pressure (SBP) and mean pulse pressure (PP) of the HS group treated with vehicle was greater than the other three groups; mean daytime SBP and PP of the HS SB group did not differ from the LS and LS SB groups. While mean SBP, DBP and MAP did not differ among the groups on the 7th day of the study, endothelium-dependent vasorelaxation was impaired specifically in the HS group; treatment with the ALK5 inhibitor prevented the high dietary salt intake-induced increases in phospho-Smad2 (S465/467) and NADPH oxidase-4 in endothelial lysates and normalized endothelial function. These findings suggest that high salt-induced endothelial dysfunction and the development of salt-dependent increases in BP were related to endothelial TGF-β signaling.
Changes in the urothelial barrier are observed in patients with cystitis, but if this leads to inflammation or occurs in response to it is currently unknown. To determine whether urothelial barrier dysfunction is sufficient to promote cystitis, we employed in situ adenoviral transduction to selectively overexpress the pore-forming tight junction-associated protein claudin-2 (CLDN-2). As expected, the expression of CLDN-2 in the umbrella cells increased the permeability of the paracellular route toward ions, but not to large organic molecules. In vivo studies of bladder function revealed higher intravesical basal pressures, reduced compliance, and increased voiding frequency in rats transduced with CLDN-2 versus controls transduced with GFP. While the integrity of the urothelial barrier was preserved in the rats transduced with CLDN-2, we found that the expression of this protein in the umbrella cells initiated an inflammatory process in the urinary bladder characterized by edema and the presence of a lymphocytic infiltrate. Taken together, these results are consistent with the notion that urothelial barrier dysfunction may be sufficient to trigger bladder inflammation and to alter bladder function.
The ascending thin limbs (ATLs) and lower descending thin limbs (DTLs) of Henle's loop in the inner medulla of the rat are highly permeable to urea and yet no urea transporters have been identified in these sections. We hypothesized that novel, yet unidentified transporters in these tubule segments could explain the high urea permeability. cDNAs encoding for SGLT1a, NaGLT1, UT-A2c, and UT-A2d were isolated and cloned from the Munich-Wistar rat inner medulla. SGLT1a is a novel amino-terminal truncated variant of the Na+-glucose transporter, SGLT1. NaGLT1 is another Na+-glucose transporter primarily located in the proximal tubules and not previously described in the thin limbs. UT-A2c and UT-A2d are novel variants of the urea transporter, UT-A2. UT-A2c is truncated at the carboxyl terminus and UT-A2d has one exon skipped. When rats underwent water restriction for 72 h, mRNA levels of SGLT1a increased in the ATLs, NaGLT1 levels increased in both ATLs and DTLs and UT-A2c increased in the ATLs. [14C]urea uptake assays performed on Xenopus oocytes heterologously expressing these proteins revealed that despite having structural differences from their full-length versions, SGLT1a, UT-A2c and UT-A2d enhanced urea uptake. NaGLT1 also facilitated urea uptake. Uptakes were Na+-independent and inhibitable by phloretin and/or phloridzin. Our data indicate that there are several alternative channels for urea in the rat inner medulla that could potentially contribute to the high urea permeabilities in the thin limb segments.
Cardiovascular disease including cardiac hypertrophy is common in patients with kidney disease, and can be partially attenuated using blockers of the renin angiotensin system (RAS). It is unknown whether cardiac microRNAs contribute to pathogenesis of cardiac hypertrophy or to the protective effect of RAS blockade in kidney disease. Using subtotal nephrectomy rat model of kidney injury, we investigated changes in cardiac microRNAs that are known to have direct target genes involved in regulation of apoptosis, fibrosis and hypertrophy. The effect of treatment with the angiotensin converting enzyme inhibitor (ACEi), ramipril on cardiac microRNAs was also investigated. Kidney injury led to a significant increase in cardiac microRNA-212 and microRNA-132 expression. Ramipril reduced cardiac hypertrophy, attenuated the increase in microRNA-212 and microRNA-132 and significantly increased microRNA-133 and microRNA-1 expression. There was altered expression of Caspase-9, B-cell lymphoma 2, transforming growth factor-beta, fibronectin 1, collagen 1a1 and forkhead box proteins O3, all known to be involved in the regulation of apoptosis, fibrosis and hypertrophy in cardiac cells, whilst being targets for the above microRNAs. ACEi treatment increased expression of microRNA-133 and microRNA-1. Inhibitory action of ACEi treatment on increased cardiac NOX1 expression after STNx surgery suggest that inhibition of oxidative stress is also one of mechanism of ACEi mediated cardioprotection. These finding suggests the involvement of microRNAs in the cardioprotective action of ACEi in acute renal injury, which is mediated through an inhibitory action on pro-fibrotic and pro-apoptotic target genes, and stimulatory action on anti-hypertrophic and anti-apoptotic target genes.
Aldosterone increases blood pressure (BP) by stimulating sodium (Na) reabsorption within the distal nephron and collecting duct (CD). Aldosterone also stimulates endothelin-1 (ET-1) production that acts within the CD to inhibit Na reabsorption via a negative feedback mechanism. We tested the hypothesis that this renal aldosterone-endothelin feedback system regulates electrolyte balance and BP by comparing the effect of a high-salt (NaCl) diet and mineralocorticoid stimulation in control and CD specific ET-1 knockout mice (CD ET-1 KO). Metabolic balance and radiotelemetric BP were measured before and after treatment with desoxycorticosterone pivalate (DOCP) in mice fed a high-salt diet with saline to drink. CD ET-1 KO mice consumed more high-salt diet and saline and had greater urine output than controls. CD ET-1 KO mice exhibited increased BP and greater fluid retention and body weight (BW) than controls on a high-salt diet. DOCP with high-salt feeding further increased BP in CD ET-1 KO mice and by the end of the study the CD ET-1 KO mice are substantially hypernatremic. Unlike controls, CD ET-1 KO mice failed to respond acutely to or escape from DOCP treatment. We conclude that local ET-1 production in the CD is required for the appropriate renal response to Na loading and lack of local ET-1 results in abnormal fluid and electrolyte handling when challenged with a high-salt diet and with DOCP treatment. Additionally, local ET-1 production is necessary, under these experimental conditions, for renal compensation to, and escape from, the chronic effects of mineralocorticoids.
Long noncoding RNAs (lncRNAs) are emerging as key species-specific regulators of cellular and disease processes. To identify potential lncRNAs relevant to acute and chronic renal epithelial injury, we performed unbiased whole transcriptome profiling of human proximal tubular epithelial cells (PTECs) in hypoxic and inflammatory conditions. RNA sequencing (RNA-seq) revealed that the protein-coding and noncoding transcriptomic landscape differed between hypoxia-stimulated and cytokine-stimulated human PTECs. Hypoxia- and inflammation-modulated lncRNAs were prioritized for focused follow up according to their degree of induction by these stress stimuli, their expression in human kidney tissue, and whether exposure of human PTECs to plasma of critically ill sepsis patients with acute kidney injury modulated their expression. For three lncRNAs--MIR210HG, linc-ATP13A4-8, and linc-KIAA1737-2--that fulfilled our criteria, we validated their expression patterns, examined their loci for conservation and synteny, and defined their associated epigenetic marks. The lncRNA landscape characterized here provides insight into novel transcriptomic variations in the renal epithelial cell response to hypoxic and inflammatory stress.
Phylogentically, the organic anion transporters 1 (OAT1) and OAT3 are closely related whereas OAT2 is more distant. Experiments with HEK293 cells stably transfected with human OAT1, OAT2, or OAT3 were performed to compare selected transport properties. Common to OAT1, OAT2, and OAT3 is their ability to transport cGMP. OAT2 interacted with prostaglandins (PGs) and cGMP uptake was inhibited by PGE2 and PGF2α with IC50 values of 40.8 µM and 12.7 µM, respectively. OAT1 (IC50 23.7 µM), OAT2 (IC50 9.5 µM), and OAT3 (IC50 1.6 µM) were potently inhibited by MK571, an established multidrug resistance protein (MRP) inhibitor. OAT2-mediated cGMP uptake was not inhibited by short-chain monocarboxylates and, as opposed to OAT1 and OAT3, not by dicarboxylates. Consequently, OAT2 showed no cGMP-glutarate exchange. OAT1 and OAT3 exhibited a pH and a chloride dependence with higher substrate uptake at acidic pH and lower substrate uptake in the absence of chloride, respectively. Such pH and chloride dependencies were not observed with OAT2. Depolarization of membrane potential by high potassium concentrations in the presence of the potassium ionophore valinomycin left cGMP uptake unaffected. In addition to cGMP, OAT2 transported urate and glutamate, but cGMP-glutamate exchange could not be demonstrated. These experiments suggest that OAT2-mediated cGMP uptake does not occur via exchange with monocarboxylates, dicarboxylates, and hydroxyl ions. The counter anion for electroneutral cGMP uptake remains to be identified.
We have previously demonstrated that the circadian clock protein Per1 coordinately regulates multiple genes involved in sodium reabsorption in renal collecting duct cells. Consistent with these results, Per1 KO mice exhibit dramatically lower blood pressure than WT mice. The proximal tubule is responsible for a majority of sodium reabsorption. Previous work has demonstrated that expression of the sodium hydrogen exchanger NHE3 oscillates with a circadian pattern and SGLT1 has been demonstrated to be a circadian target in the colon, but whether these target genes are regulated by Per1 has not been investigated in the kidney. The goal of this study was to determine if Per1 regulates the expression of NHE3, SGLT1, and SGLT2 in the kidney. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of SGLT1 and NHE3, but not SGLT2 in the renal cortex of mice. Per1 siRNA and pharmacological blockade of Per1 nuclear entry in human proximal tubule HK-2 cells yielded the same results. Examination of heterogeneous nuclear RNA (hnRNA) suggested that the effects of Per1 on NHE3 and Sglt1 expression occurred at the level of transcription. Chromatin-immunoprecipitation experiments demonstrated the interaction of Per1 and the circadian protein CLOCK with the promoters of NHE3 and SGLT1. These interactions were reduced by blockade of Per1 nuclear entry. Importantly, both membrane and intracellular protein levels of NHE3 and SGLT1 were decreased following blockade of nuclear Per1 entry. These data demonstrate a role for Per1 in the transcriptional regulation of NHE3 and SGLT1 in the kidney.
Binding of cardiac hormone, atrial natriuretic peptide (ANP) to transmembrane guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), produces the intracellular second messenger cGMP in target cells. To delineate the critical role of endocytic signal in intracellular sorting of the receptor, we have identified a FQQI (Phe790, Gln791, Gln792, and Ile793) motif in the carboxyl-terminal region of NPRA. Mouse mesangial cells (MMCs) were transiently transfected with the enhanced green fluorescence protein (eGFP)-tagged wild-type (WT) and mutant constructs of eGFP-NPRA. The mutation FQQI/AAAA, in eGFP-NPRA cDNA sequence, markedly attenuated the internalization of mutant receptors by almost 49% compared with the WT receptor. Interestingly, we show that μ1B subunit of adaptor protein-1 (AP-1, μ1B), binds directly to a phenylalanine based FQQI motif in the cytoplasmic tail of receptor. However, subcellular trafficking indicated that immunofluorescence colocalization of mutated receptor with early endosome antigen-1 (EEA-1), lysosome-associated membrane protein-1 (LAMP-1), and Rab 11 marker, was decreased by 57% into early endosome, 48% in lysosome, and 42% in recycling endosome, respectively, compared with WT receptor in MMCs. The receptor containing mutated motif (FQQI/AAAA) also produced a significantly decreased level of intracellular cGMP during subcellular trafficking than WT receptor. The co-immunoprecipitation assay confirmed a decreased level of colocalization of mutant receptor with subcellular compartments during endocytic processes. The results suggest that FQQI motif is essential for the internalization and subcellular trafficking of NPRA during the hormone signaling process in intact MMCs.
In a lentivirus-based gene delivery system, the incorporated gene is continuously expressed for a long time. In this study, we devised a simple way to knock down a specific gene in a kidney cell-specific pattern in adult mice by lentivirus-assisted transfer of shRNA. Kidney collecting duct (CD)-specific aquaporin-3 (AQP3)-knockdown mice was generated by consecutive injection of Hoxb7-Cre-expressing lentivirus (LV-Hoxb7 Cre) and loxP-AQP3 shRNA-expressing lentivirus (LV-loxP shAQP3) in adult C57BL6/J mice. LV-Hoxb7 Cre was designed to express mCherry, while LV-loxP shAQP3 was designed with a floxed EGFP tagged stop sequence, and thus EGFP would be expressed only in the absence of Cre recombination.. In mice treated with LV-Hoxb7 Cre alone, mCherry protein expression, which indicates the presence of Cre recombinase, occurred only in CD cells. However, LV-loxP shAQP3 injection alone resulted in an increase in EGFP expression in all kidney cells, indicating the transcription of the floxed region. When LV-Hoxb7 Cre and LV-loxP shAQP3 were sequentially transduced, EGFP expression was attenuated while mCherry expression was sustained in CD cells demonstrating a CD cell specific recombination of the floxed region. AQP3 expression in mice injected with LV-Hoxb7 Cre or LV-loxP shAQP3 alone did not differ, but consecutive injection of LV-Hoxb7 Cre and LV-loxP shAQP3 significantly reduced AQP3 expression in CD cells. However, the expression levels of AQP3 were not altered in other cell types. Double transduction of Cre- and loxP-based lentivirus can easily generate kidney cell-specific knockdown mice, and this method might be applicable to other species.
Podocytes are highly specialized epithelial cells located at the outer aspects of the glomerular capillary tuft and critical components of the kidney filtration barrier. To maintain their unique features podocytes express a number of proteins that are only sparsely found elsewhere in the body. In this study, we have identified four (Tmem234, Znf185, Lrrc49, and Slfn5) new highly podocyte-enriched proteins. The proteins are strongly expressed by podocytes while other parts of the kidney show only weak or no expression. Tmem234, Slfn5 and Lrrc49 are located to foot processes, whereas Znf185 is found in both foot and major processes. Expressional studies in developing kidneys show that these proteins are first expressed at the capillary stage glomerulus, the same stage when the formation of major and foot processes begins. We identified zebrafish orthologues for Tmem234 and Znf185 genes, and inactivated their expression using morpholino technology. Studies in zebrafish larvae indicate that Tmem234 is essential for the organization and functional integrity of the pronephros filtration barrier as knockdown of Tmem234 expression results in foot process effacement and proteinuria. In summary, we have identified five novel highly podocyte-enriched proteins and show that one of them, Tmem234, is essential for the normal filtration barrier in zebrafish pronephros. Identification of new molecular components of the kidney filtration barrier opens up possibilities to study their role in the glomerulus biology and diseases.
Hyperglycemia is the primary factor responsible for the microvascular, and to lesser extent, macrovascular complications. Despite this well established relationship, approximately half of all type 2 diabetic patients in the US have a HbA1c≥7.0%. In part, this is associated with the side effects, i.e. weight gain and hypoglycemia, of currently available antidiabetic agents and in part by the failure to utilize medications that reverse the basic pathophysiologic defects present in patients with type 2 diabetes. The kidney has been show to play a central role in the development of hyperglycemia by excessive production of glucose throughout the sleeping hours and enhanced reabsorption of filtered glucose by the renal tubules secondary to an increase in the threshold at which glucose spills into the urine. Recently, a new class of antidiabetic agents, the sodium-glucose cotransporter 2 (SGLT2) inhibitors, has been developed and approved for the treatment of patients with type 2 diabetes. In this review, we examine their mechanism of action, efficacy, safety, and place in the therapeutic armamentarium. Since the SGLT2 inhibitors have a unique mode of action that differs from all other oral and injectable antidiabetic agents, they can be used at all stages of the disease and in combination with all other antidiabetic medications.
Cytomegalovirus (CMV) infection is a frequent complication early post-transplant. This study examines its impact on chronic allograft changes, long-term graft loss and patient survival. We studied 594 patients who had protocol biopsies at 6 weeks, 3 and 6 months post-transplant. Chronic allograft changes were evaluated according to the updated Banff classification (IF/TA (interstitial fibrosis/tubular atrophy), vascular and glomerular lesions). Follow up data was available up to 10 years. CMV infection was diagnosed in 153 of 594 patients (26%) in the first year after transplantation, mostly within the first 3 months. Graft survival was reduced in patients with CMV (p=0.03) as well as the combined allograft/patient survival (p=0.008). Prevalence of IF/TA at 6 weeks after transplantation was already 3-fold higher in patients who experienced CMV infection later-on, compared to patients without CMV (p=0.005). In multivariate analyses CMV viremia or disease were not a significant factor for graft loss or death. In conclusion, patients with CMV infection post-transplant show more chronic allograft changes early-on –even before CMV infection– and development of IF/TA is not more prevalent in patients with CMV. Our data do not support a significant role of CMV in patient and graft outcomes.
Purinergic signaling is a major pathway in regulating bladder function, and mechanical force stimulates urothelial ATP release, which play an important role in bladder mechanotransduction. Although urothelial ATP release was first reported almost 20 years ago, the way in which release is regulated by mechanical force, and the presence of ATP converting enzymes in regulating the availability of released ATP is still not well understood. Using a set of custom designed Ussing chambers with the ability to manipulate mechanical forces applied on the urothelial tissue, we have demonstrated that it is stretch and not hydrostatic pressure which induces urothelial ATP release. The experiments reveal that urothelial ATP release is tightly controlled by stretch speed, magnitude, and direction. We have further shown that stretch induced urothelial ATP release is insensitive to temperature (4°C). Interestingly, stretch induced ATP release shows polarized distribution, with the ATP concentration in mucosal chamber (nM level) about 10 times higher than the ATP concentration in serosal chamber (sub nM level). Furthermore, we have consistently observed differential ATP lifetime kinetics in the mucosal and serosal chambers, which is consistent with our immunofluorescent localization data showing that ATP converting enzymes ENTPD3 and ALPL are expressed on urothelial basal surface, but not on the apical membrane. In summary, our data indicate that urothelial ATP release is finely regulated by stretch speed, magnitude and direction, and extracellular ATP signaling is likely to be differentially regulated by ectonucleotidase, which results in temporally and spatially distinct ATP kinetics in response to mechanical stretch.
Diabetic nephropathy (DN) is a serious complication of both type 1 and type 2 diabetes mellitus. The disease is now the most common cause of end-stage kidney disease (ESKD) in developed countries, and both the incidence and prevalence of diabetes mellitus is increasing worldwide. Current treatments are directed at controlling hyperglycemia and hypertension as well as blockade of the renin angiotensin system (RAS) with angiotensin converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs). Despite these therapies, DN progresses to ESKD in many patients. As a result, much interest is focused on developing new therapies. It has been over 2 decades since ACEIs were shown to have beneficial effects in DN independent of their blood pressure (BP) lowering actions. Since that time, our understanding of disease mechanisms in DN has evolved. In this review, we summarize major cell signaling pathways implicated in the pathogenesis of diabetic kidney disease as well as emerging treatment strategies. The goal is to identify promising targets that might be translated into therapies for the treatment of patients with diabetic kidney disease.
The aim of this study was to investigate the impact of sex and puberty stage on circadian changes in sodium excretion, sodium regulating hormones and hemodynamics. 39 healthy volunteers (9 pre-puberty boys, 10 pre-puberty girls, 10 puberty boys and 10 puberty girls) were included. They all underwent a 24-hour circadian in-patient study under standardized conditions regarding activity, diet and fluid intake. Blood samples were drawn every four hours and the urine was collected in fractions. Blood pressure and heart rate were non-invasively monitored. Atrial natriuretic peptide (ANP), angiotensin II, aldosterone, and renin were measured in blood. Results: Children in puberty had lower plasma levels of renin (p<0.05) and angiotensin II (p<0.05), and a 26 % reduction in filtered sodium without changes in sodium excretion when compared to pre-puberty children. A circadian rhythm in sodium excretion, the renin-angiotensin system, ANP and blood pressure was found with a midnight ANP peak (p<0.001), a night-time decrease in hemodynamic parameters(p<0.001), an increase in plasma renin (p<0.001) and angiotensin II (p<0.001) and a decrease in sodium excretion (p<0.001) mainly on the basis of increased sodium reabsorption (p<0.001). The timing of the changes did not depend on sex or puberty group. Conclusion: There is a circadian rhythm of sodium excretion and sodium regulation in 7-15year old children. This rhythm is similar in boys and girls. As an important new finding, puberty changes the plasma levels of renin and angiotensin II without changing the amount of sodium excreted or the day to night sodium excretion ratio.
Evidences in rodents suggest that tacrolimus-induced post-transplant hypertension is due to upregulation of the thiazide-sensitive Na+-Cl- cotransporter, NCC. Here we analyzed if a similar mechanism is involve in post-transplant hypertension in humans. From January 2013 to June 2014 all adult kidney transplant recipients receiving a kidney allograft were enrolled into a prospective cohort study. All patients received tacrolimus as part of the immunosuppressive therapy. Six months after surgery we assessed general clinical and laboratory variables, tacrolimus trough blood levels, and ambulatory 24-hour blood pressure monitoring. Urinary exosomes were extracted to perform Western blot analysis using total and phosphoNCC antibodies. A total of 52 patients, including 17 women and 35 men, were followed. At six months after transplantation, of the 35 men, 17 developed hypertension and 18 remained normotensive, while high blood pressure was observed in only 3 out 17 women. The hypertensive patients were significantly older than the normotensive group, however there were no significant differences in body weight, history of acute rejection, renal function, and tacrolimus through levels. In urinary exosomes, hypertensive patients showed higher NCC expression (1.7 ± 0.19) than normotensive (1 ± 0.13) (P=0.0096). Also, NCC phosphorylation levels were significantly higher in the hypertensive patients (1.57 ± 0.16 vs. 1 ± 0.07; P=0.0049). Our data show that there is a positive correlation between NCC expression/phosphorylation in urinary exosomes with the development of hypertension in post-transplant male patients treated with tacrolimus. Our results are consistent with the hypothesis that NCC activation plays a major role in tacrolimus-induced hypertension.
Vascular calcification (VC) is a critical complication in patients with chronic kidney disease (CKD). The effects of spironolactone (SPL), a mineralocorticoid receptor (MR) antagonist, on VC have not been fully investigated in CKD. The present in vivo study determined the protective effects of SPL on VC in CKD rats. Rats were divided into a control group and four groups of rats with adenine-induced CKD. Three groups were treated with 0, 50, and 100 mg/kg/day SPL for 8 weeks, and one group was treated with 100 mg/kg/day SPL for the last 2 weeks of the 8-week treatment period. After 8 weeks, CKD rats developed azotemia and hyperphosphatemia, with increases in the expression of serum and glucocorticoid-regulated kinase-1 and sodium-phosphate cotransporter, in inflammation and oxidative stress level, in osteogenic signaling and apoptosis, and in aortic calcification, compared with control rats. SPL dose-dependently decreased these changes in the aortas, concomitant with improvements in renal inflammation, tubulointerstitial nephritis, and kidney function. SPL neither lowered blood pressure level nor induced hyperkalemia. Treatment of CKD rats for the last 2 weeks with 100 mg/kg/day SPL attenuated VC compared with CKD rats with the same degree of kidney function and hyperphosphatemia. In conclusion, SPL dose-dependently inhibits the progression of VC by suppressing MR signaling, local inflammation, osteogenic transition, and apoptosis in the aortas of CKD rats.
It has been postulated that developmental pathways are reutilized during repair and regeneration after injury, but functional analysis of many genes required for kidney formation has not been performed in the adult organ. Mutations in SALL1 cause Townes-Brocks syndrome (TBS) and non-syndromic congenital anomalies of the kidney and urinary tract (CAKUT), both of which lead to childhood kidney failure. Sall1 is a transcriptional regulator that is expressed in renal progenitor cells and developing nephrons in the embryo. However, its role in the adult kidney has not been investigated. Using a mouse model of TBS (Sall1-TBS), we investigated the role of Sall1 in response to acute kidney injury. Our studies revealed that Sall1 is expressed in terminally differentiated renal epithelia, including the S3 segment of the proximal tubule, in the mature kidney. Sall1-TBS mice exhibited significant protection from ischemia reperfusion injury and aristolochic acid-induced nephrotoxicity. This protection from acute injury is seen in spite of the presence of slowly progressive chronic kidney disease in Sall1TBS mice. Mice containing null alleles of Sall1 are not protected from acute kidney injury, indicating that expression of a truncated mutant protein from the Sall1-TBS allele, while causative of congenital anomalies, protects the adult kidney from injury. Our studies further revealed that basal levels of the pre-conditioning factor heme oxygenase-1 are elevated in Sall1-TBS kidneys, suggesting a mechanism for the relative resistance to injury in this model. Together, these studies establish a functional role for Sall1 in the response of the adult kidney to acute injury.
More effective therapeutic strategies for the prevention and treatment of acute kidney injury (AKI) are needed to improve the high morbidity and mortality associated with this frequently encountered clinical condition. Ischemic and/or hypoxic preconditioning attenuates susceptibility to ischemic injury, which results from both oxygen and nutrient deprivation and accounts for most cases of AKI. While multiple signaling pathways have been implicated in renoprotection, this review will focus on oxygen-regulated cellular and molecular responses that enhance the kidney's tolerance to ischemia and promote renal repair. Central mediators of cellular adaptation to hypoxia are hypoxia-inducible factors (HIFs). HIFs play a crucial role in ischemic/hypoxic preconditioning through the reprogramming of cellular energy metabolism, and by coordinating adenosine and nitric oxide signaling with anti-apoptotic, oxidative stress and immune responses. The therapeutic potential of HIF activation for the treatment and prevention of ischemic injuries will be critically examined in this review.
Lack or downregulation of the dopamine D2 receptor (D2R) results in increased renal expression of injury markers and pro-inflammatory factors that is independent of blood pressure increase. This study aimed to determine the mechanisms involved in the regulation of renal inflammation by D2Rs. Silencing D2Rs in mouse renal proximal tubule cells increased the expression of the pro-inflammatory TNFα, MCP-1, and IL-6. D2R downregulation also increased Akt phosphorylation and activity, and GSK3β phosphorylation and cyclin D1 expression both downstream Akt targets, however PI3K activity was not affected. Conversely D2R stimulation decreased Akt and GSK3β phosphorylation and cyclin D1 expression. Increased phospho-Akt, in the absence of increased PI3K activity, may result from decreased Akt dephosphorylation. Inhibition of PP2A with okadaic acid reproduced the effects of D2R downregulation on Akt, GSK3β and cyclin D1. PP2A catalytic subunit and the regulatory subunit PPP2R2C co-immunoprecipitated with the D2R. Basal phosphatase activity and the expression of PPP2R2C were decreased by D2R silencing that also blunted the increase in phosphatase activity induced by D2R stimulation. Similarly, silencing PPP2R2C also increased the phosphorylation of Akt and GSK3β. Moreover, downregulation of PPP2R2C resulted in increased expression of TNFα, MCP-1, and IL-6, indicating that decreased phosphatase activity may be responsible for the D2R effect on inflammatory factors. Indeed, the increase in NFkB reporter activity induced by D2R silencing was blunted by increasing PP2A activity with protamine. Our results show that D2R controls renal inflammation, at least in part, by modulation of the Akt pathway through effects on PP2A activity/expression.
In contrast to the negative feedback of angiotensin II (Ang II) on juxtaglomerular (JG) renin, Ang II stimulates renin in the principal cells of the collecting duct (CD) in rats and mice via Ang II type 1 (AT1R) receptor, independently of blood pressure. In vitro data indicate that CD renin is augmented by AT1R activation through PKC, but the exact mechanisms are unknown. We hypothesize that Ang II stimulates CD renin synthesis through AT1R via PKC and the subsequent activation of cAMP/PKA/CREB pathway. In M-1 cells, Ang II increased cAMP, renin mRNA (3.5 fold), prorenin and renin proteins, as well as renin activity in culture media (2 fold). These effects were prevented by PKC inhibition with calphostin C, PKC-alpha dominant negative and by PKA inhibition. Forskolin-induced increases in cAMP and renin expression were prevented by calphostin C. PKC inhibition and Ca2+ depletion impaired Ang II-mediated CREB phosphorylation and upregulation of renin. Adenylate cyclase 6 (AC) siRNA remarkably attenuated the Ang II-dependent upregulation of renin mRNA. Physiological activation of AC with vasopressin increased renin expression in M-1 cells. The results suggest that the Ang II-dependent upregulation of renin in the CD depends on PKCα, which allows the augmentation of cAMP production and activation of PKA/CREB pathway via AC6. This study defines the intracellular signaling pathway involved in the Ang II-mediated stimulation of renin in the CD. This is a novel mechanism responsible for the regulation of local RAS in the distal nephron.
Human genetic linkage and association studies have nominated many genes as possible contributors to disease. Mutating or deleting these genes in a relevant disease model can validate their association with disease and potentially uncover novel mechanisms of pathogenesis. Targeted genetic mutagenesis has only recently been developed in the rat, and this technique has been applied in the Dahl salt-sensitive (S) rat to investigate human candidate genes associated with hypertension. This mini-review communicates the findings of these studies and displays how targeted genetic mutagenesis may contribute to the discovery of novel therapies for patients.
High dietary salt is common in western countries and is an important contributor to increased cardiovascular disease. Autoregulation of renal blood flow (RBF) and glomerular filtration rate (GFR) is an essential function of the renal microcirculation, which could be affected by excessive dietary salt. High salt (HS) increases renal reactive oxygen species (ROS) generation partly by the enzyme NADPH oxidase. We hypothesized that a HS diet would impair autoregulation via NADPH oxidase-dependent ROS generation. The role of NADPH-dependent ROS production on the blunted autoregulatory response with a HS diet was assessed in vitro and in vivo using the blood-perfused juxtamedullary nephron preparation and anesthetized rats, respectively. The increase in renal lipid peroxidation and p67phox expression induced by HS was prevented by apocynin treatment. Control afferent arterioles exhibited normal autoregulatory behavior in response to acute increases in renal perfusion pressure (RPP), whereas arterioles from HS rats exhibited a blunted response. Autoregulatory behavior in HS rats was restored in vitro by acute exposure to the NADPH oxidase inhibitor, apocynin. At the whole kidney level, in vivo studies showed RBF and GFR both declined in HS rats when left kidney RPP was reduced from ambient to 95 mmHg whereas control rats maintained stable GFR and RBF consistent with efficient autoregulatory behavior. Apocynin treatment improved in vivo autoregulatory behavior in HS rats and had no detectable effect in normal salt fed rats. These data support the hypothesis that impaired renal autoregulatory behavior in rats fed a HS diet is mediated by NADPH-oxidase-derived ROS.
TNF-like weak inducer of apoptosis (TWEAK) is an inflammatory cytokine that activates the Fn14 receptor. Both TWEAK an Fn14 are constitutively expressed in the kidney. TWEAK has been shown to modulate several biological responses such as inflammation, proliferation, differentiation and apoptosis that contribute to kidney injury. However, TWEAK role in fibrosis and TWEAK-activated intracellular signalling pathways remain poorly understood. We have tested the hypothesis that TWEAK can be a potent inducer of renal fibrosis by increasing TGF-β1 expression, (a well known switch in fibrosis process) through cGMP-dependent protein kinase I (PKGI) downregulation. We show that in human mesangial cells, TWEAK increased TGF-β1 expression and activity, leading to higher levels of the extracellular matrix protein fibronectin and decreased cGMP-dependent protein kinase I (PKGI) expression and activity via Ras pathway. PKGI activation with 8-BrcGMP, Ras inactivation with dominant negative Ras or Ras pathway inhibition with the Erk1/2 inhibitor PD98059, resulted in prevention of TWEAK-induced TGF-β1 upregulation. In vivo, exogenous administration of TWEAK to wild-type mice downregulated kidney PKGI and increased kidney TGF-β1 expression. These effects were blunted in H-Ras knockout mice. Together, these data demonstrate for the first time, the key role of PKGI in the TGF-β1 induction by TWEAK in kidney cells.
The small G-Proteins Rac1 and RhoA regulate actin cytoskeleton, cell shape, adhesion, migration, and proliferation. Recent studies in our laboratory showed that Nox4 NAD(P)H oxidase-derived reactive oxygen species (ROS) are involved in transforming growth factor-beta 1 (TGF-β1)-induced rat kidney myofibroblast differentiation assessed by the acquisition of an alpha-smooth muscle actin (α-SMA) phenotype and expression of an alternatively spliced fibronectin variant (Fn-EIIIA). Rac1 and RhoA are essential in signaling by some Nox homologs, but their role as effectors of Nox4 in kidney myofibroblast differentiation is not known. In the present study, we explored a link between Rac1 and RhoA and Nox4-dependent ROS generation in TGFβ1-induced kidney myofibroblast activation. TGF-β1 stimulated an increase in Nox4 protein expression, NADPH oxidase activity, and abundant α-SMA and Fn-EIIIA expression. RhoA, but not Rac1 was involved in TGF-β1-induction of Nox4 signaling of kidney myofibroblast activation. TGF-β1 stimulated active RhoA-GTP and increased Rho kinase (ROCK). Inhibition of RhoA with small interfering (si)RNA and ROCK using Y-27632 compound significantly reduced TGF-β1-induced stimulation of Nox4 protein, NADPH oxidase activity, α-SMA and Fn-EIIIA expression. Treatment with DPI, an inhibitor of NAD(P)H oxidase, did not decrease RhoA activation, but inhibited TGF-β1-induced α-SMA, Fn-EIIIA expression, indicating that RhoA is upstream of ROS generation. RhoA/ROCK also regulated polymerase [DNA-directed] delta-interacting protein 2 (Poldip2), a newly discovered Nox4 enhancer protein. Collectively, these data indicate that RhoA/ROCK is upstream of Poldip2-dependent Nox4 regulation and ROS production and induces redox signaling of kidney myofibroblast activation and may broader implications in the pathophysiology of renal fibrosis.
An increase in tubular fluid flow rate (TFF) stimulates Na reabsorption and K secretion in the cortical collecting duct (CCD) and subjects cells therein to biomechanical forces including fluid shear stress (FSS) and circumferential stretch (CS). Intracellular mitogen activated protein kinase (MAPK) and extracellular autocrine/paracrine prostaglandin E2 (PGE2) signaling regulate cation transport in the CCD and, at least in other systems, are effected by biomechanical forces. We hypothesized that FSS and CS differentially effect MAPK signaling and PGE2 release to modulate cation transport in the CCD. To validate that CS is a physiologic force in vivo, we applied the intravital microscopic approach to rodent kidneys in vivo to show that saline or furosemide injection led to a 46.5±2.0% or 170±32% increase, respectively, in distal tubular diameter. Next, murine CCD (mpkCCD) cells were grown on glass or silicone coated with collagen type IV and subjected to 0 or 0.4 dynes/cm2 of FSS or 10% CS, respectively, forces chosen based on prior biomechanical modeling of ex vivo microperfused CCDs. Cells exposed to FSS expressed a ~2-fold greater abundance of phospho(p)-ERK and p-p38 vs. static cells, while CS did not alter p-p38 and p-ERK expression compared to unstretched controls. FSS induced whereas CS reduced PGE2 release by ~40%. In conclusion, FSS and CS differentially effect ERK and p38 activation and PGE2 release in a cell culture model of the CD. We speculate that TFF differentially regulates biomechanical signaling and, in turn, cation transport in the CCD.
This study aims to understand the extent to which modulation of NKCC2 differential splicing affects NaCl delivery to the macula densa. NaCl absorption by the thick ascending limb and macula densa cells is mediated by the apical Na+-K+-2Cl- cotransporter NKCC2. A recent study has indicated that differential splicing by NKCC2 is modulated by dietary salt (Schiebl et al., Am J Physiol Renal Physiol, 2013). Given the markedly different ion affinities of its splice variants, modulation of NKCC2 differential splicing is believed to impact NaCl reabsorption. To assess the validity of that hypothesis, we have developed a mathematical model of the macula densa cell transport, and incorporated that cell model into a previously-applied model of the thick ascending limb (Weinstein, Am J Physiol Renal Physiol, 2010). The macula densa model predicts a 27.4 and 13.1~mV depolarization of the basolateral membrane (as a surrogate for activation of tubuloglomerular feedback, TGF) when luminal [NaCl] is increased from 25 to 145~mM or luminal [K+] is increased from 1.5 to 3.5~mM, respectively, consistent with experimental measurements. Simulations indicate that with luminal solute concentrations consistent with in vivo conditions near the macula densa, NKCC2 operates near its equilibrium state. Results also suggest that modulation of NKCC2 differential splicing by low salt, which induces a shift from NKCC2-A to NKCC2-B primarily in the cortical thick ascending limb and macula densa cells, significantly enhances salt reabsorption in the thick ascending limb, and reduces Na+ and Cl- delivery to the macula densa by 3.7% and 12.5%, respectively. Simulation results also predict that NKCC2 isoform shift hyperpolarizes the macula densa basolateral cell membrane, which, taken in isolation, may inhibit TGF signal release. However, excessive early distal salt delivery and renal salt loss during a low-salt diet may be prevented by an asymmetric TGF response, which may be more sensitive to flow increases.
Betaine is an important osmolyte and is, compared to other organs, much more abundant in the kidneys where it enters cells in the medulla by the Betaine-GABA-Transporter 1 (BGT1) to balance osmoregulation in the counter current system. In wildtype BGT1 (wt-BGT1)-expressing oocytes, GABA-mediated currents were diminished by preincubation of the oocytes with 100 nM PMA or 5 µM DOG, activators of the protein kinase C (PKC), while application of staurosporine prior to application of DOG restored the response to GABA. Four potential phosphorylation sites on BGT1 were mutated to alanine by site-directed mutagenesis. Three mutants (T235A, S428A, and S564A) evoked GABA currents comparable in magnitude to the currents observed in wt-BGT1-expressing oocytes, whereas GABA currents in T40A were barely detectable. Uptake of [3H]GABA was also determined in HEK293 cells expressing EGFP-tagged BGT1 with the same mutations. T235A, S428A, and S564A showed up-regulation of GABA uptake after hypertonic stress and down-regulation by PMA similar to EGFP-wt-BGT1. In contrast T40A did not respond to either hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in MDCK cells revealed that T40A was present in cytoplasm after 24 hours of hypertonic stress while the other mutants and EGFP-wt-BGT1 were in the plasma membrane. All mutants, including T40A, co-migrated with wt-BGT1 on Western blots suggesting that they are full length proteins. T40A, however, cannot be phosphorylated as revealed using a specific anti-phospho antibody and therefore T40 may be important for trafficking and insertion of BGT1 in the plasma membrane.
Recent studies show that guidance molecules that are known to regulate cell migration during development may also play an important role in adult pathophysiologic states. One such molecule, semaphorin3A (sema3A), is highly expressed after acute kidney injury (AKI) in mice and humans, but its pathophysiological role is unknown. Genetic inactivation of sema3A protected mice from ischemia/reperfusion-induced AKI, improved tissue histology, reduced neutrophil infiltration, prevented epithelial cell apoptosis, and increased cytokine and chemokine excretion in urine. Pharmacological-based inhibition of sema3A receptor binding likewise protected against ischemia/reperfusion-induced AKI. In vitro, sema3A enhanced TLR4-mediated inflammation in epithelial cells, macrophages and dendritic cells. Moreover, administration of sema3A-treated bone marrow-derived dendritic cells exacerbated kidney injury. Finally, sema3A augmented cisplatin-induced apoptosis in kidney epithelial cells in vitro via expression of DFFA-like effector a (cidea). Our data suggest that the guidance molecule sema3A exacerbates acute kidney injury via promoting inflammation and epithelial cell apoptosis.
Numerous reports have linked cytoskeleton associated proteins with the regulation of ENaC activity. The purpose of this study was to determine the effect of actin cytoskeleton disruption by cytochalasin E on ENaC activity in Xenopus 2F3 cells. Here we show cytochalasin E treatment for 60 minutes can disrupt the integrity of the actin cytoskeleton in cultured Xenopus 2F3 cells. We show by single-channel patch clamp studies and measurements of short circuit current that ENaC activity, but not its density is altered by cytochalasin E induced disruption of the cytoskeleton. In non-treated cells 8 out of 33 patches (24%) had no measurable ENaC activity while in cytochalasin E treated cells 17 out of 32 patches (53%) had no activity. Analysis of those patches that did contain ENaC activity showed channel open probability significantly decreased from 0.081 ± 0.01 in non-treated cells to 0.043 ± 0.01 in cells treated with cytochalasin E. Transepithelial current from mpkCCD cells treated with cytochalasin E, cytochalasin D, or latrunculin B for 60 minutes was decreased compared to vehicle treated cells. The subcellular expression of fodrin changed significantly and several protein elements of the cytoskeleton decreased at least two fold after 60 minutes of cytochalasin E treatment. Cytochalasin E treatment disrupted the association between ENaC and MARCKS. The results presented here suggest disruption of the actin cytoskeleton by different compounds can attenuate ENaC activity through a mechanism involving changes in the subcellular expression of fodrin, several elements of the cytoskeleton, and destabilization of the ENaC-MARCKS complex.
Angiotensin II (Ang II) acting through its type 1 (AT1) receptor stimulates total ammonia (tNH3) production by the proximal tubule. The present studies explored the role of Ang II type 2 (AT2) receptors in modulating the stimulatory effects of Ang II on tNH3 production. Mouse S2 proximal tubule segments derived from 18-h and 7-d acid loaded mice, and non-acid-loaded controls were dissected and microperfused in vitro. Adding Ang II to the luminal perfusion solution resulted in different increments in tNH3 production rates in tubules derived from 18-h versus 7-d acid-loaded mice such that the increase in tNH3 production with Ang II was higher in tubules derived from 18-h acid-loaded mice compared to those derived from control and 7-d acid-loaded mice. Adding the AT2 receptor blocker PD123319 with Ang II increased Ang II-stimulated tNH3 production in S2 segments from control and 7-d acid-loaded mice but not in those from 18-h acid-loaded mice and this increased effect of PD123319 was associated with higher AT2 receptor protein levels in brush border membranes. Studies on cultured proximal tubule cells demonstrated that 2-h exposure to pH 7.0 reduced the modulating effect of PD123319 on Ang II-simulated tNH3 production and reduced cell surface AT2 receptor levels. We concluded that AT2 receptors reduce the stimulatory effect of Ang II on proximal tubule tNH3 production and that the time-dependent impact of AT2 receptor blockade on the Ang II-stimulated tNH3 production corresponded to time-dependent changes in AT2 receptor cell surface expression in the proximal tubule.
Efficient clearance of apoptotic cells (efferocytosis) prevents inflammation and permits repair following tissue injury. Kidney injury molecule-1 (KIM-1) is a receptor for phosphatidylserine, an "eat me" signal exposed on the surface of apoptotic cells that marks them for phagocytic clearance. KIM-1 is upregulated on proximal tubule epithelial cells (PTECs) during ischemic acute kidney injury (AKI), enabling efferocytosis by surviving PTECs. KIM-1 is spontaneously cleaved at its ectodomain region to generate a soluble fragment that serves a sensitive and specific biomarker for AKI, but the biological relevance of KIM-1 shedding is unknown. Here, we sought to determine how KIM-1 shedding might regulate efferocytosis. Using cells that endogenously and exogenously express KIM-1 we found that hydrogen peroxide-mediated oxidative injury or phorbol-12-myristate-13-acetate (PMA) treatment accelerated KIM-1 shedding in a dose-dependent manner. KIM-1 shedding was also accelerated when apoptotic cells were added. Accelerated shedding or the presence of excess soluble KIM-1 in the extracellular milieu significantly inhibited efferocytosis. We identified that tumor necrosis factor alpha-converting enzyme (TACE or ADAM17) mediates both the spontaneous and PMA-accelerated shedding of KIM-1. While accelerated shedding inhibited efferocytosis, we found that spontaneous KIM-1 cleavage does not affect the phagocytic efficiency of PTECs. Our results suggest that KIM-1 shedding is accelerated by worsening cellular injury and excess soluble KIM-1 competitively inhibits efferocytosis. These findings may be important in AKI when there is severe cellular injury.
Ammoniagenesis and gluconeogenesis are prominent metabolic features of the renal proximal convoluted tubule that contribute to maintenance of systemic acid-base homeostasis. Molecular analysis of the mechanisms that mediate the coordinate regulation of the two pathways required development of a cell line that recapitulates these features in vitro. By adapting porcine renal epithelial LLC-PK1 cells to essentially glucose-free medium, a gluconeogenic subline, termed LLC-PK1-FBPase+ cells, was isolated. LLC-PK1-FBPase+ cells grow in the absence of hexoses and pentoses and exhibit enhanced oxidative metabolism and increased levels of phosphate-dependent glutaminase. The cells also express significant levels of the key gluconeogenic enzymes, fructose-1,6-bisphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK). Thus, the altered phenotype of LLC-PK1-FBPase+ cells is pleiotropic. Most importantly, when transferred to medium that mimics a pronounced metabolic acidosis (9 mM HCO3-, pH 6.9), the LLC-PK1-FBPase+ cells exhibit a gradual increase in NH4+ ion production, accompanied by increases in glutaminase and cytosolic PEPCK mRNA levels and proteins. Therefore, the LLC-PK1-FBPase+ cells retained in culture many of the metabolic pathways and pH-responsive adaptations characteristic of renal proximal tubules. The molecular mechanisms that mediate enhanced expression of the glutaminase and PEPCK in LLC-PK1-FBPase+ cells have been extensively reviewed. The present review describes novel properties of this unique cell line and summarizes the molecular mechanisms that have been defined more recently using LLC-PK1-FBPase+ cells to model the renal proximal tubule. It also identifies future studies that could be performed using these cells.
Activation of histone deacetylases (HDACs) is required for renal epithelial cell proliferation and kidney development. However, their role in renal tubular cell survival and regeneration after acute kidney injury (AKI) remains unclear. In this study, we demonstrated that all class I HDAC isoforms (1, 2, 3, and 8) were expressed in the renal epithelial cells of the mouse kidney. Inhibition of class I HDACs with MS-275, a highly selective inhibitor, resulted in more severe tubular injury in the mouse model of AKI induced by folic acid or rhabdomyolysis, as indicated by worsening renal dysfunction, increased NGAL expression, and enhanced apoptosis and caspase-3 activation. Blocking class I HDAC activity also impaired renal regeneration as evidenced by decreased expression of renal Pax-2, vimentin and proliferating cell nuclear antigen. Injury to the kidney is accompanied by increased phosphorylation of epidermal growth factor receptor (EGFR), signal transducers and activators of transcription 3 (STAT3), and Akt. Inhibition of class I HDACs suppressed EGFR phosphorylation as well as reduced its expression. MS-275 was also effective in inhibiting STAT3 and Akt phosphorylation, but this treatment did not affect their expression levels. Taken together, these data suggest that the class I HDAC activity contributes to renal protection and functional recovery and is required for renal regeneration after AKI. Further, renal EGFR signaling is subject to regulation by this class of HDACs.
Energy depletion increases the renal production of 2',3'-cAMP (a positional isomer of 3',5'-cAMP that opens mitochondrial permeability transition pores) and 2',3'-cAMP is converted to 2'-AMP and 3'-AMP, which in turn are metabolized to adenosine. Because the enzymes involved in this "2',3'-cAMP-adenosine pathway" are unknown, we examined whether 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) participates in the renal metabolism of 2',3'-cAMP. Western blotting and real time PCR demonstrated expression of CNPase in rat glomerular mesangial, preglomerular vascular smooth muscle and endothelial, proximal tubular, thick ascending limb and collecting duct cells. Real time PCR established the expression of CNPase in human glomerular mesangial, proximal tubular and vascular smooth muscle cells; and the level of expression of CNPase was greater than that for phosphodiesterase 4 (major enzyme for the metabolism of 3',5'-cAMP). Over-expression of CNPase in rat preglomerular vascular smooth muscle cells increased the metabolism of exogenous 2',3'-cAMP to 2'-AMP. Infusions of 2',3'-cAMP into isolated CNPase wildtype (+/+) kidneys increased renal venous 2'-AMP, and this response was diminished by 63% in CNPase knockout (-/-) kidneys; whereas the conversion of 3',5'-cAMP to 5'-AMP was similar in CNPase +/+ versus -/- kidneys. In CNPase +/+ kidneys, energy depletion (metabolic poisons) increased kidney tissue levels of adenosine and its metabolites (inosine, hypoxanthine, xanthine and uric acid) without accumulation of 2',3'-cAMP. In contrast, in CNPase -/- kidneys, energy depletion increased kidney tissue levels of 2',3'-cAMP and abolished the increase in adenosine and its metabolites. Conclusion: Kidneys express CNPase, and renal CNPase mediates in part the renal 2',3'-cAMP-adenosine pathway. Key Words: 2',3'-cyclic adenosine monophosphate; 2'-adenosine monophosphate; 3'-adenosine monophosphate; adenosine; 2',3'-cyclic nucleotide 3'-phosphodiesterase; CNPase
Enhanced tubular reabsorption of salt is important in the pathogenesis of obesity-related hypertension but the mechanisms remain poorly defined. To identify changes in regulation of salt transporters in the kidney, C57BL/6 mice were fed 40% (high fat, HFD) or 12% (control, CD) fat diets for 14 weeks. Compared with controls, HFD mice had significantly greater elevations in weight, blood pressure, and serum insulin and leptin levels. Examining sodium transporter expression, NKCC2 was unchanged in whole kidney and reduced in the cortex; NCC and α- and -ENaC were unchanged; β-ENaC was reduced. Phosphorylation of NCC was unaltered. Activating phosphorylation of NKCC2 at S126 was increased 2.5-fold. Activation of SPAK/OSR-1 was increased in kidneys from HFD mice and enhanced phosphorylation of NKCC2 at T96/T101 was evident in the cortex. Increased activity of NKCC2 in vivo was confirmed with diuretic studies. HFD mice had reduced activating phosphorylation of AMPK in the renal cortex. In vitro, activation of AMPK led to a reduction in pSPAK/pOSR1 in MEF-AMPK+/+ cells, but no effect was seen in MEF-AMPK-/- cells, indicating an AMPK-mediated effect. Activation of the WNK/SPAK/OSR1 pathway with low sodium chloride solution invoked a greater elevation in pSPAK/pOSR1 in MEF-AMPK-/- cells than MEF-AMPK+/+ cells, consistent with a negative regulatory effect of AMPK on SPAK/OSR1 phosphorylation. In conclusion, this study identifies increased phosphorylation of NKCC2 on S126 as a hitherto unrecognized mediator of enhanced sodium reabsorption in obesity, and identifies a new role for AMPK in regulating the activity of SPAK/OSR1.
This study is aimed at characterizing medullary interstitial progenitor cells and to examine their capacity to induce tubular epithelial cell migration and proliferation. We have isolated a progenitor cell side population from a primary medullary interstitial cell line. We show that the medullary progenitor cells (MPCs) express CD24, CD44, CXCR7, CXCR4, nestin, and PAX7. MPCs are CD34 negative, which indicates that they are not bone marrow-derived stem cells. MPCs survive more than 50 passages, and when grown in epithelial differentiation medium develop phenotypic characteristics of epithelial cells. Inner medulla collecting duct (IMCD3) cells treated with conditioned medium from MPCs show significantly accelerated cell proliferation and migration. Conditioned medium from PGE2-treated MPCs induce tubule formation in IMCD3 cells grown in 3D matrigel. Moreover, most of the medullary progenitor cells express the pericyte marker PDGFR-b. Our study shows that the medullary interstitium harbors a side population of progenitor cells that can differentiate to epithelial cells and can stimulate tubular epithelial cell migration and proliferation. The findings of this study suggest that medullary pericyte/progenitor cells may play a critical role in collecting duct cell injury repair.
Adriamycin (ADR)-induced nephropathy in animals is an experimental analogue of human focal segmental glomerulosclerosis (FSGS), which presents as severe podocyte injury and massive proteinuria and has a poorly understood mechanism. The current study was designed to test the hypothesis that the peroxisome proliferator-activated receptor- coactivator 1α (PGC-1α)/mitochondria axis is involved in ADR-induced podocyte injury. Using MPC5 immortalized mouse podocytes, the ADR dose-dependently induced the downregulation of nephrin and podocin, cell apoptosis, and mitochondrial dysfunction based on the increase in mitochondrial reactive oxygen species (ROS) production, a decrease in mitochondrial DNA (mtDNA) copy number, and the reduction of mitochondrial membrane potential (MMP) and ATP content. Moreover, ADR treatment also remarkably reduced the expression of PGC-1α, an important regulator of mitochondrial biogenesis and function, in podocytes. Strikingly, PGC-1α overexpression markedly attenuated mitochondrial dysfunction, reduction of nephrin and podocin, and the apoptotic response in podocytes following ADR treatment. Moreover, the downregulation of PGC-1α and mitochondria disruption in podocytes were also observed in rat kidneys with ADR administration, suggesting that the PGC-1α/mitochondria axis is relevant to the in vivo ADR-induced podocyte damage. Taken together, these novel findings suggest that dysfunction of the PGC-1α/mitochondria axis is highly involved in ADR-induced podocyte injury. Targeting PGC-1α may be a novel strategy for treating ADR nephropathy and FSGS disease.
Hyperphosphatemia contributes to increased cardiovascular mortality through vascular calcification (VC) in patients with chronic kidney disease (CKD). Malnutrition and inflammation are also closely linked to increased risk of cardiovascular death in CKD. However, the effects of Pi overload on inflammation and malnutrition remain to be elucidated. The aim of the present study was to investigate the effects of dietary phosphate loading on the interaction among inflammation, malnutrition and VC in CKD. We used control rats fed normal diets and adenine-induced CKD rats fed diets with different phosphate concentrations ranging from 0.3% to 1.2% for 8 weeks. CKD rats showed dietary phosphate concentration-dependent increases in serum and tissue levels of tumor necrosis factor-α, and urinary and tissue levels of oxidative stress markers, and developed malnutrition (decrease in body weight, serum albumin and urinary creatinine excretion), VC and premature death without affecting kidney function. Treatment with 6% lanthanum carbonate blunted almost all changes induced by phosphate overload. Regression analysis showed that serum phosphate levels closely correlated with the extent of inflammation, malnutrition and VC. Also, in cultured human vascular smooth muscle cells, high-phosphate medium directly increased the expression of tumor necrosis factor-α in advance of the increase in osteochondrogenic markers. Our data suggest that dietary phosphate overload induces systemic inflammation and malnutrition, accompanied by VC and premature death in CKD, and inhibition of phosphate loading through dietary or pharmacological interventions, or anti-inflammatory therapy may be a promising treatment for the prevention of malnutrition-inflammation-atherosclerosis syndrome.
Using dual cell patch clamp recording, we examined pericyte, endothelial and myoendothelial cell-to-cell communication in descending vasa recta. Graded current injections into pericytes or endothelia yielded input resistances of 220 ± 21 and 128 ± 20 MOhm, respectively (P < 0.05). Injection of positive or negative current into an endothelial cell depolarized and hyperpolarized adjacent endothelial cells, respectively. Similarly, current injection into a pericyte depolarized and hyperpolarized adjacent pericytes. During myoendothelial studies, current injection into a pericyte or an endothelial cell yielded small, variable, but significant change of membrane potential in heterologous cells. Membrane potentials of paired pericytes or paired endothelia were highly correlated and nearly identical. Paired measurement of resting potentials in heterologous cells were also correlated, but with slight hyperpolarization of the endothelium relative to the pericytes, -55.2 ± 1.8 vs -52.9 ± 2.2 mV (P < 0.05). During dual recordings, angiotensin II or bradykinin stimulated temporally identical variations of pericyte and endothelial membrane potential. Similarly, voltage clamp depolarization of pericytes or endothelial cells induced parallel changes of membrane potential in the heterologous cell types. We conclude that the DVR endothelial syncytium is of lower resistance than the pericyte syncytium, and that high resistance myoendothelial coupling also exists. The myoendothelial communication between pericytes and endothelium maintains near identity of membrane potentials at rest and during agonist stimulation. Finally, endothelia membrane potential lies slightly below pericyte membrane potential, suggesting a tonic role for the former to hyperpolarize the latter and provide a brake on vasoconstriction.
LMX1B is a transcription factor of the LIM-homeodomain type and is implicated in the development of diverse structures such as limbs, kidneys, eyes and the brain. Furthermore, LMX1B has been implicated in nail-patella syndrome, which is predominantly characterized by malformation of limbs and nails, and in 30% of patients, nephropathy, including renal fibrosis, is observed. Since no reports were available that studied the link between LMX1B expression and renal interstitial fibrosis, we explored if LMX1B affects typical markers of fibrosis, e.g., extracellular matrix components, profibrotic factors, and apoptosis as the final detrimental consequence. We recently showed that LMX1B acts as a negative regulator of transforming growth factor-βl, collagen-III, fibronectin, cleaved caspase-3, and cell apoptosis rate in a renal tubular epithelial cell system under hypoxic conditions. Here we confirmed these results in unilateral ureteral obstructed rats. Furthermore, LMX1B was distinctly expressed throughout the glomerulus and the tubule lining, including the epithelial cells. Knockdown of LMX1B aggravated the expression of fibrosis markers, oxidative stress, and apoptosis compared with the already increased levels due to unilateral ureteral obstruction, whereas overexpression attenuated these effects. In conclusion, reduced LMX1B levels clearly represent a risk factor for renal fibrosis, whereas overexpression affords some level of protection. In general, LMX1B may be considered to be a negative regulator of the fibrosis index, transforming growth factor-βl, collagen-III, fibronectin, cleaved caspase-3, cell apoptosis, reactive oxygen species and malon dialdehyde (r= -0.756, -0.698, -0.921, -0.923, -0.843, -0.794, -0.883, -0.825; each P<0.01).
The amount of sodium and potassium in the diet promotes significant changes in endothelial cell function. In the present study, a series of in vitro and in vivo experiments determined the role of sodium and potassium in the regulation of two Pleckstrin Homology domain-containing intracellular signaling molecules - phospholipase C gamma-1 (PLC-1) and Epithelial and endothelial tyrosine kinase/Bone marrow tyrosine kinase on chromosome X (Bmx) - and agonist-generated calcium signaling in the endothelium. Extracellular [K+] regulated the levels of activated PLC-1, Bmx and carbachol-stimulated intracellular Ca2+ mobilization in human endothelial cells. Additional experiments confirmed that high-conductance calcium-activated potassium channels and phosphatidylinositol 3-kinase mediated these effects. The content of sodium and potassium in the diet also regulated Bmx levels in endothelial cell and activated PLC-1 levels in rats in vivo. The effects of dietary potassium on Bmx was more pronounced in rats on the high-salt diet, compared to rats on the low-salt diet. These studies elucidated an endothelial cell signaling mechanism regulated by electrolytes, further demonstrating an integral relationship between endothelial cell function and dietary sodium and potassium content.
Unconjugated bilirubin is an endogenous circulating antioxidant, bound to albumin, and therefore is retained in the vascular compartment. Bilirubin has well-documented neurotoxic effects in infants, however, current evidence indicates mildly elevated bilirubin is associated with protection from cardiovascular disease and all-cause mortality in adults. Recent clinical studies show mildly elevated bilirubin is associated with protection from kidney damage and dysfunction, in addition to cardiovascular events and all-cause mortality in patients undergoing hemodialysis. This is the first review to examine the clinical evidence and summarize the potential mechanisms of action that link bilirubin to protection from kidney damage, subsequent kidney failure and dialysis related mortality. With this understanding, it is hoped that new therapies will be developed to prevent renal dysfunction and mortality from cardiovascular disease in at risk individuals.
Oxidative stress promotes vascular dysfunction in chronic kidney disease (CKD). We utilized the cutaneous circulation to test the hypothesis that reactive oxygen species derived from NADPH oxidase and xanthine oxidase impair nitric oxide (NO)-dependent cutaneous vasodilation in CKD. Twenty subjects, 10 stage 3 and 4 patients with CKD (61±4 years; 5 male/5 female; eGFR: 39 ± 4 ml•min-1•1.73m-2) and 10 healthy controls (55±2 years; 4 male/6 female; eGFR: >60 ml•min-1•1.73m-2) were instrumented with 4 intradermal microdialysis fibers for the delivery of 1) Ringers solution (Control), 2) 10µM Tempol (scavenge superoxide), 3) 100µM apocynin (NAD(P)H oxidase inhibition), and 4) 10µM allopurinol (xanthine oxidase inhibition). Skin blood flow was measured via laser Doppler flowmetry during standardized local heating (42°C). 10mM L-NAME was infused to quantify the NO-dependent portion of the response. Cutaneous vascular conductance (CVC) was calculated as a percentage of the maximum CVC achieved during sodium nitroprusside infusion at 43°C. Cutaneous vasodilation was attenuated in patients with CKD (77±3 vs. 88±3 %, p=0.01), but augmented with Tempol and apocynin (Tempol: 88±2 (p=0.03), apocynin: 91±2 % (p=0.001). The NO-dependent portion of the response was reduced in patients with CKD (41±4 vs. 58±2 %, p=0.04), but improved with Tempol and apocynin (Tempol: 58±3 (p=0.03), apocynin: 58±4 % (p=0.03). Inhibition of xanthine oxidase did not alter cutaneous vasodilation in either group (p>0.05). These data suggest that NAD(P)H oxidase is a source of reactive oxygen species and contributes to microvascular dysfunction in patients with CKD.
Sodium-glucose cotransporter 2 (SGLT2) inhibitors showed glucose lowering effect in type 2 diabetes patients through inducing renal glucose excretion. Detailed analysis of the mechanism of glucosuric effect of SGLT2 inhibition, however, has been hampered by limitations of clinical study. Here, we investigated the mechanism of urinary glucose excretion using non-human primates with SGLT inhibitors tofogliflozin and phlorizin, both in vitro and invivo. In cells overexpressing cynomolgus monkey SGLT2 (cSGLT2), both tofogliflozin and phlorizin competitively inhibited uptake of the substrate (AMG, α-methyl-D-glucopyranoside). Tofogliflozin was found to be a selective cSGLT2 inhibitor, inhibiting cSGLT2 more strongly than did phlorizin, with selectivity toward cSGLT2 1000 times that toward cSGLT1; phlorizin was found to be a non-selective cSGLT1/2 inhibitor. In a glucose titration study in cynomolgus monkeys under conditions of controlled plasma drug concentration, both tofogliflozin and phlorizin increased fractional excretion of glucose (FEG) by up to 50% under hyperglycemic conditions. By fitting the titration curve using a newly introduced method that avoids variability in estimating the threshold of renal glucose excretion, we found that tofogliflozin and phlorizin lowered the threshold and extended the splay in a dose-dependent manner without significantly affecting the tubular transport maximum for glucose (TmG). Our results demonstrate the contribution of SGLT2 to renal glucose reabsorption (RGR) in cynomolgus monkeys and demonstrate that competitive inhibition of cSGLT2 exerts a glucosuric effect by mainly extending splay and lowering threshold without affecting TmG.
Bilateral ureteral obstruction (BUO) is associated with renal damage and impaired ability to concentrate urine and is known to induce alterations in an array of kidney proteins. The aim of this study was to identify acute proteomic alterations induced by BUO. Rats were subjected to BUO for 2, 6, or 24 h. Mass spectrometry-based proteomics was performed on renal inner medulla, and protein changes in the obstructed group were identified. Significant changes were successfully identified for 109 proteins belonging to different biological classes. Interestingly, proteins belonging to the cytoskeleton and proteins related to cytoskeletal regulation were found to be biologically enriched in BUO using online-accessible tools. Western blots confirmed the selected results demonstrating acute downregulation of proteins belonging to all 3 cytoskeletal components. The microfilament protein β-actin and the intermediate filament proteins Pankeratin and Vimentin were all downregulated. B-tubulin, an important microtubular protein, was found to be significantly downregulated after 24 h. Also, there was significant upregulation of Cofilin, an actin-binding protein known to be upregulated in other nephropathy models. Furthermore, both upregulation and downregulation of cytoskeletal motor and regulatory proteins were observed. These findings were confirmed by immunohistochemistry, which clearly showed alterations in labeling in the inner medulla. Interestingly, we were able to confirm selected results in mpkCCD cells exposed to mechanical stretch. Our findings add to the knowledge of BUO-induced acute changes in the renal cytoskeleton, and suggest that these molecular changes are partly mediated by increased stretch of the cells during obstruction.
Modulation of NCC activity is essential to adjust K+ excretion in the face of changes in dietary K+ intake. We used previously characterized genetic mouse models to assess the role of SPAK and WNK4 in the modulation of NCC by K+ diets. SPAK knockin and WNK4 knockout mice were placed on normal, low or high K+ diets for four days. Low K+ diet decreased and high K+-citrate diet increased plasma aldosterone levels, but both diets were associated with increased phosphorylation of NCC (pNCC; T44/48/53) and pSPAK/OSR1 (S383/S325). The effect of the low K+ diet on SPAK phosphorylation persisted in the WNK4 knockout and SPAK knockin mice, while the effects of angiotensin II on NCC and SPAK were lost in both mouse colonies, suggesting that for NCC activation by angiotensin II integrity of the WNK4/SPAK pathway is required, while for low K+ diet SPAK phosphorylation occurred despite the absence of WNK4, suggesting the involvement of another WNK kinase. Additionally, because NCC activation also occurred in SPAK knockin mice, it is possible that loss of SPAK was compensated by OSR1. The positive effect of high K+ diet was observed when the accompanying anion was citrate, while high KCl diet reduced NCC phosphorylation. However, the effect of high K+-citrated diet was aldosterone-dependent and neither metabolic alkalosis induced by bicarbonate, nor citrate administration in the absence of K+ increased NCC phosphorylation, suggesting that it was not due to the citrate-induced metabolic alkalosis. Thus, accompanying anion might modulate the NCC response to high potassium diet.
The kidney is one of the major loci for the expression of cystathionine β-synthase (CBS) and cystathionine -lyase (CTH). While CBS-deficient (Cbs-/-) mice display homocysteinemia/methioninemia and severe growth retardation, and rarely survive beyond the first 4 weeks, CTH-deficient (Cth-/-) mice show homocysteinemia/cystathioninemia but develop with no apparent abnormality. This study examined renal amino acid reabsorption in those mice. Although both 2-week-old Cbs-/- and Cth-/- mice had normal renal architecture, their serum/urinary amino acid profiles largely differed from wild-type mice. The most striking feature was marked accumulation of Met and cystathionine in serum/urine/kidney samples of Cbs-/- and Cth-/- mice, respectively. Levels of some neutral amino acids (Val, Leu, Ile, and Tyr) that were not elevated in Cbs-/- serum were highly elevated in Cbs-/- urine, and urinary excretion of other neutral amino acids (except Met) was much higher than expected from their serum levels, demonstrating neutral aminoaciduria in Cbs-/- (not Cth-/-) mice. Because the bulk of neutral amino acids is absorbed via a B0AT1 transporter and Met has the highest substrate affinity for B0AT1 than other neutral amino acids, hypermethioninemia may cause hyperexcretion of neutral amino acids.
Acute kidney injury (AKI) is an independent risk factor of the development of chronic kidney disease (CKD). Kidney fibrosis is a typical feature of CKD and is characterized as an expansion of interstitium due to increases in extracellular matrix molecules and interstitial cells caused by accumulations of extrarenal cells and by the proliferation or differentiation of intrarenal cells. However, the role of bone marrow-derived cells (BMDCs) in AKI-induced kidney fibrosis remains to be defined. Here, we investigated the role of BMDCs in kidney fibrosis following ischemia/reperfusion injury (IRI)-induced AKI in GFP-expressing bone marrow (BM) chimeric mice. IRI resulted in severe fibrotic changes in kidney tissues and dramatically increased interstitial cell numbers. Furthermore, GFP-expressing BMDCs accounted for over 80% of interstitial cells in fibrotic kidneys. Interstitial GFP-expressing cells expressed α-smooth muscle actin (a myofibroblast marker), fibroblast-specific protein-1 (a fibroblast marker), collagen III, and F4/80 (a macrophage marker). Over 20% of interstitial cells were bromodeoxyuridine (BrdU)-incorporating (proliferating) cells and of these 80% cells were GFP-expressing BMDCs. Daily treatment of IRI mice with apocynin (a NADPH oxidase inhibitor which functions as an antioxidant) from the day after surgery until sacrifice slightly inhibited these changes with a small reduction of fibrosis. Taken together, our findings show that BMDCs make a major contribution to IRI-induced fibrosis due to their infiltration, subsequent differentiation, and proliferation in injured kidneys, suggesting that BMDCs be considered an important target for the treatment of kidney fibrosis.
The emerging role of TRPC6 as a central contributor to various pathologic processes affecting podocytes has generated interest in the development of therapeutics to modulate its function. Recent insights into the regulation of TRPC6 have revealed protein kinase G (PKG) as a potent negative modulator of TRPC6 conductance and associated signaling via its phosphorylation at 2 highly conserved amino acid residues, threonine 69 (Thr69 in murines; Thr70 in humans) and serine 321 (Ser321 in murines; Ser322 in humans). Here, we tested the role of PKG in modulating TRPC6-dependent responses in primary and conditionally immortalized mouse podocytes. TRPC6 was phosphorylated at Thr69 in non-stimulated podocytes but this declined upon angiotensin II (Ang II) stimulation or overexpression of a constitutively active calcineurin phosphatase (CA-Cn). Ang II induced podocyte motility in an in vitro wound-assay, and this was reduced 30-60% in cells overexpressing a phosphomimetic mutant TRPC6 (TRPC6T70E/S322E) or activated PKG (p<0.05). Pretreatment of podocytes with the PKG agonists SNAP (NO-donor), 8Br-cGMP, Bay 41-2772 (soluble guanylate cyclase activator), or phosphodiesterase 5 inhibitor 4-{[3',4'-(Methylenedioxy)benzyl]amino}[7]-6-methoxyquinazoline (PDE5i), attenuated Ang II-induced Thr69 de-phosphorylation and also inhibited TRPC6-dependent podocyte motility by 30-60%. These data reveal that PKG activation strategies including PDE5 inhibition ameliorate Ang II-induced podocyte dysmotility by targeting TRPC6 in podocytes, highlighting the potential therapeutic utility of these approaches to treat hyperactive TRPC6-dependent glomerular disease.
The inorganic phosphate (Pi) concentration of mammalian cerebrospinal fluid (CSF) is about one-half that of plasma, a phenomenon also shown here in the spiny dogfish, Squalus acanthias. The objective of the present study was to characterize the possible role of the choroid plexus (CP) in determining CSF [Pi]. The large sheet-like IVth CP of the shark was mounted in Ussing chambers where unidirectional 33Pi fluxes revealed potent active transport from CSF to blood side under short-circuited conditions. The flux ratio was 8:1 with an average transepithelial resistance of 87 ± 17.9 x cm2 and electrical potential difference of +0.9 ± 0.17 mV, CSF side positive. The active Pi absorption from CSF was inhibited by 10 mM arsenate, 0.2 mM ouabain, Na+-free medium, and by increasing [K+] from 5 mM to 100 mM. Li+ stimulated transport 2-fold compared to Na+-free medium. Phosphonoformic acid (1 mM) had no effect on active Pi transport. RT-PCR revealed both PiT1 and PiT2 (SLC20 family) gene expression, but no NaPiII (SLC34 family) expression, in the shark CP. PiT2 immunoreactivity was shown by immunoblot, and localized by immunohistochemistry in (or near) the CP apical microvillar membranes of both shark and rat. PiT1 appeared to be localized primarily to the vascular endothelial cells. Together, the data indicate that the CP actively removes Pi from the CSF. This process has transport properties consistent with a PiT-2, Na+-dependent transporter which is located in the apical region of the CP epithelium.
(Pro)renin receptor (PRR) is predominantly expressed in the distal nephron where it is activated by angiotensin II (AngII), resulting in increased renin activity in the renal medulla thereby amplifying the de novo generation and action of local AngII. The goal of the present study was to test the role of cycloxygenase-2 (COX-2) in meditating AngII-induced PRR expression in the renal medulla in vitro and in vivo. Exposure of primary rat inner medullary collecting duct (IMCD) cells to AngII induced sequential increases in COX-2 and PRR protein expression. When the cells were pretreated with a COX-2 inhibitor NS-398, Ang II-induced upregulation of PRR protein expression was almost completely abolished, in parallel with the changes in medium active renin content. The inhibitory effect of NS-398 on the PRR expression was reversed by adding exogenous PGE2. A 14-day AngII infusion elevated renal medullary PRR expression and active and total renin content in parallel with increased urinary renin, all of which were remarkably suppressed by the COX-2 inhibitor celecoxib. In contrast, plasma and renal cortical active and total renin content were suppressed by AngII treatment, an effect that was unaffected by COX-2 inhibition. Systolic blood pressure (SBP) was elevated with AngII infusion, which was attenuated by the COX-2 inhibition. Overall, the results obtained from in vitro and in vivo studies have established a crucial role of COX-2 in mediating upregulation of renal medullary PRR expression and renin content during AngII hypertension.
Integrin αvβ8 is most abundantly expressed in kidney, brain and female reproductive organs, and its cognate ligand is latent transforming growth factor-β (LTGFβ). Kidney αvβ8 localizes to mesangial cells, and global Itgb8 deletion results in embryonic lethality due to impaired placentation and cerebral hemorrhage. To circumvent the lethality, and better define kidney αvβ8 function, Cre-lox technology was used to generate mesangial-specific Itgb8-null mice. PDGFBR-Cre mice crossed with a reporter strain revealed functional Cre recombinase activity in a predicted mesangial pattern. However, mating between two different PDGFBR-Cre or Ren1d-Cre strains with Itgb8flox/- mice consistently resulted in incomplete recombination, with no renal phenotype in mosaic offspring. Induction of a renal phenotype with Habu snake venom, a reversible mesangiolytic agent, caused exaggerated glomerular capillary microaneurysms and delayed recovery in Cre+/-PDGFRBflox/- mice compared to Cre+/-PDGFRBflox/+ controls. To establish the mechanism, in vitro studies were conducted in Itgb8-null versus Itgb8-expressing mesangial cells and fibroblasts, which revealed β8-regulated adhesion to Arg-Gly-Asp (RGD) peptides within mesangial-conditioned matrix, as well as β8-dependent migration on RGD-containing LTGFβ or vitronectin matrix. We speculate that kidney αvβ8 indirectly controls glomerular capillary integrity through mechanical tension generated by binding RGD peptides in mesangial matrix, and healing after glomerular injury may be facilitated by mesangial cell migration, which is guided by transient β8 interaction with RGD ligands.
Background: Acute kidney injury (AKI) is associated with dysregulated iron metabolism, which may play a significant role in cellular injury. The effect of hemodialysis (HD) on iron metabolism in AKI therapy has not been well defined. Methods: The effects of HD on iron parameters were tested in control rats and bilateral nephrectomy (BNx) rats. The BNx rats were divided into the following 3 groups: 1) the Sham-operated group (BNx-Sham), 2) the BNx group, and 3) the HD group (BNx-HD), which received HD therapy 40-45 hours after BNx. Sections of the liver or spleen were stained with Berlin blue to examine the accumulation of iron. The mRNA levels of hepcidin and ferroportin1 in the spleen and liver were also quantified using RT-PCR. Results: In the BNx group, the plasma iron and hematocrit levels were decreased, and hepcidin levels were increased. The iron staining in the spleen in the BNx group was significantly more intense than that in the BNx-Sham group; however, after an HD session, splenic iron staining diminished to the level of the Sham group along with an increase in plasma iron and a decrease in hepcidin. Conclusions: BNx moved iron from hemoglobin and the plasma to the spleen, which is associated with an increase in plasma hepcidin. A single HD session accelerated the release of iron from the spleen, and the increased plasma iron was linked to the removal of hepcidin. Our data suggested that hepcidin might dynamically modulate the iron metabolism in BNx as well as in HD.
Lower urinary tract symptoms (LUTS) become prevalent with aging and affect millions; however, therapy is often ineffective because etiology is unknown. Existing assays of LUT function in animal models are often invasive, however a non-invasive assay is required to study symptom progression and determine genetic correlates. Here we present a spontaneous voiding assay which is simple, reproducible, quantitative and non-invasive. Young females from eight strains of inbred mice - 129S1/SvImJ, A/J, C57BL/6J, NOD/ShiLtJ, NZO/H1LtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ - were tested for urination patterns on filter paper. Repeat testing at different times of the day showed minimal within individual and within strain variation, but all parameters (spot number, total volume, percentage area in primary void, corner voiding and center voiding) exhibited significant variation between strains. Calculation of the intraclass correlation coefficient, an estimate of broad sense heritability (H2), for each time of day and for each voiding parameter, revealed highly significant heritability (spot number, 61%; percentage urine in primary void, 90%; total volume, 94% [afternoon data]). Cystometrograms confirmed strong strain-specific urodynamic characteristics. Behavior/voiding correlation analysis showed no correlation with anxiety phenotypes. Diagnostically, the assay revealed LUTS in several systems, including demonstration of voiding abnormalities in older C57BL/6J mice (18-24 months), in a model of protamine sulfate induced urothelial damage and in a model of sucrose-induced diuresis. This assay may be used to derive pathophysiological LUT readouts from mouse models. Voiding characteristics are heritable traits, opening the way for genetic studies of LUTS using outbred mouse populations.
Older literature suggested that the plasma sodium concentration is not individual - that it is neither intrinsic to an individual nor reproducible, longitudinally. We recently observed that plasma sodium concentration is heritable. Because demonstrable heritability requires individuality of the relevant phenotype, we hypothesized that plasma sodium concentration was substantially individual. In two large health plan-based cohorts, we demonstrated individuality of the plasma sodium concentration over a ten-year interval; intraclass correlation coefficient (ICC) averaged 0.4 - 0.5. Individuality of plasma sodium increased significantly with age. Plasma sodium individuality was equal to or only slightly less than that for plasma glucose, but was less than the individuality for creatinine. Individuality of plasma sodium was further confirmed by comparing the Pearson correlation coefficient for within-individual vs. between-individual pairs of sodium determinations, and via application of the agreement index. Furthermore, the distribution of all sodium determinations for all participants within a population was similar to the distribution for the mean sodium concentration for individuals within that population. Therefore, the near-normal distribution of plasma sodium measurements within a population is likely not attributable to assay-specific factors but rather to genuine and durable biological variability in osmotic set-point. In aggregate, these data strongly support the individuality of the plasma sodium concentration. They further indicate that serial plasma sodium values for any given individual tend to cluster around a patient-specific set-point, and that these set-points vary among individuals.
The renin-angiotensin-aldosterone system and cardiac natriuretic peptides (atrial and B-type natriuretic peptide, ANP / BNP) are opposing control mechanisms for arterial blood pressure. Accordingly, an inverse relationship between the plasma concentrations of renin (PRC) and ANP exists in most circumstances. However, PRC and ANP levels are both elevated in renovascular hypertension. Because ANP can directly suppress renin release, we used ANP knockout mice (ANP-/-) to investigate whether high ANP levels attenuate the increase in PRC in response to renal hypoperfusion, thus buffering renovascular hypertension. ANP-/- mice were hypertensive and had reduced PRC compared with that in ANP+/+ under control conditions. Unilateral renal artery stenosis (2-kidney, 1-clip, 2k1c) for 1 week induced similar increases in the blood pressure and in the PRC in both genotypes. Unexpectedly, the plasma BNP concentrations in ANP-/- significantly increased in response to 2k1c, potentially compensating for the lack of ANP. In fact, in mice lacking guanylyl cyclase A (GC-A-/-), which is the common receptor for both ANP and BNP, the renovascular hypertension was markedly augmented compared with that in GC-A+/+. However, the higher blood pressure of GC-A-/- was not caused by a disinhibition of the renin system because the PRC and renal renin synthesis were significantly lower in GC-A-/- than in GC-A+/+. Thus, natriuretic peptides buffer renal vascular hypertension via renin-independent effects, such as vasorelaxation. The latter possibility is supported by experiments in isolated perfused mouse kidneys, in which physiological concentrations of ANP and BNP elicited renal vasodilatation and attenuated renal vasoconstriction in response to angiotensin II.
Dual renin-angiotensin system (RAS) blockade in diabetic nephropathy is no longer feasible because of the profit/side effect imbalance. (Pro)renin receptor ((P)RR) blockade with HRP has been reported to exerts beneficial effects in various diabetic models in a RAS-independent manner. To what degree (P)RR blockade adds benefit on top of RAS blockade is still unknown. Here we treated diabetic TGR(mREN2)27 rats, a well-established nephropathy model with high prorenin levels (allowing continuous (P)RR stimulation in vivo), with HRP on top of renin inhibition with aliskiren. Aliskiren alone lowered blood pressure and exerted renoprotective effects, as evidenced by reduced glomerulosclerosis, diuresis, proteinuria, albuminuria, and urinary aldosterone levels, and diminished renal (P)RR and AT1 receptor expression. It also suppressed plasma and tissue RAS activity, and suppressed cardiac ANP and BNP expression. HRP, when given on top of aliskiren, did not alter the effects of renin inhibition on blood pressure, RAS activity or aldosterone. Yet, it counteracted the beneficial effects of aliskiren in the kidney, induced hyperkalemia and increased plasma plasminogen activator-inhibitor 1, renal cyclo-oxygenase-2 and the cardiac collagen content. All these effects have been linked to (P)RR stimulation, suggesting that HRP might in fact act as a partial agonist. Therefore, the use of HRP on top of RAS blockade in diabetic nephropathy is not advisable.
Metabolic and functional abnormalities in the kidney precede or coincide with the initiation of overt hypertension in the Dahl salt-sensitive SS rat. However, renal histological injury in SS rats is mild before the development of overt hypertension. We performed electron microscopy analysis in 7 week old SS rats and salt-insensitive consomic SS.13BN rats and Sprague-Dawley (SD) rats fed a 4% NaCl diet for 7 days. Long mitochondria (>2μm) accounted for a significantly smaller fraction of mitochondria in medullary thick ascending limbs in SS rats (4% ± 1%) than in SS.13BN rats (8% ± 1%, P<0.05 vs. SS) and SD rats (9% ± 1%, P<0.01 vs. SS), consistent with previous findings of mitochondrial functional insufficiency in the medulla of SS rats. Long mitochondria in proximal tubules, however, were more abundant in SS rats than in SS.13BN and SD rats. The width of endoplasmic reticulum, an index of endoplasmic reticulum stress, was significantly greater in medullary thick ascending limbs of SS rats (107 ± 1 nm) than SS.13BN rats (95 ± 2 nm, P<0.001 vs. SS) and SD rats (74 ± 3 nm, P<0.01 vs. SS or SS.13BN). The tubules examined were indistinguishable under light microscopy. These data indicate that ultrastructural abnormalities occur in the medullary thick ascending limbs of SS rats prior to the development of histological injury in renal tubules, providing a potential structural basis contributing to subsequent development of overt hypertension.
Extracellular vesicles have been isolated in various body fluids including urine. The cargo of urinary extracellular vesicles (uEVs) is composed of proteins and nucleic acids reflecting the physiological and possibly the pathophysiological state of cells lining the nephron. Because urine is a non-invasive and readily available biofluid, the discovery of uEVs has opened a new field of biomarker research. Their potential use as diagnostic, prognostic, or therapeutic biomarkers for various kidney diseases, including glomerulonephritis, acute kidney injury, tubular disorders and polycystic kidney disease is currently being explored. Some challenges, however, remain. These challenges include the need to standardize isolation methods, normalization between samples, and validation of candidate biomarkers. Also, the development of a high-throughput platform to isolate and analyze uEVs, for example an enzyme-linked immunosorbent assay, is desirable. Here, we review recent studies on uEVs dealing with kidney physiology and pathophysiology. Furthermore, we discuss new and exciting developments regarding vesicles, including their role in cell-to-cell communication and the possibility to use vesicles as therapy for kidney disorders.
Glomerular matrix accumulation is a hallmark of diabetic renal disease. The serine/threonine protein kinase C β1 (PKCβ1) mediates glucose-induced Akt S473 phosphorylation, RhoA activation, transforming growth factor β1 (TGFβ1) upregulation, and finally leads to matrix upregulation in mesangial cells (MCs). It has been reported that glucose-induced PKCβ1 activation is dependent on caveolin-1 and the presence of intact caveolae in MCs, however, whether activated PKCβ1 regulate caveolin-1 expression and phosphorylation is unknown. Here, we show that although caveolin-1 protein level had no significant change, PKCβ specific inhibitor LY333531 blocked caveolin-1 Y14 phosphorylation in high glucose (HG)-treated MCs and in the renal cortex of diabetic rats. Src specific inhibitor SU6656 prevented HG-induced association between PKCβ1 and caveolin-1, and PKCβ1 membrane translocation, whereas PKCβ1 siRNA failed to block Src activation, indicating that Src kinase is the upstream of PKCβ1 activation. Although PKCβ-specific inhibitor LY333531 blocked PKCβ1 membrane translocation, it had no effect on PKCβ1/caveolin-1 association, suggesting that PKCβ1 activation requires interaction of caveolin-1 and PKCβ1. PKCβ1-mediated Akt S473 phosphorylation, RhoA activation and fibronectin upregulation in response to HG were prevented by Src specific inhibitor SU6656 and nonphosphorylatable mutant caveolin-1 Y14A. In conclusion, Src activation by HG mediates PKCβ1/caveolin-1 association and PKCβ1 activation, which assists in caveolin-1 Y14 phosphorylation by Src kinase. The downstream effects including Akt S473 phosphorylation, RhoA activation and fibronectin upregulation require caveolin-1 Y14 phosphorylation. Caveolin-1 is thus important mediator of the profibrogenic process in diabetic renal disease.
DW1029M is a botanical extract consisting of Morus bark (MB) and Puerariae radix (PR), produced by Dong-Wha Pharmaceutical, Inc. Co. for nephroprotective drug development; it has been in phase II clinical trials in Korea. In our mechanistic investigations, we found that DW1029M inhibits advanced glycation end products (AGEs), rat lens aldose reductase (RLAR), and transforming growth factor β1(TGFβ1) signaling, all of which are implicated in diabetic complications such as diabetic nephropathy and diabetic retinopathy. DW1029M inhibits AGE formation via Fe2+ ion chelation. The extract contains 13 active constituents that inhibit AGE formation, eight that inhibit RLAR activity, and one inhibitor of TGFβ1 signaling. Our results suggest DW1029M protects against diabetic nephropathy via blockade of AGE formation, RLAR activity, and TGFβ1 signaling.
Acute kidney injury (AKI) is associated with mitochondrial fragmentation, which contributes to mitochondrial damage and tubular cell apoptosis. Mitochondrial fragmentation involves the cleavage of both mitochondrial outer and inner membranes. Cleavage of the outer membrane results from Drp-1-mediated fission activation and Bak-promoted fusion arrest, but the molecular mechanism of inner membrane cleavage remains elusive. OMA1-mediated proteolysis of OPA1, a key inner membrane fusion protein, was recently suggested to account for inner membrane cleavage during cell stress. In this study, we have determined the role of OMA1 in OPA1 proteolysis and mitochondrial fragmentation in experimental models of ischemic AKI. In ATP-depletion injury, knockdown of OMA1 suppressed OPA1 proteolysis, mitochondrial fragmentation, cytochrome c release and consequent apoptosis in renal proximal tubular cells. In mice, OMA1-deficiency prevented ischemic AKI as indicated by better renal function, less tubular damage, and lower apoptosis. OPA1 proteolysis and mitochondrial injury during ischemic AKI were ameliorated in OMA1-deficient mice. Thus OMA1-mediated OPA1 proteolysis plays an important role in the disruption of mitochondrial dynamics in ischemic AKI.
Cystogenesis and tubulogenesis are basic building blocks for many epithelial organs, including the kidney. Most researchers have used two-dimensional (2D) cell culture to investigate signaling pathways downstream of hepatocyte growth factor (HGF). We hypothesize that three-dimensional (3D) collagen-grown Madin-Darby canine kidney (MDCK) cells, which form cysts and then tubulate in response to hepatocyte growth factor (HGF), is a much more in vivo-like system for the identification of novel tubulogenes. Using a canine microarray containing over 20,000 genes, 2,417 genes were identified as potential tubulogenes that were differentially regulated exclusively in 3D-grown MDCK cells. Among these, 840 were dependent on MAPK signaling. Importantly, this work shows that many putative tubulogenes previously identified via microarray analysis of 2D cultures, including by us, do not change in 3D-culture and vice versa. The use of a 3D-culture system allowed for the identification of novel MAPK-dependent and independent genes that regulate early renal tubulogenesis in vitro, e.g. matrix metalloproteinase 1 (MMP1). Knockdown of MMP1 led to defects in cystogenesis and tubulogenesis in 3D-grown MDCK cells, most likely due to problems establishing normal polarity. We suggest that data obtained from 2D-cultures, even those using MDCK cells treated with HGF, should not be automatically extrapolated to factors important for cystogenesis and tubulogenesis. Instead, 3D-culture that more closely replicates the biological environment, and is therefore a more accurate model for identifying tubulogenes, is preferred. Results from the present analysis will be used to build a more accurate model of the signaling pathways that control cystogenesis and tubulogenesis.
Podocytes are key cells in the glomerular filtration barrier with a major role in the development of diabetic nephropathy. Podocytes are insulin-sensitive cells and have a functionally active local renin-angiotensin system. The presence and activity of angiotensin-converting enzyme 2 (ACE2), the main role of which is cleaving pro-fibrotic and pro-inflammatory angiotensin-II into angiotensin-(1-7), has been demonstrated in podocytes. Conditionally immortalized mouse podocytes were cultured with insulin in the presence and absence of albumin. We found that insulin increases ACE2 gene and protein expression, by real-time PCR and western blot respectively, and enzymatic activity within the podocyte and these increases were maintained over time. Furthermore, insulin favored an "anti-angiotensin-II" regarding ACE/ACE2 gene expression balance and decreased fibronectin gene expression as a marker of fibrosis in the podocytes, all studied by real-time PCR. Likewise insulin incubation seemed to protect podocyte from cell death, studied by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. However, all these effects disappeared in the presence of albumin, which may mimic albuminuria, a main feature in DN pathophysiology. Our results suggest that modulation of renin-angiotensin system balance, fibrosis and apoptosis by insulin in the podocyte may be an important factor in preventing the development and progression of diabetic kidney disease but the presence of albuminuria seems to block these beneficial effects.
Renal ammonia metabolism is a fundamental element of acid-base homeostasis, comprising a major component of both basal and physiologically-altered renal net acid excretion. Over the past several years, a fundamental change in our understanding of the mechanisms of renal epithelial cell ammonia transport has occurred, replacing the previous model which was based upon diffusion equilibrium for NH3 and trapping of NH4+ with a new model in which specific and regulated transport of both NH3 and NH4+ across renal epithelial cell membranes via specific membrane proteins is required for normal ammonia metabolism. A major advance has been the recognition that members of a recently recognized transporter family, the Rhesus glycoprotein family, mediate critical roles in renal and extrarenal ammonia transport. The erythroid-specific Rhesus glycoprotein, Rh A Glycoprotein (Rhag), was the first member of this family recognized as an ammonia-specific transporter. Subsequently, the nonerythroid Rh glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), were cloned and identified as ammonia transporters. They are expressed in specific cell populations and membrane domains in distal renal epithelial cells, where they facilitate ammonia secretion. In this review, we discuss the distribution of Rhbg and Rhcg in the kidney, the regulation of their expression and activity in physiologic disturbances, the effects of genetic deletion on renal ammonia metabolism, and the molecular mechanisms of Rh glycoprotein-mediated ammonia transport.
During the 1st trimester of human pregnancy, the maternal systemic circulation undergoes remarkable vasodilation. The kidneys participate in this vasodilatory response resulting in marked increases in renal plasma flow (RPF) and glomerular filtration rate (GFR). Comparable circulatory adaptations are observed in conscious gravid rats. Administration of the corpus luteal hormone, relaxin (RLN), to non-pregnant rats and humans elicits vasodilatory changes like those of pregnancy. Systemic and renal vasodilation are compromised in midterm pregnant rats by neutralization or elimination of circulating RLN, and in women conceiving with donor eggs who lack a corpus luteum and circulating RLN. Although RLN exerts both rapid (minutes) and sustained (hours to days) vasodilatory actions through different molecular mechanisms, a final common pathway is endothelial nitric oxide. In preeclampsia (PE), maternal systemic and renal vasoconstriction leads to hypertension and modest reduction in GFR exceeding that of RPF. Elevated level of circulating soluble vascular endothelial growth factor receptor-1 secreted by the placenta is implicated in the hypertension and disruption of glomerular fenestrae and barrier function, the former causing reduced Kf and the latter proteinuria. Additional pathogenic factors are discussed. Last, potential clinical ramifications include RLN replacement in women conceiving with donor eggs and its therapeutic use in PE. Another goal has been to apply knowledge gained from investigating circulatory adaptations in pregnancy to identifying and developing novel therapeutic strategies for renal and cardiovascular disease in the non-pregnant population. So far, one candidate to emerge is RLN and its potential therapeutic use in heart failure.
Chronic inflammation promotes the progression of diabetic nephropathy (DN). However, the role of TNFα remains unclear. The objectives of the present study are to examine whether TNFα inhibition with a soluble TNF receptor 2 (TNFR2) fusion protein, i.e. Etanercept (ETN), improves early stage of DN in type 2 diabetic model of the KK-Ay mouse, and to also investigate which TNF pathway, TNFR1 or TNFR2, predominantly involves in the progression of this disease. Methods: ETN was injected intraperitoneally to mice for 8 weeks. Renal damage was evaluated by immunohistochemistry, western blotting, and/or real-time PCR. In vitro, mouse tubular proximal cells were stimulated by TNFα and/or high glucose (HG), and treated by ETN. ETN dramatically improved not only albuminuria but also glycemic control. Renal mRNA and/or protein levels of TNFR2, but not TNFα and TNFR1, in the ETN-treated KK-Ay mice were significantly decreased compared with the no-treated KK-Ay mice. The mRNA levels of ICAM-1, VCAM-1, and MCP-1 and the number of F4/80 positive cells were all decreased after treatment. The numbers of cleaved caspase 3 and TUNEL positive cells in the no-treated mice were very few, and were not different from the ETN-treated mice. In vitro, stimulation by TNFα or HG markedly increased both TNFRs mRNA levels, unlike in the case of in vivo. Furthermore, ETN partly recovered TNFα, but not HG, induced TNFRs mRNA levels. It appears that ETN may improve the progression of early stage of DN predominantly through the inhibition of the anti-inflammatory action of the TNFα-TNFR2 pathway.
COX-2 has an established role in postnatal kidney development. 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is recently identified as an endogenous inhibitor of COX-2, limiting the production of COX-2-derived prostanoids in several pathological conditions. The present study was undertaken to examine the regulation of renal 15-PGDH expression during postnatal kidney development in rats, in comparison with COX-2. qRT-PCR and immunoblotting demonstrated that 15-PGDH mRNA and protein in the kidney were present in neonates, peaked in the 2nd postnatal weeks, and then declined sharply to very low level in adulthood. Immunostaining demonstrated that at the 2nd postnatal week, renal 15-PGDH protein was predominantly found in the proximal tubule stained positive for NHE3 whereas COX-2 protein was restricted to macular densa and adjacent thick ascending limbs stained positive for AQP2. Interestingly, in the 4th week of postnatal week, 15-PGDH protein was redistributed to thick ascending limbs stained positive for NKCC2. After 6 weeks of age, 15-PGDH protein was found in the granules in subsets of the proximal tubule. Overall, these results support a possibility that 15-PGDH may regulate postnatal kidney development through interaction with COX-2.
Ang (1-7) contributes to the blood pressure (BP) lowering effect of angiotensin receptor blockers (ARBs) in male experimental animals. Females have greater Ang (1-7) concentrations than males; however, the contribution of Ang (1-7) to ARB-mediated decreases in BP in females is unknown. The current study tested the hypothesis that female SHR have a larger Ang (1-7) contribution to the BP lowering effects of the ARB candesartan than male SHR. 12 wk old male and female SHR were randomized to receive: candesartan (0.5 mg/kg/day; 7 days); candesartan plus Ang II (200 ng/kg/min; 7 days); the Ang (1-7) antagonist A-779 (48 µg/kg/hr) plus candesartan and Ang II. Candesartan decreased basal BP in males and females (Baseline vs Candesartan: 142±2 vs. 122±3 and 129±1 vs. 115±1 mm Hg, respectively; p<0.05); however, the decrease was greater in males. Ang II increased BP in males in the presence of candesartan (149±2 mm Hg; p<0.05); candesartan blocked Ang II-induced increases in BP in females (116±1 mm Hg). Pre-treatment with A-779 abolished candesartan-mediated decreases in BP in females, but not males. A-779 also exacerbated Ang II-induced proteinuria (26±6 vs. 77±11 µg/kg/day; respectively; p<0.05) and nephrinuria (20±5 vs. 202±58 µg/kg/day; respectively; p<0.05) in candesartan-treated female SHR, with no effect in males. In conclusion, females are more sensitive to the BP lowering effect of ARBs during Ang II infusion, while males are more sensitive under basal conditions. In addition, Ang (1-7) has a greater contribution to ARB-mediated decreases in BP, protein and nephrin excretion in females relative to males.
Both experimental and clinical studies suggest that any potential treatment of polycystic kidney disease should start early and last for a long time to be effective, with unavoidable side reactions and considerable costs. The aim of the present study was to test how low-doses of Rapamycin (RAPA, 0.15 mg/Kg/4 days a week i.p.), Tolvaptan (TOLV, 0.005% in the diet) or AEZ131, a novel ERK inhibitor (AEZ, 30 mg/kg/thrice a week by gavage), alone and in association, affect the progression of polycystic renal disease in PCK rats. Rats were treated for 8 weeks, starting at 4-6 weeks of age. The efficacy of low-doses of such drugs in inhibiting their respective targets was confirmed by immunoblotting studies. Compared to control rats, RAPA determined a significant reduction in cyst volume density (CVD, -19% vs CON), numerically similar in TOLV treated rats (-18%, NS), whereas AEZ was not effective. RAPA+TOLV determined a significantly lower CVD (-49% vs CON), associated with a striking decrease in CREB phosphorylation, and similar data were detected in RAPA+AEZ rats (-42%), whereas the association TOLV+AEZ had virtually no effect. RAPA administration significantly lessened body weight gain, whereas TOLV determined mild increase in diuresis and a significant increase in cAMP urinary excretion. Histological data of tubular proliferation were in full agreement with the data of CVD. In conclusion, this study demonstrates that the association of low-doses of RAPA, TOLV and AEZ slows the progression of PKD with limited side effects, suggesting the use of combined therapies also in clinical trials.
Aging nephropathy is characterized by podocyte depletion, accompanied by progressive glomerulosclerosis. Replacement of terminally differentiated podocytes by local stem/progenitor cells is likely a critical mechanism for their regeneration. Recent studies have shown that cells of renin lineage (CoRL), normally restricted to the kidney's extra-glomerular compartment, might serve this role following an abrupt depletion in podocyte number. To determine the effects of aging on the CoRL reserve, and if CoRL moved from an extra- to the intra- glomerular compartment during aging, genetic cell fate mapping was performed in aging Ren1cCre x Rs-ZsGreen reporter mice. Podocyte number decreased, and glomerular scarring increased with advanced age. CoRL number decreased in the juxta-glomerular compartment with age. There was a paradoxical increase in CoRL in the intra-glomerular compartment at 52 and 64 weeks of age, where a subset co-expressed the podocyte proteins nephrin, podocin and synaptopodin. Transmission EM studies showed that a subset of labeled CoRL in the glomerulus displayed foot processes, which attached to the GBM. No CoRL in the glomerular compartment stained for renin. These results suggest that despite a decrease in the reserve, a subpopulation of CoRL move to the glomerulus following chronic podocyte depletion in aging nephropathy, where they acquire a podocyte-like phenotype. This suggests that they might serve as adult podocyte stem/progenitors cells under these conditions, albeit in insufficient numbers to fully replace podocytes depleted with age.
The present study was conducted to determine whether and how store-operated Ca2+ entry (SOCE) in glomerular mesangial cells (MCs) was altered by high glucose (HG) and diabetes. Human MCs were treated with either normal glucose or HG for different time periods. Cyclopiazonic acid-induced SOCE was significantly greater in the MCs with 7 day HG treatment and the response was completely abolished by GSK-7975A, a selective inhibitor of store-operated Ca2+ channel. Similarly, the inositol 1,4,5-trisphosphate-induced store-operated Ca2+ currents were significantly enhanced in the MCs treated with HG for 7 days and the enhanced response was abolished by both GSK-7975A and La3+. In contrast, receptor-operated Ca2+ entry in MCs was significantly reduced by HG treatment. Western blot showed that HG increased the expression levels of STIM1 and Orai1 in cultured MCs. A significant HG effect occurred at a concentration as low as 10 mM, but required a minimum of 7 days. The HG effect in cultured MCs was recapitulated in renal glomeruli/cortex of both type I and II diabetic rats. Furthermore, quantitative real time RT-PCR revealed that a 6 day HG treatment significantly increased mRNA expression level of STIM1. However, the expressions of STIM2 and Orai1 transcripts were not affected by HG. Taken together, these results suggest that HG/diabetes enhanced SOCE in MCs by increasing STIM1/Orai1 protein expressions. HG upregulates STIM1 by promoting its transcription, but increases Orai1 protein through a post-transcriptional mechanism.
The T-cell immunoglobulin mucin 1, also known as kidney injury molecule-1 modulates CD4+ T-cell responses and is also expressed by damaged proximal tubules within the kidney. Both Th subset imbalance (Th1/Th2/Th17), and regulatory T cell and B-cell alterations contribute to the pathogenesis of autoimmune disease. This study investigated the effects of an inhibitory anti-T-cell immunoglobulin mucin 1 antibody (RMT1-10) in lupus-prone MRL-Faslpr mice. MRL-Faslpr mice were treated with RMT1-10 or a control antibody intraperitoneally twice weekly from three mo of age for sixteen wks. RMT1-10 treatment significantly improved survival, limited the development of lymphadenopathy and skin lesions, preserved renal function and decreased proteinuria, reduced serum anti-DNA antibody levels and attenuated renal leukocyte accumulation. Th1 and Th17 cellular responses systemically and intrarenally were reduced, but regulatory T and B cells were increased. RMT1-10 treatment also reduced glomerular immunoglobulin and C3 deposition, suppressed cellular proliferation and apoptosis. Urinary excretion and renal expression of kidney injury molecule-1 was reduced, reflecting diminished interstitial injury. As RMT1-10 attenuated established lupus nephritis, manipulating immune system T-cell immunoglobulin mucin 1 may represent a therapeutic strategy in autoimmune diseases affecting the kidney.
One third of diabetes mellitus patients develop diabetic nephropathy and with underlying mechanisms unknown it is imperative that diabetic animal models resemble human disease. The present study investigated the susceptibility to develop diabetic nephropathy in four commonly used and commercially available mouse strains with type 1 diabetes in order to determine the suitability of each strain. Type 1 diabetes was induced in C57Bl/6, NMRI, BALB/c and 129Sv mice by alloxan and conscious glomerular filtration rate, proteinuria and oxidative stress levels were measured in control and diabetic animals at baseline and after five and ten weeks. Histological alterations were analyzed using periodic acid-Schiff staining. Diabetic C57Bl/6 displayed increased glomerular filtration rate, i.e. hyperfiltration, whereas all other parameters remained unchanged. Diabetic NMRI developed the most pronounced hyperfiltration as well as increased oxidative stress and proteinuria, but without glomerular damage. Diabetic BALB/c did not develop hyperfiltration, but presented with pronounced proteinuria, increased oxidative stress and glomerular damage. Diabetic 129Sv displayed proteinuria and increased oxidative stress without glomerular hyperfiltration or damage. However, all strains displayed intra-strain correlation between oxidative stress and proteinuria. In conclusion, diabetic C57Bl/6 and NMRI both developed glomerular hyperfiltration, but neither presented with histological damage, although NMRI developed low-degree proteinuria. Thus, these strains may be suitable when investigating the mechanism causing hyperfiltration. Neither BALB/c nor 129Sv developed hyperfiltration although both developed pronounced proteinuria. However, only BALB/c developed detectable histological damage. Thus, BALB/c may be suitable when studying the roles of proteinuria and histological alterations for the progression of diabetic nephropathy.
A mathematical model of renal hemodynamics is used to assess the individual contributions of the tubuloglomerular feedback (TGF) mechanism and the myogenic response to glomerular filtration rate regulation in the rat kidney. The model represents an afferent arteriole segment, glomerular filtration, and a short loop of Henle. The afferent arteriole model exhibits myogenic response, which is activated by hydrostatic pressure variations to induce changes in membrane potential and vascular muscle tone. The tubule model predicts tubular fluid and Cl00; transport. Macula densa Cl00; concentration is sensed as the signal for TGF, which acts to constrict or dilate the afferent arteriole. With this configuration, the model afferent arteriole maintains stable glomerular filtration rate within a physiologic range of perfusion pressure (80-180 mmHg). The contribution of TGF to overall autoregulation is significant only within a narrow band of perfusion pressure values (80-110 mmHg). Model simulations of ramp-like perfusion pressure perturbations agree well with findings by Flemming et al. (J Am Soc Nephrol 12:2253- 2262, 2001), which indicate that changes in vascular conductance is markedly sensitive to pressure velocity. That asymmetric response is attributed to the rate-dependent kinetics of the myogenic mechanism. Moreover, simulations of renal autoregulation in diabetes mellitus predict that, due to the impairment of the voltage-gated Ca2+ channels of the afferent arteriole smooth muscle cells, the perfusion pressure range in which SNGFR remains stable is reduced by ~70%, and that TGF gain is reduced by nearly 40%, consistent with experimental findings.
Renal blood flow (RBF) responses to arginine vasopressin (AVP) were tested in anesthetized wild-type (WT) and CD38-/- mice that lack the major calcium mobilizing second messenger cyclic ADP ribose. AVP (3-25 ng) injected iv produced dose-dependent decreases in RBF, reaching a maximum of 25±2% below basal RBF in WT and 27±2% in CD38-/- mice with 25 ng of AVP. Renal vascular resistance (RVR) increased 75±6% and 78±6% in WT and CD38-/- mice. Inhibition of NO synthase with L-NAME increased the maximum RVR response to AVP to 308±76% in WT and 388±81% in CD38-/- (P<0.001 for both). Cyclooxygenase inhibition with indomethacin increased the RVR response to 125±15% in WT and 120±14% in CD38-/- mice (P<0.001, <0.05). Superoxide suppression with tempol inhibited the RVR response to AVP by 38% in both strains (p<0.005), but was ineffective when administered after L-NAME. The rate of RBF recovery (relaxation) after AVP was slowed by L-NAME and indomethacin (P<0.001, <0.005), but was unchanged by tempol. Vascular responses to AVP were abolished by an AVP V1a receptor antagonist. A V2 receptor agonist or antagonist had no effect on AVP-induced renal vasoconstriction. The results indicate that renal vasoconstriction by AVP in the mouse is strongly buffered by vasodilatory actions of NO and prostanoids. The vasoconstriction depends on V1a receptor activation without involvement of CD38 or concomitant vasodilatation by V2 receptors. The role of superoxide is to enhance the contractile response to AVP, most likely by reducing the availability of NO rather than directly stimulating intracellular contraction signaling pathways.
Over-activation of hypoxia inducible factor (HIF)-1α is implicated as a pathogenic factor in chronic kidney diseases (CKD). However, controversy exists regarding the roles of HIF-1α in CKD. Additionally, although hypoxia and HIF-1α activation is observed in various CKD and HIF-1α has been shown to stimulate fibrogenic factors, there is no direct evidence whether HIF-1α is an injurious or protective factor in chronic renal hypoxic injury. The present study determined whether knocking down the HIF-1α gene can attenuate or exaggerate kidney damage using a chronic renal ischemic model. Chronic renal ischemia was induced by unilaterally clamping the left renal artery for 3 weeks in Sprague-Dawley rats. HIF-1α shRNA or control vectors were transfected into the left kidneys. Experimental groups: sham + control vector, clip + control vector, and clip + HIF-1α shRNA. Enalapril was used to normalize blood pressure one week after clamping the renal artery. HIF-1α protein levels were remarkably increased in clipped kidneys, and this increase was blocked by shRNA. Morphological examination showed that HIF-1α shRNA significantly attenuated injury in clipped kidneys: glomerular injury indices were 0.71 ± 0.04, 2.50 ± 0.12, and 1.34 ± 0.11, and the percentage of globally damaged glomeruli 0.02%, 34.3 ± 5.0%, and 6.3 ± 1.6% in sham, clip and clip + shRNA groups, respectively. The protein levels of collagen and α-smooth muscle actin also dramatically increased in clipped kidneys, but this effect was blocked by HIF-1α shRNA. Conclusion: long-term over-activation of HIF-1α is a pathogenic factor in chronic renal injury associated with ischemia/hypoxia.
Mesenchymal stem cells (MSCs) ameliorate injury and accelerate repair in many organs, including the kidney, although the reparative mechanisms and interaction with macrophages have not been elucidated. This study investigated the reparative potential of human bone marrow-derived MSCs and traced their homing patterns following administration to mice with ischemia/reperfusion (IR) injury using whole body bioluminescence imaging. The effect of MSCs on macrophage phenotype following direct and indirect co-culture was assessed using qPCR. Human cytokine production was measured using multiplex arrays. After IR, MSCs homed to injured kidneys where they afforded protection indicated by decreased proximal tubule kidney injury molecule-1 expression, blood urea nitrogen and serum creatinine levels. SDS-PAGE and immunofluorescence labeling revealed MSCs reduced collagen α1(I) and IV by day 7 post-IR. Gelatin zymography confirmed that MSC treatment significantly increased matrix metalloproteinase-9 activity in IR kidneys, which contributed to a reduction in total collagen. Following direct and indirect co-culture, macrophages expressed genes indicative of an anti-inflammatory 'M2' phenotype. MSC-derived human GM-CSF, EGF, CXCL1, IL-6, IL-8, MCP-1, PDGF-AA and CCL5 were identified in culture supernatants. In conclusion, MSCs home to injured kidneys and promote repair, which may be mediated by their ability to promote M2 macrophage polarization.
Water-handling epithelia are sensitive to the osmotic environment. In this study, the effects of a hypo-osmotic challenge on carbachol (CCh)-induced fluid secretion was investigated using an ex vivo submandibular gland perfusion technique and intracellular pH and Ca2+ measurements. The osmolality of the perfusion solution was altered to examine the response of the gland to a hypotonic challenge. The flow rate was increased by 34% with a 30% hypotonic solution (225 mOsm), although the Ca2+ response was unchanged. Lowering the external Cl- by 50% abolished this increase in the 30% hypotonic solution. Furthermore, bumetanide, an inhibitor of the Na+-K+-2Cl- co-transporter, completely inhibited the fluid secretion increase caused by the 30% hypotonic solution, and both the total amount of fluid and the flow rate were identical to those of the isotonic solution. This finding was confirmed by measuring Na+-K+-2Cl- co-transporter, bumetanide-dependent NH4+ transport; Na+-K+-2Cl-- transport was up-regulated more than 40% by a 30% hypotonic challenge. Therefore, the increase in CCh-induced fluid secretion in response to hypotonic conditions can be attributed to a large extent to the specific activation of the Na+-K+-2Cl- co-transporter.
Dietary potassium loading results in rapid kaliuresis, natriuresis and diuresis associated with reduced phosphorylation (-p) of the distal tubule Na+-Cl- cotransporter (NCC). Decreased NCC-p inhibits NCC mediated Na+ reabsorption and shifts Na+ downstream for reabsorption by epithelial Na+ channels (ENaC) which can drive K+ secretion. Whether the signal is initiated by ingesting potassium or a rise in plasma [K+] is not understood. We tested the hypothesis, in male rats, that an increase in plasma [K+] is sufficient to reduce NCC-p and drive kaliuresis. After an overnight fast, a single 3 hr 2% potassium (2%K) containing meal increased plasma [K+] from 4.0±0.1 to 5.2±0.2 mM, increased urinary K+, Na+, and volume excretion, decreased NCC-p by 60%, and marginally reduced cortical Na+-K+-2Cl- cotransporter (NKCC) phosphorylation 25% (P=0.055). When plasma [K+] was increased by tail vein infusion of KCl to 5.5±0.1 mM over 3 hr, significant kaliuresis and natriuresis ensued, NCC-p decreased by 60% and STE20/SPS1-related proline alanine-rich kinase (SPAK) phosphorylation was marginally reduced 35% (P=0.052). The following were unchanged at 3 hr by either the potassium-rich meal or KCl infusion: Na+/H+ exchanger 3 (NHE3), NHE3-p, NKCC, ENaC subunits, renal outer medullary K+ channel. In summary, raising plasma [K+] by intravenous infusion to a level equivalent to that observed after a single potassium-rich meal triggers renal kaliuretic and natriuretic responses, independent of K+ ingestion, likely driven by decreased NCC-p and activity sufficient to shift sodium reabsorption downstream to where Na+ reabsorption and flow drive K+ secretion.
We used an unbiased approach of gene expression profiling to determine differential gene expression of all the macromolecular modulators (MMs) considered to be involved in stone formation, in hyperoxaluric rats, with and without treatment with NADPH oxidase inhibitor apocynin. Male rats were fed rat chow or chow supplemented with 5% w/w hydroxy-L-proline (HLP) with or without apocynin-supplemented water. After 28 days, rats were euthanized and their kidneys explanted. Total RNA was isolated and microarray analysis was conducted using the Illumina bead array readerTM Gene ontology analysis and the pathway analyses of the genes were done using Database for Annotation, Visualization of Integrated Discovery enrichment analysis tool. Quantitative RT-PCR of selected genes was carried out to verify the microarray results. Expression of selected gene products was confirmed using immunohistochemistry. Administration of HLP led to crystal deposition. Apocynin treatment resulted in near complete absence of crystals. Genes encoding for fibronectin, CD 44, fetuin B, osteopontin, and matrix-gla protein, were up-regulated while those encoding for heavy chains of inter-alpha-inhibitor 1, 3 and 4, calgranulin B, prothrombin, and Tamm-Horsfall protein were down-regulated. HLP-fed rats receiving apocynin had a significant reversal in gene expression profiles, those that were up-regulated came down while those that were down-regulated stepped up. Clearly, there are two types of MMs, one is down-regulated while the other up-regulated during hyperoxaluria and crystal deposition. Apparently gene and protein expressions of known macromolecular modulators of CaOx crystallization are likely regulated by ROS produced in part, through the activation of NADPH oxidase.
Tissue hypoxia has been demonstrated, in both the renal cortex and medulla, during the acute phase of reperfusion after ischemia induced by occlusion of the aorta upstream from the kidney. But there are also recent clinical observations indicating relatively well preserved oxygenation in the non-functional transplanted kidney. To test whether severe acute kidney injury can occur in the absence of widespread renal tissue hypoxia, we measured cortical and inner medullary tissue PO2, and total renal oxygen delivery (DO2) and consumption (VO2) during the first 2 h of reperfusion after 60 min of occlusion of the renal artery in anesthetized rats. To perform this experiment, we employed a new method for measuring kidney DO2 and VO2 that relies on implantation of fluorescence optodes in the femoral artery and renal vein. We were unable to detect reductions in renal cortical or inner medullary tissue tissue oxygen tension (PO2) during reperfusion after ischemia localized to the kidney. This is likely explained by the observation that VO2 (-57%) was reduced by at least as much as DO2 (-45%), due to a large reduction in glomerular filtration (-94%). However, localised tissue hypoxia, as evidence by pimonidazole adduct immunohistochemistry, was detected in kidneys subjected to ischemia and reperfusion, particularly in, but not exclusive to, the outer medulla. Thus, cellular hypoxia, particularly in the outer medulla, may still be present during reperfusion even when reductions in tissue PO2 are not detected in the cortex or inner medulla.
Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease worldwide. The purpose of this study is to investigate whether WldS (slow Wallerian degeneration; also known as Wld) gene plays a renoprotective role during the progression of DN. Diabetes was induced in eight-week-old male wild-type (WT) and C57BL/WldS mice by streptozotocin (STZ) injection. Blood and urinary variables including blood glucose, glycated hemoglobin (GHb), insulin, urea nitrogen and albumin/creatinine ratio were assessed 4, 7 and 14 weeks after STZ injection. Periodic acid-Schiff (PAS) staining, Masson staining and silver staining were performed for renal pathological analyses. In addition, the renal ultrastructure was observed by electron microscope. The activities of p38 and ERK signaling in renal cortical tissues were evaluated by western blot. NAD+/NADH ratio and NADPH oxidase activity were also measured. Moreover, the expressions of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and IL-6 were examined. We provide experimental evidence demonstrating that the WldS gene is expressed in kidney cells and protects against early stage of diabetes-induced renal dysfunction and extracellular matrix accumulation through delaying the reduction of NAD+/NADH ratio, inhibiting the activation of p38 and ERK signaling and suppressing oxidative stress as evidenced by the decreased NADPH oxidase activity and lower expression of TNF-α, IL-1 and IL-6.
A large body of research has contributed to our understanding of the pathophysiology of diabetic nephropathy. Yet, many questions remain regarding the progression of a disease that accounts for nearly half the patients entering dialysis yearly. Several murine models of diabetic nephropathy secondary to Type 2 Diabetes Mellitus (T2DM) do exist, and some are more representative than others, but all have limitations. In this study, we aimed to identify a new mouse model of diabetic nephropathy secondary to T2DM in a previously described T2DM model, MKR (MCK-KR-hIGF-IR) mouse. In this mouse model, T2DM develops as a result of functional inactivation of insulin-like growth factor-1 receptor (IGF-1R) in the skeletal muscle. These mice are lean, with marked insulin resistance, hyperinsulinemia, hyperglycemia, and dyslipidemia, and thus, are representative of non-obese human T2DM. We show that the MKR mice, when under stress (high fat diet or unilateral nephrectomy), develop progressive diabetic nephropathy with marked albuminuria and meet the histopathological criteria as defined by the Animal Models of Diabetic Complications Consortium. Finally, these MKR mice are fertile and are on a common background strain, making it a novel model to study the progression of diabetic nephropathy.
The arginine vasopressin (AVP) type 2 receptor (V2R) is unique among AVP receptor subtypes in signaling through cAMP. Its key function is in the kidneys, facilitating the urine-concentrating mechanism through the AVP/V2-type receptor/aquaporin 2 system in the medullary and cortical collecting ducts. Recent clinical and research observations strongly support the existence of extrarenal V2R. The clinical importance of extrarenal V2R spans widely from stimulation of coagulation factor in the endothelium, to as yet untested potential therapeutic targets. These include V2R-regulated membranous fluid turnover in the inner ear, V2R-regulated mitogensis and apoptosis in certain tumor tissue, and numerous other cell types where the physiological role of V2R still requires further research. Here, we review current evidence on the physiological and pathophysiological functions of renal and extrarenal V2R. These functions of the V2R are important, not only in rare diseases with loss or gain of function of the V2R, but also in relation to the recent use of non-peptide V2R antagonists to treat hyponatremia and possibly retard the growth of cysts and development of renal failure in autosomal dominant polycystic kidney disease. The main functions of the V2R are reabsorption of water and urea in the principal cells of the collecting duct, and vasodilation and stimulation of coagulation factor properties, mainly seen with pharmacological doses of dDAVP. The AVPR2 gene is located on the X chromosome, in a region with high probability of escape from inactivation; this may lead to phenotypic gender differences, with females expressing higher levels of transcript than males.
Mitochondrial dysfunction is increasingly recognized as contributing to glomerular diseases, including those secondary to mitochondrial DNA (mtDNA) mutations and deletions. Mitochondria maintain cellular redox and energy homeostasis and are a major source of intracellular reactive oxygen species (ROS) production. Mitochondrial ROS accumulation may contribute to stress-induced mitochondrial dysfunction and apoptosis and thereby to glomerulosclerosis. In mice, deletion of the gene encoding Mpv17 is associated with glomerulosclerosis, but the underlying mechanism remains poorly defined. Here we report that Mpv17 localizes to mitochondria of podocytes and its expression is reduced in several glomerular injury models and in human focal segmental glomerulosclerosis (FSGS), but not in minimal change disease (MCD). Using models of mild or severe nephrotoxic serum nephritis (NTSN) in Mpv17+/+ wildtype (WT) and Mpv17-/- knockout mice we found that Mpv17-deficiency resulted in increased proteinuria (mild NTSN) and renal insufficiency (severe NTSN) compared with WT. These lesions were associated with increased mitochondrial ROS generation and mitochondrial injury such as oxidative DNA damage. In vitro, podocytes with loss of Mpv17 function were characterized by increased susceptibility to apoptosis and ROS injury including decreased mitochondrial function, loss of mtDNA content and change in mitochondrial configuration. In summary, the inner mitochondrial membrane protein Mpv17 in podocytes is essential for the maintenance of mitochondrial homeostasis and protects podocytes against oxidative stress-induced injury both in vitro and in vivo.
Canonical transient receptor potential-6 (TRPC6) channels have been implicated in the pathogenesis of kidney disease and in the regulation of vascular smooth muscle tone, podocyte function, and a variety of processes in other cell types. The question of whether their gating is intrinsically mechanosensitive has been controversial. We examined activation of two alleles of TRPC6 transiently expressed in CHO-K1 cells: the wild-type human TRPC6 channel, and TRPC6-N143S, an allele identified in a family with autosomal dominant familial focal and segmental glomerulosclerosis (FSGS). We observed that both channel variants carried robust cationic currents that could be evoked by application of membrane-permeable analogs of diacylglycerol (DAG) or by the P2Y receptor agonist ATP. The amplitudes and characteristics of currents evoked by the DAG analog or ATP were indistinguishable in cells expressing the two TRPC6 alleles. By contrast, hypoosmotic stretch evoked robust currents in wild-type TRPC6 channels, but had no discernible effect on currents in cells expressing TRPC6-N143S, indicating that the mutant form lacks mechanosensitivity. Co-expression of TRPC6-N143S with wild-type TRPC6 or TRPC3 channels did not alter stretch-evoked responses compared to when TRPC3 channels were expressed by themselves, indicating that TRPC6-N143S does not function as a dominant-negative. These data indicate that mechanical activation and activation evoked by DAG occur through fundamentally distinct biophysical mechanisms, and they provide support for the hypothesis that protein complexes containing wild-type TRPC6 subunits can be intrinsically mechanosensitive.
The renal glomerulus forms a selective filtration barrier that allows the passage of water, ions and small solutes into the urinary space while restricting the passage of cells and macromolecules. The three layers of the glomerular filtration barrier include the vascular endothelium, glomerular basement membrane (GBM) and podocyte epithelium. Podocytes are capable of internalizing albumin and are hypothesized to clear proteins that traverse the GBM. The present study followed the fate of FITC-labeled albumin to establish the mechanisms of albumin endocytosis and processing by podocytes. Confocal imaging and total internal reflectance fluorescence microscopy of immortalized human podocytes showed FITC-albumin endocytosis occurred preferentially across the basal membrane. Inhibition of clathrin-mediated endocytosis and caveolae-mediated endocytosis demonstrated that the majority of FITC-albumin entered podocytes through caveolae. Once internalized, FITC-albumin co-localized with EEA1 and LAMP1, endocytic markers, and with the neonatal Fc receptor, a marker for transcytosis. After pre-loading podocytes with FITC-albumin, the majority of loaded FITC-albumin was lost over the subsequent 60 min of incubation. A portion of the loss of albumin occurred via lysosomal degradation as pre-treatment with leupeptin, a lysosomal protease inhibitor, partially inhibited the loss of FITC-albumin. Consistent with transcytosis of albumin, pre-loaded podocytes also progressively released FITC-albumin into the extracellular media. These studies confirm the ability of podocytes to endocytose albumin and provide mechanistic insight into cellular mechanisms and fates of albumin handling in podocytes.
Hyperuricemia is associated with kidney complications including glomerulosclerosis and mesangial cell (MC) proliferation by poorly understood mechanisms. The present study investigated the underlying mechanisms that mediate uric acid (UA)-induced MC proliferation. A rat MC line, HBZY-1, was treated with various concentrations of UA in the presence or absence of a specific extracellular regulated protein kinases1/2 (ERK1/2) inhibitor (U0126), apocynin. UA dose-dependently stimulated MC proliferation as shown by increased DNA synthesis and number of cells in the S and G2 phases in parallel with the up-regulation of cyclin A2 and cyclin D1. In addition, UA time-dependently promoted MC proliferation and significantly increased phosphorylation of ERK1/2, but not c-Jun N-terminal kinase and p38 MAPK in MCs as assessed by immunoblotting. Inhibition of ERK1/2 signaling via U0126 markedly blocked UA-induced MC proliferation. More importantly, UA induced intracellular reactive oxygen species (ROS) production of MCs dose dependently, which was completely blocked by apocynin, a specific NADPH oxidase inhibitor. Toll-like receptor (TLR)2 and TLR4 signaling had no effect on NADPH-derived ROS and UA-induced MC proliferation. Interestingly, pretreatment with apocynin inhibited ERK1/2 activation, the up-regulation of cyclin A2 and cyclin D1 and MC proliferation. In conclusion, UA-induced MC proliferation was mediated by NADPH/ROS/ERK1/2 signaling pathway. This novel finding not only reveals the mechanism of UA-induced MC cell proliferation, but also provides some potential targets for future treatment of UA-related glomerular injury.
Our previous studies support the protective effect of cGMP and cGMP-dependent protein kinase I (PKG-I) pathway on the development of renal fibrosis. Therefore, in the present studies, we determined whether pharmacologically or genetically increased PKG activity attenuates renal fibrosis in a unilateral ureteral obstruction (UUO) model and also examined the mechanisms involved. To increase PKG activity, we used the phosphodiesterase 5 (PDE5) inhibitor-sildenafil and PKG transgenic mice. UUO model was induced in wild type or PKG-I transgenic mice by ligating the left lateral ureteral and the renal fibrosis was observed after 14 days of ligation. Sildenafil was administered into wild type UUO mice for 14 days. In vitro, macrophage and proximal tubular cell function was also analyzed. We found that sildenafil treatment or PKG transgenic mice had significantly reduced UUO-induced renal fibrosis, which was associated with reduced TGF-β signaling and reduced macrophage infiltration into kidney interstitial. In vitro data further demonstrated that both macrophages and proximal tubular cells were important sources of UUO-induced renal TGF-β levels. The interaction between macrophages and tubular cells contributes to TGF-β-induced renal fibrosis. Taken together, these data suggest that increasing PKG activity ameliorates renal fibrosis in part through regulation of macrophage and tubular cell function, leading to reduced TGF-β-induced fibrosis.
Genetic hypercalciuric stone-forming (GHS) rats demonstrate increased intestinal Ca absorption, increased bone resorption and reduced renal tubular Ca reabsorption leading to hypercalciuria, and all form kidney stones. GHS have increased vitamin D receptors (VDR) at these sites of Ca transport. Injection of 1,25(OH)2D3 (1,25D) leads to a greater increase in uCa in GHS than in control Sprague-Dawley (SD), possibly due to the additional VDR. In GHS the increased uCa persists on a low Ca diet (LCD) suggesting enhanced bone resorption. We tested the hypothesis that LCD, coupled to inhibition of bone resorption by alendronate (alen), would eliminate the enhanced 1,25D-induced hypercalciuria in GHS. SD and GHS were fed LCD and half were injected daily with 1,25D. After 8d all were also given alen until sacrifice at d16. At 8d, 1,25D increased uCa in SD and to a greater extent in GHS. At 16d, alen eliminated the 1,25D-induced increase in uCa in SD. However, in GHS alen decreased, but did not eliminate, the 1,25D-induced hypercalciuria, suggesting maximal alen cannot completely prevent the 1,25D-induced bone resorption in GHS, perhaps due to increased VDR. There was no consistent effect on mRNA expression of renal transcellular or paracellular Ca transporters. Urine CaP and CaOx SS increased with 1,25D alone in both SD and GHS. Alen eliminated the increase in CaP SS in SD but not in GHS. If these results are confirmed in humans with IH, the use of bisphosphonates, such as alendronate, may not prevent the decreased bone density observed in these patients.
Besides glomerulus tubulo-interstitium is often concomitantly affected in certain diseases, e.g., diabetic nephropathy, and activation of renin-angiotensin system, to a certain extent, worsens its outcome because of perturbations in hemodynamics and possibly tubulo-glomerular feedback. Certain studies suggest that pathobiology of tubulo-interstitial is influenced by small GTPases, e.g., Rap1. We investigated effect of Ang II on inflammatory cytokines, while at the same time focusing on upstream effector of Rap1, i.e., Epac1, and some of the downstream tubular transport molecules, i.e., NHE3. Ang II treatment of LLC-PK1 cells decreased Rap1a GTPase activity in a time- and dose-dependent manner. While Ang II treatment led to an increased membrane translocation of NHE3, which was reduced with Epac1 and PKA activators. Ang II-induced NHE3 translocation was notably reduced with transfection of Rap1a dominant positive mutants, i.e., Rap1a-G12V or Rap1a-T35A. While transfection of cells with dominant negative Rap1a mutants, i.e., Rap1a-S17A, or with Epac1 mutant, i.e., EPAC-cAMP, normalized the Ang II-induced translocation of NHE3. In addition, Ang II treatment led to an increased expression of inflammatory cytokines, i.e., IL-1β, IL-6, IL-8 and TNF-α, which was reduced with Rap1a-G12V or Rap1a-T35A transfection; while it reverted to previous comparable levels following transfection of Rap1a-S17A or EPAC-cAMP. Ang II-induced expression of cytokines was reduced with the treatment of NHE3 inhibitor, S3226, or with Epac1 and PKA activators. These data suggest that this novel Epac1-Rap1a-NHE3 pathway conceivably modulates the Ang II-induced expression of inflammatory cytokines, and this information may aid in developing strategies to reduce tubulo-intertstitial inflammation in renal diseases.
The vacuolar H+-ATPase (V-ATPase) mediates ATP-driven H+ transport across membranes.. This pump is present at the apical membrane of proton-secreting cells, such as kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of PKA and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases co-regulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, co-localizing with the brush border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of V-ATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely co-regulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress.
We have developed a whole-kidney model of the urine concentrating mechanism and renal autoregulation. The model represents the tubuloglomerular feedback (TGF) and myogenic mechanisms, which together affect the resistance of the afferent arteriole and thus glomerular filtration rate. TGF is activated by fluctuations in macula densa [Cl-] and myogenic mechanism by changes in hydrostatic pressure. The model was used to investigate the relative contributions of medullary blood flow autoregulation and inhibition of transport in the proximal convoluted tubule to pressure natriuresis in both diuresis and antidiuresis. The model predicts that medullary blood flow autoregulation, which only affects the interstitial solute composition in the model, has negligible influence on the rate of NaCl excretion. However, it exerts a significant effect on urine flow, particularly in the antidiuretic kidney. This suggests that interstitial washout has significant implications for the maintenance of hydration status but little direct bearing on salt excretion, and that medullary blood flow may only play a signalling role for stimulating a pressure-natriuresis response. Inhibited reabsorption in the model proximal convoluted tubule is capable of driving pressure natriuresis when the known actions of vasopressin on the collecting duct epithelium are taken into account.
Extracellular ATP may contribute to Ca2+ signaling in podocytes during tubuloglomerular feedback (TGF) and possibly as a result of local tissue damage. TRPC6 channels are Ca2+-permeable cationic channels that have been implicated in the pathophysiology of podocyte diseases. Here we show using whole-cell recordings that ATP evokes robust activation of TRPC6 channels in mouse podocyte cell lines and in rat podocytes attached to glomerular capillaries in ex vivo glomerular explants. The ED50 for ATP is around 10 μM, is maximal at 100 μM, and currents were blocked by the P2 antagonist suramin. In terms of maximal currents that can be evoked, ATP is the strongest activator of podocyte TRPC6 that we have characterized to date. Smaller currents were observed in response to ADP, UTP, and UDP. ATP-evoked currents in podocytes were abolished by TRPC6 knockdown and by pretreatment with 10 μM SKF-96365 or 50 μM La3+. ATP effects were also abolished by inhibiting G protein signaling and by the PLC/PLA2 inhibitor D-609. ATP effects on TRPC6 were also suppressed by knockdown of the slit diaphragm scaffolding protein podocin, and also by tempol, a membrane-permeable quencher of reactive oxygen species. Modulation of podocyte TRPC6 channels, especially in foot processes, could provide a mechanism for regulation of glomerular function by extracellular nucleotides, possibly leading to changes in permeation through slit diaphragms. These results raise the possibility that sustained ATP signaling could contribute to foot process effacement, Ca2+-dependent changes in gene expression, and/or detachment of podocytes.
Microvascular rarefaction, or loss of microvascular density, is increasingly implicated in the progression from acute ischemic kidney injury to chronic kidney disease. Microvascular dropout results in chronic tissue hypoxia, interstitial inflammation and fibrosis. There is currently no therapeutic intervention for microvascular rarefaction. We hypothesize that capillary dropout begins with ischemic damage to endothelial mitochondria due to cardiolipin peroxidation, resulting in loss of cristae and the failure to regenerate ATP upon reperfusion. SS-31 is a cell-permeable peptide that targets the inner mitochondrial membrane and binds selectively to cardiolipin. It was recently shown to inhibit cardiolipin peroxidation by cytochrome c peroxidase activity, and it has been shown to protect mitochondrial cristae in proximal tubular cells during ischemia, and accelerated ATP recovery upon reperfusion. We found mitochondrial swelling and loss of cristae membranes in endothelial and medullary tubular epithelial cells after 45 minutes ischemia in the rat. The loss of cristae membranes limited the ability of these cells to regenerate ATP upon reperfusion and led to loss of vascular integrity and tubular cell swelling. SS-31 prevented mitochondria swelling and protected cristae membranes in both endothelial and epithelial cells. By minimizing endothelial and epithelial cell injury, SS-31 prevented "no-reflow" after ischemia and significantly reduced the loss of peritubular capillaries and cortical arterioles, interstitial inflammation, and fibrosis at four weeks after ischemia. These results suggest that mitochondria protection represents an upstream target for pharmacological intervention in microvascular rarefaction and fibrosis.
Tubulointerstitial injury plays an important role in the development and progression of chronic kidney disease (CKD). Kidney injury molecule-1 (KIM-1) is induced in damaged proximal tubules both in acute renal injury and in CKD. However, the dynamics of KIM-1 in CKD and effects of KIM-1 expression on disease progression are unknown. Here we aimed to determine the associations between tubular KIM-1 expression level, renal function and inflammation in CKD. The relationships between the levels of KIM-1 and clinicopathological parameters were analyzed in patients with progressive and non-progressive Immunoglobulin A nephropathy (IgAN). KIM-1 expression was increased in IgAN patients and its expression significantly correlated with the decrease of renal function. KIM-1 was particularly evident at the site with reduced capillary density and KIM-1 positive tubules were surrounded by infiltrates of inflammatory cells. Using in vitro cell models, we showed that cellular stressors, including hypoxia induced KIM-1 expression. KIM-1 expressing cells produced more chemokine/cytokine when cultured under hypoxic conditions. Furthermore, we showed that tubular cells with KIM-1 expression can regulate the immune response of inflammatory cells through secretion of chemotactic factors. These data suggest that KIM-1 expressing epithelial cells may play a role in the pathogenesis of tubulointerstitial inflammation during chronic renal injury through secretion of chemokines/cytokines.
In the current study, an extended two-pore theory is presented where the porous pathways are continuously distributed according to small- and large-pore mean radii (uS and uL) and standard deviations (sS and sL). Experimental glomerular sieving data for Ficoll are analyzed using the model. In addition, several theoretical findings are presented along with analytical solutions to many of the equations used in distributed pore modeling. The results of the data analysis reveal a small-pore population in the glomerular capillary wall with a mean-radius of 36.6Å with a wide arithmetic standard deviation being ~5Å and a large-pore radius of 98.6Å with an even wider standard deviation of ~44Å. The small-pore radius obtained in the analysis is close to that of human serum albumin (35.5Å). By reanalyzing the data and setting the distribution spread of the model constant, we discovered that a narrow distribution is compensated by an increased mean-pore radius and a decreased pore area to diffusion length ratio (A0/x). The wide distribution of pore-sizes obtained in the current analysis, even when considering electrostatic hindrance due to the negatively charged barrier, is inconsistent with the high selectivity to proteins typically characterizing the glomerular filtration barrier. We therefore hypothesize that a large portion of the variance in the distribution of pore-sizes obtained is due to the molecular 'flexibility' of Ficoll, implying that the true variance of the pore-system is lower than that obtained using flexible probes. This would also, in part, explain the commonly noted discrepancy between A0/x and the filtration coefficient Kf.
Purpose: The role of inflammation in oxalate induced nephrolithiasis is debated. Our gene expression study indicated an increase in Interleukin-2 Receptor beta (IL-2Rβ) mRNA in response to oxalate. Herein, we evaluated IL-2Rβ expression and its down-stream signaling pathway in HK2 cells in an effort to understand the mechanisms of oxalate nephrotoxicity. Materials and Methods: HK-2 cells were exposed to oxalate for various time points in the presence or absence of SB203580, a specific p38 MAP kinase inhibitor. Gene expression data were analyzed by Ingenuity Pathway Analysis software. mRNA expression was quantitated via Real Time-PCR and changes in protein expression/kinase activation were analyzed by western blot. Results: Exposure of HK-2 cells to oxalate resulted in increased transcription of IL-2Rβ mRNA and increased protein levels. Oxalate treatment also activated IL-2Rβ signaling pathway (JAK1/STAT5 phosphoryylation). Moreover, the increase in IL-2Rβ protein was dependent upon p38-MAPK activity. Conclusions: These results suggest that oxalate-induced activation the IL-2Rβ pathway may lead to a plethora of cellular changes, the most common of which is the induction of inflammation. These results suggest a central role for p38-MAPK pathway in mediating the effects of oxalate in renal cells, and additional studies may provide the key to unlocking novel biochemical targets in stone disease.
Toll-like receptor-4 (TLR-4) has been increasingly recognized to play a critical role in the pathogenesis of ischemia-reperfusion injury (IRI) of renal grafts. This review provides a detailed overview of the new understanding of the involvement of TLR-4 in ischemia-reperfusion injury of renal grafts and its clinical significance in renal transplantation. TLR-4 responds not only to exogenous microbial motifs but can also recognise molecules which are released by stressed and necrotic cells, as well as degraded products of endogenous macromolecules. Up-regulation of TLR-4 is found in tubular epithelial cells, vascular endothelial cells and infiltrating leukocytes during renal ischemia-reperfusion injuries, which was induced by massive release of endogenous damage-associated molecular pattern molecules such as HMGB-1. Activation of TLR-4 promotes the release of pro-inflammatory mediators, facilitates leukocyte migration and infiltration, activates the innate and adaptive immune system and potentiates renal fibrosis. TLR-4 inhibition serves as the target of pharmacological agents, which could attenuate ischemia reperfusion injury and associated delayed graft function and allograft rejection. There is evidence in the literature showing that targeting TLR-4 could improve long-term transplantation outcomes. Given the pivotal role of TLR-4 in ischemia reperfusion injury and associated delayed graft function and allograft rejection, inhibition of TLR-4 using pharmacological agents could be beneficial for long-term graft survival.
The physiological roles of ANG-(3-4) (Val-Tyr), a potent ANG II-derived peptide, remain largely unknown. The present study: (i) investigates whether ANG-(3-4) modulates ouabain-resistant Na+-ATPase resident in proximal tubule cells, and (ii) verifies whether its possible action on pumping activity -considered the fine tuner of Na+ reabsorption in this nephron segment- depends on blood pressure. ANG-(3-4) inhibited Na+-ATPase activity in membranes of spontaneously hypertensive rats (SHR) at nanomolar concentrations, with no effect in Wistar-Kyoto rats (WKY) or on (Na++K+)-ATPase. PD123319 (10-7 M) and PKA(5-24) (10-6 M), an AT2R antagonist and a specific PKA inhibitor respectively, abrogated this inhibition, indicating that AT2R and PKA are central in this pathway. Despite the lack of effect of ANG-(3-4) when assayed alone in WKY rats, the peptide (10-8 M) completely blocked stimulation of Na+-ATPase induced by 10-10 M ANG II in normotensive rats through a mechanism that also involves AT2R and PKA. Tubular membranes from WKY rats had higher levels of AT2R/AT1R heterodimers, which remain associated in 10-10 M ANG II and dissociate to a very low dimerization state upon addition of 10-8 M ANG-(3-4). This lower level of heterodimers was that found in SHR, and heterodimers did not dissociate when the same concentration of ANG-(3-4) was present. Oral administration of ANG-(3-4) (50 mg/kg body mass) increased [Na+]ur and UNaV with a simultaneous decrease in systolic arterial pressure in SHR, but not in WKY rats. Thus, the influence of ANG-(3-4) on Na+ transport and its hypotensive action depend on receptor association and on blood pressure.
Background: Acute Kidney Injury is common, serious with no specific treatment. Ischemia-reperfusion is a common cause of acute kidney injury. Clinical trials suggest that pre-operative erythropoietin or remote ischemic preconditioning may have a renoprotective effect. Study Design: Using a porcine model of warm-ischemia-reperfusion induced acute kidney injury (40 min bilateral cross-clamping of renal arteries, 48h reperfusion) we examined the renoprotective efficacy of erythropoietin (1000 iu/kg i.v.) or remote ischemic preconditioning (3x cycles, 5 min inflation/deflation to 200mm Hg of a hind-limb sphygmomanometer cuff). Results: Ischemia-reperfusion induced significant kidney injury at 24 and 48h (2, 1df, >10 for 6/7 histopathological features). At 2h, a panel of biomarkers including plasma creatinine, NGAL and IL-1β, and urinary albumin:creatinine could be used to predict histopathological injury. Ischemia-reperfusion increased cell proliferation and apoptosis in the renal cortex but, for pre-treated groups, the apoptotic cells were predominantly intratubular rather than interstitial. At 48h reperfusion, plasma IL-1β and the number of subcapsular cells in G2-M arrest were reduced after preoperative erythropoietin, but not after remote ischemic preconditioning. Conclusions: These data suggest an intra-renal mechanism acting within cortical cells that may underpin a renoprotective function for pre-operative EPO and, to a limited extent, remote ischemic preconditioning. Despite equivocal longer-term outcomes in clinical studies investigating EPO as a renoprotective agent in AKI, optimal clinical dosing and administration have not been established. Our data suggest further clinical studies on the potential renoprotective effect of EPO and remote ischemic preconditioning are justified
Picrotoxin, an antagonist for -aminobutyric acid receptor subtype A (GABAA), was used to investigate the role of GABAA receptors in nociceptive and non-nociceptive reflex bladder activities and pudendal inhibition of these activities in cats under α-chloralose anesthesia. Acetic acid (AA, 0.25%) was used to irritate the bladder and induce nociceptive bladder overactivity, while saline was used to distend the bladder and induce non-nociceptive bladder activity. To modulate the bladder reflex pudendal nerve stimulation (PNS) was applied at multiple threshold (T) intensities for inducing anal sphincter twitching. AA irritation significantly (p<0.01) reduced bladder capacity to 34.3±7.1% of the saline control capacity; while PNS at 2T and 4T significantly (p<0.01) increased AA bladder capacity to 84.0±7.8% and 93.2±15.0%, respectively, of the saline control. Picrotoxin (0.4 mg, i.t.) did not change AA bladder capacity but completely removed PNS inhibition of AA-induced bladder overactivity. Picrotoxin (i.v.) only increased AA bladder capacity at a high dose (0.3 mg/kg) but significantly (p<0.05) reduced 2T PNS inhibition at low doses (0.01-0.1 mg/kg). During saline cystometry, PNS significantly (p<0.01) increased bladder capacity to 147.0±7.6% at 2T and 172.7±8.9% at 4T of control capacity; and picrotoxin (0.4 mg, i.t. or 0.03-0.3 mg/kg, i.v.) also significantly (p<0.05) increased bladder capacity. However, picrotoxin treatment did not alter PNS inhibition during saline infusion. These results indicate that spinal GABAA receptors have different roles in controlling nociceptive and non-nociceptive reflex bladder activities and in PNS inhibition of these activities.
The dopaminergic and sympathetic systems interact to regulate blood pressure. Our previous studies showed that a regulation of α1-adrenergic receptor function by D1-like dopamine receptors in vascular smooth muscle cells. Because renalase could regulate circulating epinephrine levels and dopamine production in renal proximal tubules (RPTs), we tested the hypothesis that D1-like receptors regulate renalase expression in kidney. The effect of D1-like receptor stimulation on renalase expression and function was measured in immortalized RPT cells from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHRs). We found that the D1-like receptor agonist, fenoldopam (10-7-10-5mol/L), increased renalase protein expression and function in WKY RPT cells but decreased them in SHR cells. Fenoldopam also increased renalase mRNA levels in WKY but not in SHR cells. In contrast, fenoldopam increased the degradation of renalase protein in SHR cells but not in WKY cells. The regulation of renalase by the D1-like receptor was mainly via the D5 receptor because silencing of the D5 but not D1 receptor by antisense oligonucleotides blocked the stimulatory effect of the D1-like receptor on renalase expression in WKY cells. Moreover, inhibition of PKC, by the PKC inhibitor 19-31, blocked the stimulatory effect of fenoldopam on renalase expression while stimulation of PKC, by a PKC agonist (PMA), increased renalase expression, indicating that PKC is involved in the process. Our studies suggest that the D5 receptor positively regulates renalase expression in WKY but not SHR RPT cells; aberrant regulation of renalase by the D5 receptor may be involved in the pathogenesis of hypertension
MicroRNAs (miRNAs) are small, non-coding regulatory RNAs that act as posttranscriptional repressors by binding to the 3' untranslated region (3'UTR) of target genes. They require processing by Dicer, an RNase III enzyme, to become mature regulatory RNAs. Previous work from our laboratory revealed critical roles for miRNAs in nephron progenitors at mid-gestation. To interrogate roles for miRNAs in the early metanephric mesenchyme, which gives rise to nephron progenitors as well as the renal stroma during kidney development, we conditionally ablated Dicer function in this lineage. Despite normal ureteric bud outgrowth and condensation of the metanephric mesenchyme to form nephron progenitors, early loss of miRNAs in the metanephric mesenchyme resulted in severe renal dysgenesis. Nephron progenitors are initially correctly specified in the mutant kidneys, with normal expression of several transcription factors known to be critical in progenitors, including Six2, Pax2, Sall1 and Wt1. However, there is premature loss of the nephron progenitor marker Cited1, marked apoptosis, and increased expression of the pro-apoptotic protein Bim shortly after the initial inductive events in early kidney development. Subsequently, there is a failure in ureteric bud branching and nephron progenitor differentiation. Taken together, our data demonstrate a previously undetermined requirement for miRNAs during early kidney organogenesis, and indicate a crucial role for miRNAs in regulating the survival of this lineage.
The type IIa sodium-dependent phosphate co-transporter (Npt2a) plays a critical role in re-absorption of inorganic phosphate (Pi) by renal proximal tubular cells. Pi abnormalities during early stages of sepsis have been reported, but the mechanisms regulating Pi homeostasis during acute inflammation are poorly understood. We examined the regulation of Pi metabolism and renal Npt2a expression during lipopolysaccharide (LPS)-induced inflammation in mice. Dose-response and time-course studies with LPS showed significant increases of plasma Pi and intact parathyroid hormone (iPTH) levels and renal Pi excretion; while renal calcium excretion was significantly decreased. There was no difference in plasma 1,25-dihydroxyvitamin D levels, but the induction of plasma intact fibroblast growth factor 23 levels peaked 3 h after LPS treatment. Western blotting, immunostaining, and quantitative real-time PCR showed that LPS administration significantly decreased Npt2a protein expression in the brush border membrane (BBM) 3 h after injection, but there was no change in renal Npt2a mRNA levels. Moreover, tumor necrosis factor-alpha injection also increased plasma iPTH and decreased renal BBM Npt2a expression. Importantly, we revealed that parathyroidectomized rats had impaired renal Pi excretion and BBM Npt2a expression in response to LPS. These results suggest that the down-regulation of Npt2a expression in renal BBM through induction of plasma iPTH levels alter Pi homeostasis during LPS-induced acute inflammation.
Albuminuria is associated with metabolic syndrome and diabetes. It correlates with the progression of chronic kidney disease, particularly with tubular atrophy. The fatty acid load on albumin significantly increases in obesity, presenting a proinflammatory environment to the proximal tubules. However, little is known about changes in the redox milieu during fatty acid overload and how redox-sensitive mechanisms mediate cell death. Here, we show that albumin with fatty acid impurities or conjugated with palmitate, but not albumin itself compromised mitochondrial and cell viability, membrane potential and respiration. Fatty acid overload led to a redox imbalance which deactivated the antioxidant protein peroxiredoxin 2 and caused a peroxide-mediated apoptosis through the redox-sensitive pJNK/caspase-3 pathway. Transfection of tubular cells with peroxiredoxin 2 was protective and mitigated apoptosis. Mitochondrial fatty acid entry and ceramide synthesis modulators suggested that mitochondrial β oxidation but not ceramide synthesis may modulate lipotoxic effects on tubular cell survival. These results suggest that albumin overloaded with fatty acids but not albumin itself changes the redox environment in the tubules, inducing a peroxide-mediated redox-sensitive apoptosis. Thus mitigating circulating fatty acid levels may be an important factor in both preserving redox balance and preventing tubular cell damage in proteinuric diseases.
Acute kidney injury (AKI) is a complication of sepsis and leads to a high mortality rate. Human and animal studies suggest that mitochondrial dysfunction plays an important role in sepsis-induced multi-organ failure; however, the specific mitochondrial targets damaged during sepsis remain elusive. We used a clinically relevant cecal ligation and puncture (CLP) murine model of sepsis and assessed renal mitochondrial function using high-resolution respirometry, renal microcirculation using intravital microscopy and renal function. CLP caused a time-dependent decrease in mitochondrial complex I and II/III respiration and reduced ATP. By 4 hours after CLP, activity of manganese superoxide dismutase (MnSOD) was decreased by 50% and inhibition was sustained through 36 hours. These events were associated with increased mitochondrial superoxide generation. We then evaluated whether the mitochondria-targeted antioxidant Mito-TEMPO could reverse renal mitochondrial dysfunction and attenuate sepsis-induced AKI. Mito-TEMPO (10 mg/kg) given at 6 hours post CLP decreased mitochondrial superoxide levels, protected complex I and II/III respiration, and restored MnSOD activity by 18 hours. Mito-TEMPO also improved renal microcirculation and glomerular filtration rate. Importantly, even delayed therapy with a single dose of Mito-TEMPO significantly increased 96-hour survival rate from 40% in untreated septic mice to 80%. Thus, sepsis causes sustained inactivation of three mitochondrial targets that can lead to increased mitochondrial superoxide. Importantly, even delayed therapy with Mito-TEMPO alleviated kidney injury, suggesting that it may be a promising approach to treat septic AKI.
Epidemiological studies reveal that children born with a solitary functioning kidney (SFK) have a greater predisposition to develop renal insufficiency and hypertension in early adulthood. A congenital SFK is present in patients with unilateral renal agenesis or unilateral multicystic kidney dysplasia, leading to both structural and functional adaptations in the remaining kidney, which act to mitigate the reductions in glomerular filtration rate and sodium excretion that would otherwise ensue. To understand the mechanisms underlying the early development of renal insufficiency in children born with a SFK, we established a model of fetal uninephrectomy (uni-x) in sheep, a species which similar to humans complete nephrogenesis prior to birth. This model results in a 30% reduction in nephron number rather than 50%, due to compensatory nephrogenesis in the remaining kidney. Similar to children with a congenital SFK, uni-x sheep demonstrate a progressive increase in arterial pressure and a loss of renal function with ageing. This review summarizes the compensatory changes in renal hemodynamics and tubular sodium handling which drive impairments in renal function and highlights the existence of sex differences in the functional adaptations following the loss of a kidney during fetal life.
The degradation of angiotensin (Ang) II by angiotensin converting enzyme 2 (ACE2), leading to the formation of Ang 1-7, is an important step in the regulation of the renin-angiotensin-aldosterone system (RAAS), and one which is significantly altered in the diabetic kidney. This study examines the role of ACE2 in hyperfiltration associated with diabetes. Streptozotocin diabetes was induced in male c57bl6 mice and ACE2 KO mice. C57bl6 mice were further randomised to receive the selective ACE2 inhibitor, MLN-4760. After two weeks of study, animals were subjected to micropuncture studies. Renal reserve was further assessed in c57bl6 mice and ACE2 KO mice following exposure to a high protein diet. The induction of diabetes in wild-type mice was associated with increased renal ACE2 activity, hyperfiltration and renal hypertrophy. On micropuncture, diabetes was associated with increased tubular free-flow and stop-flow pressure, enhanced TGF reactivity and an increased maximal response indicative of increased glomerular hydrostatic capillary pressure. Each of these increases were prevented in diabetic ACE2 KO mice and diabetic mice treated with a selective ACE2 inhibitor for seven days. ACE2 KO mice also failed to increase their creatinine clearance in response to a high protein diet. Our studies suggest that ACE2 plays a key role in the recruitment of renal reserve and hyperfiltration associated with diabetes.
Mutations in the electrogenic Na+/HCO3 cotransporter (NBCe1) that cause pRTA, glaucoma and cataracts in patients are recessive. Parents and siblings of these affected individuals seem asymptomatic though their tissues should make some mutant NBCe1 protein. Biochemical studies with AE1 and NBCe1 indicate that both Slc4 members form dimers. However, the physiologic implications of dimerization have not yet been fully explored. Here, human NBCe1A dimerization is demonstrated by biomolecular fluorescence complementation (BiFC). An EYFP fragment (1-158, EYFPN) or (159-238, EYFPC) was fused to the N- or C-terminus of NBCe1A and mix-and-matched expressed in Xenopus oocyte. The EYFP fluorescent signal was observed only when both EYFP fragments are fused to the N-terminus of NBCe1A (EYFPN-N-NBCe1A w/ EYFPC-N-NBCe1A) and the electrophysiology data demonstrated this EYFP-NBCe1A co-expressed pair have WT transport function. These data suggest NBCe1A forms dimers and that N-termini from the two monomers are in close proximity, likely pair up, to form a functional unit. To explore the physiologic significance of NBCe1 dimerization, we chose two severe NBCe1 mutations (6.6 and 20% WT function individually): S427L (naturally occurring) and E91R (for N-terminal structure studies). When we co-expressed S427L and E91R, we measured 50% wild type function which can only occur if the S427L-E91R heterodimer is the functional unit. We hypothesize that the dominant negative effect of heterozygous NBCe1 carrier should be obvious if the mutated residues are structurally crucial to the dimer formation. The S427L-E91R heterodimer complex allows the monomers to structurally complement each other resulting in a dimer with wild-type like function.
Anti-glomerular basement membrane glomerulonephritis (anti-GBM GN) is a Th1- and Th17-predominant autoimmune disease. Galectin-9 (Gal-9), identified as the ligand of Tim-3, functions in diverse biological processes and leads to the apoptosis of CD4+Tim-3+ T cells. It is still unclear how Gal-9 regulates the functions of Th1 and Th17 cells and prevents renal injury in anti-GBM GN. In this study, Gal-9 was administered to anti-GBM GN mice for 7 days. We found that Gal-9 retarded the increase of Scr, ameliorated renal tubular injury and reduced the formation of crescents. The infiltration of Th1 and Th17 cells into the spleen and kidneys significantly decreased in Gal-9-treated nephritic mice. The reduced infiltration of Th1 and Th17 cells might be associated with the down-regulation of CCL-20, CXCL-9 and CXCL-10 mRNAs in the kidney. In parallel, the blood levels of IFN- and IL-17A declined in Gal-9-treated nephritic mice at day 21 and day 28. In addition, an enhanced Th2 cell-mediated immune response was observed in the kidneys of nephritic mice after a 7-day injection of Gal-9. In conclusion, the protective role of Gal-9 in anti-GBM GN is associated with the inhibition of Th1 and Th17 cell-mediated immune responses and enhanced Th2 immunity in the kidney.
The Na+-K+-2Cl- co-transporter NKCC2 is exclusively expressed in the renal thick ascending limb (TAL), where it exists as 3 main splice isoforms, NKCC2B, NKCC2A and NKCC2F, with the latter two predominating. NKCC2A is expressed in both medullary and cortical TAL, but NKCC2F localizes to the medullary TAL. The biochemical characteristics of the isoforms have been extensively studied by ion uptake studies in Xenopus oocytes, but the functional consequences of alternative splicing remain unclear. We develop a charge-difference model of an NKCC2-transfected oocyte. The model closely recapitulated existing data from ion-uptake experiments. This allowed the reconciliation of different apparent Km values reported by various groups, which have hitherto either been attributed to species differences or remained unexplained. Instead, simulations showed that apparent Na+ and Cl- dependencies are influenced by the ambient K+ or Rb+ bath concentrations which differed between experimental protocols. At steady state, under bath conditions similar to the outer medulla, NKCC2F mediated greater Na+ reabsorption than NKCC2A. Furthermore, Na+ reabsorption by the NKCC2F-transfected oocyte was more energy efficient, as quantified by JNKCC/JPump. Both the increased Na+ reabsorption and the increased efficiency were eroded as osmolarity decreased towards levels observed in the cortical TAL. This supports the hypothesis that the NKCC2F is a medullary specialization of NKCC2, and demonstrates the utility of modeling in analyzing the functional implications of ion uptake data at physiologically relevant steady states.
Renal repair begins soon after the kidney suffers ischemia-reperfusion injury (IRI); however, its molecular pathways are not fully understood. Clusterin (CLU) is a chaperone protein with cytoprotective functions in renal IRI. This study investigated the role of CLU in renal repair after IRI. IRI was induced in the left kidneys of wild type (WT) C57BL/6J (B6) versus CLU knockout (KO) B6 mice by clamping the renal pedicles for 28 - 45 min at the body temperature of 32oC. The renal repair was assessed by histology and confirmed by renal function. Gene expression was examined using PCR array. Here, we showed that following IRI, renal tubular damage and CLU expression in WT kidneys were induced at day 1, reached the maximum at day 3 and significantly diminished at day 7 along with normal function, whereas the tubular damage in CLU KO kidneys steadily increased from initiation of insult to the end of experiment when renal failure occurred. Renal repair in WT kidneys was positively correlated with an increase in Ki67+ proliferative tubular cells and survival from IRI. The functions of CLU in renal repair and renal tubular cell proliferation in cultures were associated with up-regulation of a panel of genes that could positively regulate cell cycle progression and DNA damage repair, which might promote cell proliferation but not involve cell migration. In conclusion, these data suggest that CLU is required for renal tissue regeneration in the kidney repair phase after IRI, which are associated with promotion of tubular cell proliferation.
This study tested the hypothesis that P2Y12 receptor blockade with clopidogrel preserves renal autoregulatory ability during angiotensin II (Ang II)-induced hypertension. Clopidogrel was administered orally to male Sprague-Dawley rats chronically infused with Ang II. Following 14 days of treatment, whole kidney autoregulation of renal blood flow was assessed in vivo in pentobarbital anesthetized rats using an ultrasonic flow probe placed around the left renal artery. In Ang II-vehicle treated rats, decreasing arterial pressure over a range from 160-100 mmHg resulted in a 25±5% decrease in renal blood flow demonstrating significant loss of autoregulation with an autoregulatory index (AI) of 0.66±0.15. However, clopidogrel treatment preserved autoregulatory behavior in Ang II rats to levels indistinguishable from normotensive sham rats (AI, 0.04±0.14). Compared to normotensive sham-vehicle rats, Ang II infusion increased renal CD-3 positive T-cell infiltration by 66±6%, induced significant thickening of the preglomerular vessels, glomerular basement membrane (GBM), increased glomerular collagen I deposition, tubulo-interstitial fibrosis and damage to proximal tubular brush border and increased protein excretion. Clopidogrel significantly reduced renal infiltration of T-cells by 39±9%, prevented interstitial artery thickening, Ang II-induced damage to GBM, deposition of collagen I, and tubulo-interstitial fibrosis, despite maintenance of hypertension. These data demonstrate that systemic P2Y12 receptor blockade with clopidogrel protects against impairment of autoregulatory behavior and renal vascular injury in Ang II-induced hypertension possibly by reducing renal T-cell infiltration.
This study gives a three-dimensional (3D) structural analysis of rat nephrons and their connections to collecting ducts. A total of 4500, 2.5-µm-thick serial sections from the renal surface to the papillary tip were obtained from three kidneys of Wistar rats. Digital images were recorded and aligned into three image stacks and traced from image to image. Short-loop nephrons (SLNs), long-loop nephrons (LLNs), and collecting ducts (CDs) were reconstructed in 3D. We identified a well-defined boundary between the outer stripe and the inner stripe of the outer medulla corresponding to the transition of descending thick limbs to descending thin limbs and between the inner stripe and the inner medulla, i.e. the transition of ascending thin limbs into ascending thick limbs of LLNs. In all nephrons a mosaic pattern of proximal tubule (PT) cells and descending thin limb (DTL) cells was observed at the transition between the PT and the DTL. The course of the LLNs revealed tortuous proximal "straight" tubules and winding of the DTLs within the outer half of the inner stripe. The localization of loop bends of SLNs in the inner stripe of the outer medulla and the bends of LLNs in the inner medulla reflected the localization of their glomeruli, i.e. the deeper the glomerulus, the deeper the bend. Each collecting duct drained approximately 3 to 6 nephrons with a different pattern than previously established in mice. This information will provide basis for evaluation of structural changes within nephrons as a result of physiological or pharmaceutical intervention.
Adenylyl cyclases (AC) catalyze formation of cAMP, a critical component of G-protein coupled receptor signaling. So far, nine distinct membrane-bound AC isoforms (AC1-9) and one soluble AC (sAC) have been identified and, except for AC8, all of them are expressed in the kidney. While the role of ACs in renal cAMP formation is well established, we are just beginning to understand the function of individual AC isoforms, particularly with regard to hormonal regulation of transporter and channel phosphorylation, membrane abundance, and trafficking. This review focuses on the role of different AC isoforms in regulating renal water and electrolyte transport in health as well as potential pathologic implications of disordered AC isoform function. In particular, we focus on modulation of transporter and channel abundance, activity and phosphorylation, with emphasis on studies employing genetically modified animals. As will be described, it is now evident that specific AC isoforms can exert unique effects in the kidney that may have important implications in our understanding of normal physiology as well as disease pathogenesis.
Angiotensin converting enzyme 2 (ACE2) is located in several tissues and is highly expressed in renal proximal tubules, where it degrades the vasoconstrictor angiotensin II (Ang-II) to Ang-(1-7). Accumulating evidence supports protective roles of ACE2 in several disease states, including diabetic nephropathy. A disintegrin and metalloprotease (ADAM) 17, is involved in the shedding of several transmembrane proteins, including ACE2. Our previous studies showed increased renal ACE2, ADAM17 expression, and urinary ACE2 in type 2 diabetic mice. The aim of the present study was to determine the effect of insulin on ACE2 shedding and ADAM17 in type 1 diabetic Akita mice. Results demonstrate increased renal ACE2 and ADAM17 expression, increased urinary ACE2 fragments ( 70 kDa) and albumin excretion in Akita diabetic mice. Immunostaining revealed co-localization of ACE2 with ADAM17 in renal tubules. Renal proximal tubular cells treated with ADAM17 inhibitor showed reduced ACE2 shedding into the media confirming ADAM17-mediated shedding of ACE2. Treatment of Akita mice with insulin implants for 20 weeks normalized hyperglycemia, and decreased urinary ACE2 and albumin excretion. Insulin also normalized renal ACE2, ADAM17, but had no effect on tissue inhibitors of metalloproteinase 3 (TIMP3) protein expression. There was a positive linear correlation between urinary ACE2 and albuminuria, blood glucose, plasma creatinine, glucagon and triglycerides. This is the first report showing an association between hyperglycemia, cardiovascular risk factors and increased shedding of urinary ACE2 in Akita diabetic mice. Urinary ACE2 could be used as biomarker for diabetic nephropathy and as an index of intrarenal ACE2 status.
Blood oxygen level dependent (BOLD) MRI data of kidney, while indicative of tissue oxygenation level (pO2), is in fact influenced by multiple confounding factors, such as R2, perfusion, oxygen permeability, and hematocrit. We aim to explore the feasibility of extracting tissue pO2 from renal BOLD data. A method of two steps was proposed: first, a Monte Carlo simulation to estimate blood oxygen saturation (SHb) from BOLD signals and second, an oxygen transit model to convert SHb to tissue pO2. The proposed method was calibrated and validated with 20 pigs (12 before and after furosemide injection) in which BOLD-derived tissue pO2 was compared to microprobe-measured values. The method was then applied to 9 healthy human subjects (age 25.7±3.0 yrs) in whom BOLD was performed before and after furosemide. For the 12 pigs before furosemide injection, the proposed model estimated renal tissue pO2 with errors of 2.3±5.2 mmHg (5.8%±13.4%) in cortex and -0.1±4.5 mmHg (1.7%±18.1%) in medulla, compared with microprobe measurements. After injection of furosemide, the estimation errors were 6.9±3.9 mmHg (14.2%±8.4%) for cortex and 2.6±4.0 mmHg (7.7%±11.5%) for medulla. In the human subjects, BOLD-derived medullary pO2 increased from 16.0±4.9 mmHg (SHb 31%±11%) at baseline to 26.2±3.1 mmHg (SHb 53%±6%) at 5 min after furosemide injection, while cortical pO2 did not change significantly at ~58 mmHg (SHb 92%±1%). Our proposed method, validated with a porcine model, appears promising for estimating tissue pO2 from renal BOLD MRI data in human subjects.
Epigenetics plays a key role in the pathogenesis of diabetic nephropathy (DN), although the precise regulatory mechanism is still unclear. Here, we examined the role of endoplasmic reticulum (ER) stress in histone H3 lysine 4 (H3K4) methyltransferase SET7/9-induced monocyte chemoattractant protein-1 (MCP-1) expression in the kidneys of db/db mice. Our results indicate that the expression of MCP-1 significantly increased in the kidneys of db/db mice in a time-dependent manner. An increased chromatin mark associated with an active gene (H3K4me1) at MCP-1 promoters accompanied this change in expression. The expression of SET7/9 and the recruitment to these promoters were also elevated. SET7/9 gene silencing with siRNAs significantly attenuated the expression of H3K4me1 and MCP-1. Furthermore, expression of signaling regulator glucose-regulated protein 78 (GRP78), a monitor of ER stress, significantly increased in the kidneys of db/db mice. The expression of ATF6α and spliced X-box binding protein 1 (XBP1s), ER stress-inducible transcription factors, and the recruitment of XBP1s to the SET7/9 promoters were also increased. XBP1s gene silencing with siRNAs significantly attenuated the expression of SET7/9, H3K4me1, and MCP-1. The chaperone betaine not only effectively down-regulated the GRP78, ATF6α and XBP1s expression levels but also markedly decreased the SET7/9, H3K4me1, and MCP-1 levels. Luciferase reporter assay demonstrated that XBP1s participated in ER stress-induce SET7/9 transcription, Taken together, these results reveal that ER stress can trigger the expression of MCP-1, in part through the XBP1s-mediated induction of SET7/9.
The post-transcriptional regulation of gene expression occurs through cis RNA regulatory elements by the action of trans factors, which are represented by non-coding RNAs (especially microRNAs) and the turnover and translation regulatory RNA binding-proteins (TTR-RBPs). These multifactorial proteins are a group of heterogeneous RBPs primarily implicated in controlling the decay and translation rates of target mRNAs. TTR-RBPs usually shuttle between cellular compartments (nucleus, cytoplasm) in response to various stimuli and undergo post-translational modifications such as phosphorylation or methylation to ensure their proper subcellular localization and function. TTR-RBPs are emerging as key regulators of a wide variety of genes influencing kidney physiology and pathology. This review summarizes the current knowledge of TTR-RBPs that influence renal metabolism. We will discuss the role of TTR-RBPs as regulators of kidney ischemia, fibrosis and matrix remodeling, angiogenesis, membrane transport, immunity, vascular tone, hypertension, acid-base balance, as well as anemia, bone mineral disease, and vascular calcification.
Iodinated contrast media (CM) have adverse effects which may result in contrast induced acute kidney injury. Oxidative stress is believed to play a role in CM induced kidney injury. We test the hypothesis that oxidative stress and reduced nitric oxide in tubules are consequences of CM-induced direct cell damage, and that increased local oxidative stress may increase tubulo-glomerular feedback. Rat thick ascending limbs (TAL) were isolated and perfused. Superoxide and nitric oxide were quantified using fluorescence techniques. Cell death rate was estimated using propidium iodide and trypan blue. The function of macula densa and tubulo-glomerular feedback responsiveness were measured in isolated perfused juxta-glomerular apparatuses (JGA) of rabbits. The expression of genes related to oxidative stress and the activity of superoxide dismutase (SOD) were investigated in the renal medulla of rats which received CM. CM increased superoxide concentration and reduced NO bioavailability in TAL. Propidium iodide fluorescence and trypan blue uptake increased more in CM perfused TAL than in controls, indicating increased rate of cell death. There were no marked acute changes in the expression of genes related to oxidative stress in medullary segments of Henle's loop. SOD activity did not differ between CM and control groups. The tubulo-glomerular feedback in isolated JGA was increased by CM. Tubular cell damage and accompanying oxidative stress in our model are consequences of CM-induced direct cell damage, which also modifies the tubulo-vascular interaction at the macula densa, and may therefore contribute to disturbances of renal perfusion and filtration.
Resveratrol is suggested to have beneficial cardiovascular and renoprotective effects. Resveratrol increases endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) synthesis. We hypothesized resveratrol acts as an acute renal vasodilator, mediated through increased NO production and scavenging of reactive oxygen species (ROS). In anesthetized rats, we found 5.0 mg/kg b.w. resveratrol increased renal blood flow (RBF) by 8% (from 6.98±0.42 to 7.54±0.17 ml/min/gkw n=8 p<0.002), and decreased renal vascular resistance (RVR) by 18% from 15.00±1.65 to 12.32±1.20 ARU (p<0.002). To test the participation of NO, we administered 5.0 mg/kg b.w. resveratrol, before and after 10 mg/kg b.w. of the NOS inhibitor, L-NAME. L-NAME reduced the increase in RBF to resveratrol by 54% (from 0.59±0.05 to 0.27±0.06 ml/min/gkw, n=10 p<0.001). To test the participation of ROS, we gave 5.0 mg/kg b.w. resveratrol before and after 1 mg/kg b.w. tempol, a superoxide dismutase mimetic. Resveratrol increased RBF 7.6% (from 5.91±0.32 to 6.36±0.12 ml/min/gkw n=7 p<0.001) and decreased RVR 19% (from 18.83±1.37 to 15.27±1.37 ARU). Tempol blocked resveratrol-induced increase in RBF (from 0.45±0.12 to 0.10±0.05 ml/min/gkw, n=7 p<0.03) and the decrease in RVR post-tempol was 44% of the control response (3.56±0.34 vs. 1.57±0.21 ARU n=7 p<0.006). We also tested the role of endothelium-derived prostanoids. Two days of 10 mg/kg b.w. indomethacin pretreatment did not alter basal blood pressure or RBF. Resveratrol-induced vasodilation remained unaffected. We conclude intravenous resveratrol acts as an acute renal vasodilator, partially mediated by increased NO production/NO bioavailability and superoxide scavenging, but not by inducing vasodilatory cyclooxygenase products.
The aim of this study was to assess the renoprotective effect of renal human liver-type fatty acid binding protein (hL-FABP) and angiotensin II (Ang II) type 1a receptor (AT1a) loss in renal injury caused by renin-angiotensin system (RAS) activation. We established hL-FABP chromosomal transgenic mice (L-FABP+/-AT1a+/+), crossed the L-FABP+/-AT1a+/+ with AT1a knock-down homo mice (L-FABP-/-AT1a-/-), generated L-FABP+/-AT1a hetero mice (L-FABP+/-AT1a+/-). After the back-cross of these cubs, L-FABP+/-AT1a-/- were obtained. In order to activate the renal RAS, wild type mice (L-FABP-/-AT1a+/+), L-FABP+/-AT1a+/+, L-FABP-/-AT1a+/-, L-FABP+/- AT1a+/-, L-FABP-/-AT1a-/- , and L-FABP+/-AT1a-/- were administered high dose systemic Ang II infusion plus a high salt diet for 28 days. In the L-FABP-/-AT1a+/+, RAS activation (L-FABP-/-AT1a+/+RAS) caused hypertension and tubulointerstitial damage. In the L-FABP+/-AT1a+/+RAS, tubulointerstitial damage was significantly attenuated compared with L-FABP-/-AT1a+/+RAS. In the AT1a partial knockout (AT1a+/-) or complete knockout (AT1a-/-) mice, reduction of AT1a expression led to a significantly lower degree of renal injury compared with L-FABP-/-AT1a+/+RAS or L-FABP+/-AT1a+/+RAS mice. Renal injury in L-FABP+/-AT1a+/-RAS mice was significantly attenuated compared with L-FABP-/-AT1a+/-RAS mice. In both L-FABP-/-AT1a-/-RAS and L-FABP+/-AT1a-/-RAS mice, renal damage was rarely found. The degrees of renal hL-FABP expression and urinary hL-FABP levels increased by RAS activation and gradually decreased along with reduction of AT1a expression levels. In conclusion, in this mouse model, renal hL-FABP expression and a decrease in AT1a expression attenuated tubulointerstitial damage due to RAS activation.
In angiotensin II (Ang II)-dependent hypertension, the augmented intrarenal Ang II constricts the renal microvasculature and stimulates Rho kinase (ROCK), which modulates vascular contractile responses. Rho may also stimulate angiotensinogen (AGT) expression in preglomerular vascular smooth muscle cells (VSMCs) but this has not been established. Therefore, the aims of this study were to determine the direct interactions between Rho and Ang II in regulating AGT and other renin-angiotensin system (RAS) components and to elucidate the roles of the ROCK/nuclear factor kappa B (NFB) axis in the Ang II-induced AGT augmentation in primary cultures of preglomerular VSMCs. We first demonstrated that these preglomerular VSMCs express renin, AGT, angiotensin converting enzyme, and Ang II type 1 (AT1) receptors. Furthermore incubation with Ang II (100 pmol/L for 24 hr) increased AGT mRNA (1.42 ± 0.03, ratio to control) and protein (1.68 ± 0.05, ratio to control) expression levels, intracellular Ang II levels and NFB activity. In contrast, the Ang II treatment did not alter AT1a and AT1b mRNA levels in the cells. Treatment with H-1152 (ROCK inhibitor, 10 nmol/L) and ROCK1 siRNA suppressed the Ang II-induced AGT augmentation and the upregulation and translocalization of p65 into nuclei. Functional studies showed that ROCK exerted a greater influence on afferent arterioles responses to Ang II in rats subjected to chronic Ang II infusions. These results indicate that ROCK is involved in NFB activation and the ROCK/NFB axis contributes to Ang II-induced AGT upregulation, leading to intracellular Ang II augmentation.
We examined how the presence of a fixed level of basal renal oxygen consumption (VO2basal; oxygen used for processes independent of sodium transport) confounds the utility of the ratio of sodium reabsorption (TNa+) to total renal VO2 (VO2basal) as an index of the efficiency of oxygen utilization for sodium reabsorption. We performed a systematic review and additional experiments in anesthetized rabbits to obtain the best possible estimate of the fractional contribution of VO2basal to VO2basal; under physiological conditions (basal % renal VO2). Estimates of basal % renal VO2 from 24 studies varied from 0% to 81.5%. Basal % renal VO2 varied with the fractional excretion of sodium (FENa+) in the 14 studies in which FENa+ was measured under control conditions. Linear regression analysis predicted a basal % renal VO2 of 12.7% to 16.5% when FENa+ = 1% (r2 = 0.48, P = 0.001). Experimentally induced changes in TNa+ altered TNa+/VO2total in a manner consistent with theoretical predictions. We conclude that, because VO2basal represents a significant proportion of VO2total, TNa+/VO2total can change markedly when TNa+ itself changes. Therefore, caution should be shown when TNa+/VO2total is interpreted as a measure of the efficiency of oxygen utilization for sodium reabsorption, particularly under experimental conditions where TNa+ or VO2total changes.
Cyclic AMP is a key mediator of collecting duct (CD) Na and water reabsorption. Studies performed in vitro have suggested that CD adenylyl cyclase 3 (AC3) partly mediates vasopressin actions; however, the physiological role of CD AC3 has not been determined. To assess this, mice were developed with CD-specific disruption of AC3 (CD AC3 KO). Inner medullary CDs from these mice exhibited 100% target gene recombination and had reduced angiotensin II, but not vasopressin, induced cAMP accumulation. However, there were no differences in urine volume, urinary urea excretion or urine osmolality between KO and control mice during normal water intake or varying degrees of water restriction in the presence or absence of chronic vasopressin administration. There were no differences between CD AC3 KO and control mice in arterial pressure or urinary Na or K excretion during a normal or high salt diet, while plasma renin and vasopressin concentrations were similar between the two genotypes. Patch-clamp analysis of split-open cortical CDs revealed no difference in epithelial Na channel activity in the presence or absence of vasopressin. Compensatory changes in AC6 were not responsible for the lack of a renal phenotype in CD AC3 KO mice since combined CD AC3/AC6 KO mice had similar arterial pressure and renal Na and water handling as compared to CD AC6 KO mice. In summary, these data do not support a significant role for CD AC3 in the regulation of renal Na and water excretion in general or vasopressin regulation of CD function in particular.
The H,K-ATPase type 2 (HKA2) also known as the "non-gastric" or "colonic" H,K-ATPase is broadly expressed and its presence in the kidney has puzzled experts in the field of renal ion transport systems for many years. One of the most important and robust characteristics of this transporter is that it is strongly stimulated after dietary K+ restriction. This result prompted many investigators to propose that it should play a role in allowing the kidney to efficiently retain K+ under K+ depletion. However, the apparent absence of a clear renal phenotype in HKA2-null mice has led to the idea that this transporter is an epiphenomenon. This review summarizes past and recent findings regarding the functional, structural and physiological characteristics of the H,K-ATPase type 2. The findings discussed in this review suggest that, as in the storybook, the ugly duckling of the X,K-ATPase family is actually a swan.
Treatment of renal ischemia-reperfusion (IR) injury with recombinant human erythropoietin (rhEPO) reduces acute kidney injury and improves function. We aimed to investigate whether progression to chronic kidney disease associated with acute injury was also reduced by rhEPO treatment, using in vivo and in vitro models. Rats were subjected to bilateral 40min renal ischemia, and kidneys were studied at 4, 7 and 28 days post-reperfusion for renal function, tubular injury and repair, inflammation and fibrosis. Acute injury was modulated using rhEPO (1000 or 5000IU/kg, intraperitoneally) at time of reperfusion. Renal tubular epithelial cells or fibroblasts in culture were subjected to hypoxia or oxidative stress, with or without rhEPO (200IU/mL), and fibrogenesis studied. The results of the in vivo model confirmed functional and structural improvement with rhEPO at 4 days post-IR (p<0.05). At 7 days post-IR, fibrosis and myofibroblast stimulation were increased with IR with and without rhEPO (p<0.01). However, at 28 days post-IR, renal fibrosis and myofibroblast numbers were significantly greater with IR plus rhEPO (p<0.01) compared with IR only. Mechanistically, rhEPO stimulated pro-fibrotic transforming growth factor-β, oxidative stress (marker 8-hydroxy-deoxyguanosine), and phosphorylation of the signal transduction protein extra-cellular signal regulated kinase. In vitro, rhEPO protected tubular epithelium from apoptosis, but stimulated epithelial-mesenchymal transition and also protected and activated fibroblasts, particularly with oxidative stress. In summary, although rhEPO was protective of renal function and structure in acute kidney injury, the supraphysiological dose needed for renoprotection contributed to fibrogenesis and stimulated chronic kidney disease in the long-term.
Epithelial Na+ channel (ENaC) subunits (α, β and ) found in functional complexes are translated from mature mRNAs that are similarly processed by inclusion of 13 canonical exons. We examined whether individual exons 3 through 12, encoding the large extracellular domain, are required for functional channel expression. Human ENaCs with an in-frame deletion of a single α subunit exon were expressed in Xenopus oocytes and their functional properties were examined by two-electrode voltage clamp. With the exception of exon 11, deletion of an individual exon eliminated channel activity. Channels lacking α subunit exon 11 were hyperactive. Oocytes expressing this mutant exhibited 4-fold greater amiloride-sensitive whole cell currents than cells expressing wild type channels. A parallel 5-fold increase in channel open probability was observed with channels lacking α subunit exon 11. These mutant channels also exhibited a lost Na+ self-inhibition, whereas we found similar levels of surface expression of mutant and wild type channels. In contrast, in-frame deletions of exon 11 from either the β or subunit led to a significant loss of channel activity, in association with a marked decrease in surface expression. Our results suggest that exon 11 within the three human ENaC genes encode structurally homologous, yet functionally diverse domains and that exon 11 in the α subunit encodes a module that regulates channel gating.
Using patch clamp, we induced depolarization of descending vasa recta (DVR) pericytes or endothelia and tested whether it was conducted to distant cells. Membrane potential was measured with the fluorescent voltage dye, di-8-ANEPPS, or with a second patch clamp electrode. Depolarization of an endothelial cell induced responses in other endothelia within a millisecond and was slowed by gap junction blockade with heptanol. Endothelial response to pericyte depolarization was poor, implying high resistance myo-endothelial coupling. In contrast, dual patch clamp of neighboring pericytes revealed syncytial coupling. At high sampling rate, the spread of depolarization between pericytes and endothelia occurred in 9 ± 2 or 12 ± 2 microseconds, respectively. Heptanol (2 mM) increased the overall input resistance of the pericyte layer to current flow and prevented transmission of depolarization between neighboring cells. The fluorescent tracer, Lucifer yellow (LY), when introduced through ruptured patches, spread between neighboring endothelia in 1 to 7 seconds, depending on location of the flanking cell. LY diffused to endothelial cells on the ipsilateral but not contralateral side of the DVR wall and minimally between pericytes. We conclude that both DVR pericytes and endothelia are part of individual syncytia. The rate of conduction of membrane potential exceeds that for diffusion of hydrophilic molecules by orders of magnitude. Gap junction coupling of adjacent endothelial cells may be spatially oriented to favor longitudinal transmission along the DVR axis.
Basement membrane abnormalities have often been observed in the kidney cysts of polycystic kidney disease (PKD) patients and animal models. There is abnormal deposition of extracellular matrix molecules, including laminin-α3,β3,2 (laminin-332), in human autosomal dominant PKD (ADPKD). Knockdown of PKD1 paralogs in zebrafish leads to dysregulated synthesis of the extracellular matrix, suggesting that altered basement membrane assembly may be a primary defect in ADPKD. In this study, we demonstrate that laminin-332 is aberrantly expressed in cysts and precystic tubules of human autosomal recessive (ARPKD) kidneys as well as in the kidneys of PCK rats, an orthologous ARPKD model. There was aberrant expression of laminin 2 as early as postnatal (PN) day 2 and elevated laminin-332 protein in PN day 30, coinciding with the formation and early growth of renal cysts in PCK kidneys. We also show that a kidney cell line derived from orpk mice, another model of ARPKD, exhibited abnormal lumen-deficient and multi-lumen structures in matrigel culture. These cells had increased proliferation rates and altered expression levels of laminin-332 compared to their rescued counterparts. A function-blocking polyclonal antibody to laminin-332 significantly inhibited their abnormal proliferation rates and rescued their aberrant phenotype in matrigel culture. Furthermore, abnormal laminin-332 expression in cysts originating from collecting ducts and proximal tubules as well as in precystic tubules was observed in a human end-stage ADPKD kidney. Our results suggest that abnormal expression of laminin-332 contributes to aberrant proliferation of cyst epithelial cells and cyst growth in genetic forms of PKD.
We used the patch-clamp technique to examine the effect of angiotensin II (AngII) on the basolateral K channels in the thick ascending limb (TAL) of the rat kidney. Application of AngII increased the channel activity and the current amplitude of the basolateral 50 pS K channel. The stimulatory effect of AngII on the K channels was completely abolished by losartan, an inhibitor of type 1 angiotensin receptor (AT1R), but not by PD123319, an AT2R antagonist. Moreover, inhibition of phospholipase C (PLC) and protein kinase C (PKC) also abrogated the stimulatory effect of AngII on the basolateral K channels in the TAL. This suggests that the stimulatory effect of AngII on the K channels was induced by activating PLC and PKC pathways. Western blotting demonstrated that AngII increased the phosphorylation of c-Src in tyrosine residue 416, an indication of c-Src activation. This effect was mimicked by PKC stimulator but abolished by calphostin C. Moreover, inhibition of NADPH oxidase (NOX) also blocked the effect of AngII on c-Src tyrosine phosphorylation. The role of Src-family protein tyrosine kinase (SFK) in mediating the effect of AngII on the basolateral K channel was further suggested by the experiments in which inhibition of SFK abrogated the stimulatory effect of AngII on the basolateral 50 pS K channel. We conclude that AngII increases basolateral 50 pS K channels activity via AT1R and that activation of AT1R stimulates SFK by a PLC-PKC-NOX-dependent mechanism.
Diabetic nephropathy (DN) is one of the most important diabetic microangiopathies. Epithelial to mesenchymal transition (EMT) plays an important role in DN. The physiological role of microRNA-21 (miR-21) was closely linked to EMT. However, it remained elusive whether Tongxinluo (TXL) ameliorated renal structure and function by regulating miR-21-Induced EMT in DN. This study was to determine the effect of TXL on miR-21-induced renal tubular EMT and to explore the relationship between miR-21 and TGF-β1/smads signals. Real-time RT-PCR, cell transfection and in situ hybridization (ISH) and laser confocal microscopy were used, respectively. Here, we revealed that TXL dose-dependently lowered miR-21 expression in tissue, serum and cell. Over-expression of miR-21 can enhance a-SMA expression and decrease E-cadherin expression by upregulating smad3/p-smad3 expression and downregulating smad7 expression. Interestingly, TXL also increased E-cadherin expression and decreased a-SMA expression by regulating miR-21 expression. More importantly, TXL decreased col-IV, FN, GBM, GA and albumin creatinine ratio (ACR), whereas, increased creatinine clearance ratio (Ccr). The results demonstrated that TXL ameliorated renal structure and function by regulating miR-21-Induced EMT, which was one of the mechanisms to protect DN, and that miR-21 may be one of the novel therapeutic target for TXL in diabetic nephropathy.
PTBAs are a new class of HDAC inhibitors that accelerate recovery and reduce post-injury fibrosis after ischemia-reperfusion-induced acute kidney injury. However, unlike the more common scenario in which patients present with protracted and less clearly defined onset of renal injury, this model of acute kidney injury gives rise to a clearly defined injury that begins to resolve over a short period of time. In these studies we show for the first time that treatment with the PTBA analogue, 4-methyl-thiobutanate (M4PTB), accelerates recovery and reduces post-injury fibrosis in a progressive model of acute kidney injury and renal fibrosis that occurs after aristolochic acid ingestion in mice. These effects are apparent when M4PTB treatment is delayed 4 days after the initiating injury and are associated with increased proliferation and decreased G2/M arrest of regenerating renal tubular epithelial cells. In addition, there is reduced peri-tubular macrophage infiltration and decreased expression of the macrophage chemokines CX3Cl1 and CCL2. Since macrophage infiltration plays a role in promoting kidney injury, and since renal tubular epithelial cells show defective repair and a marked increase in maladaptive G2/M arrest after aristolochic acid injury, these findings suggest M4PTB may be particularly beneficial in reducing injury and enhancing intrinsic cellular repair even when administered days after aristolochic acid ingestion.
Chronic metabolic acidosis (CMA) might result in a decrease in bone mass in vivo based on its reported in vitro inhibition of bone mineralization, bone formation or stimulation of bone resorption, but such data, in the absence of other disorders, have not been reported. CMA also results in negative nitrogen balance which might decrease skeletal muscle mass. This study analysed the net in vivo the effects of CMA's cellular and physico-chemical processes on bone turnover, trabecular and cortical bone density and bone microarchitecture using both pQCT and CT. CMA induced by NH4Cl administration (15 mEq/kg bw/ day) in intact and ovariectomized (OVX) rats resulted in stable CMA (mean {HCO3-]p = 10 mmol/L). CMA decreased osteocalcin and increased TRAP5b in intact and OVX animals. CMA decreased total volumetric bone density (vBMD) after 6 and 10 weeks (week 10: intact normal +2.1±0.9% vs. intact acidosis -3.6±1.2%, p < 0.001), an effect attributable to a decrease in cortical thickness and cortical vBMD (no significant effect on cancellous vBMD, week 10) and an increase in endosteal bone resorption (increased endosteal circumference). Trabecular bone volume (BV/TV) decreased significantly in both CMA groups at 6 and 10 weeks associated with a decrease in trabecular number. CMA significantly decreased muscle cross-sectional area in the proximal hind-limb at 6 and 10 weeks. In conclusion, chronic metabolic acidosis induces a large decrease in cortical bone mass (a prime determinant of bone fragility) in intact and OVX rats and impairs bone microarchitecture characterized by a decrease in trabecular number.
Healthy kidneys are continuously exposed to an array of physical forces as they filter the blood: shear stress along the inner lumen of the tubules, distension of the tubular walls in response to changing fluid pressures, and bending moments along both the cilia and microvilli of individual epithelial cells that comprise the tubules. Dysregulation of kidney homeostasis via underlying medical conditions such as hypertension, diabetes, or glomerulonephritis fundamentally elevates the magnitudes of each principle force in the kidney and leads to fibrotic scarring and eventual loss of organ function. The purpose of this review is to summarize the progress made characterizing the response of kidney cells to pathological levels of mechanical stimuli. In particular, we examine important, mechanically responsive signaling cascades and explore fundamental changes in renal cell homeostasis after cyclic strain or fluid shear stress exposure. Elucidating the effects of these disease-related mechanical imbalances on endogenous signaling events in kidney cells presents the best chance of unequivocally understanding the fibrotic process.
Lithium is the most common cause of nephrogenic diabetes insipidus (Li-NDI). Hydrochlorothiazide (HCTZ) combined with amiloride is the mainstay treatment in Li-NDI. The paradox antidiuretic action of HCTZ in Li-NDI is generally attributed to increased sodium and water uptake in proximal tubules as a compensation for increased volume loss due to HCTZ inhibition of the NaCl-co-transporter (NCC), but alternative actions for HCTZ have been suggested. Here, we investigated whether HCTZ exerted an NCC-independent effect in Li-NDI. In polarized mouse cortical collecting duct (mpkCCD) cells, HCTZ treatment attenuated the Li-induced downregulation of the Aquaporin-2 (AQP2) water channel abundance. In these cells, amiloride reduces cellular Li influx through the epithelial sodium channel ENaC. HCTZ also reduced Li influx, but to a lower extent. HCTZ increased AQP2 abundance on top of that of amiloride and did not affect the ENaC-mediated transcellular voltage. MpkCCD cells did not express NCC mRNA or protein. These data indicated that in mpkCCD cells, HCTZ attenuated lithium-induced downregulation of AQP2 independent from NCC and ENaC. Treatment of Li-NDI NCC knockout mice with HCTZ revealed a significantly reduced urine volume, unchanged urine osmolality and increased cortical AQP2 abundance as compared to Li-treated NCC knockout mice. HCTZ treatment further resulted in reduced blood Li levels, creatinine clearance, and alkalinized urinary pH. Our in vitro and in vivo data indicate that part of the antidiuretic effect of HCTZ in Li-NDI is NCC-independent and may involve a tubuloglomerular feedback response mediated reduction in glomerular filtration rate due to proximal tubular carbonic anhydrase inhibition.
This study was performed to test the hypothesis that angiotensin II (Ang II) contributes to the hypertension and renal functional alterations induced by a decrease of COX2 activity during nephrogenic period. It was also examined whether renal functional reserve and renal response to volume overload and high sodium intake are reduced in 3-4 and 9-11 months old male and female rats treated with vehicle or a COX2 inhibitor during nephrogenic period (COX2np). Our data show that this COX2 inhibition induces an Ang II-dependent hypertension that is similar in male and female rats. Renal functional reserve is reduced in COX2np-treated rats since their renal response to an increase in plasma aminoacids levels is abolished, and their renal ability to eliminate a sodium load is impaired (P<0.05). This reduction in renal excretory ability is similar in both sexes during aging but does not induce the development of a sodium-sensitive hypertension. However, the prolonged high sodium intake at 9-11 months of age leads to a greater proteinuria in male than in female (114±12 mg/min vs. 72±8 mg/min, P<0.05) COX2np-treated rats. Renal hemodynamic sensitivity to acute increments in Ang II is unaltered in both sexes and at both ages in COX2np-treated rats. In summary, these results indicate that the reduction of COX2 activity during nephrogenic period programs for the development of an Ang II-dependent hypertension, reduces renal functional reserve to a similar extent in both sexes, and increases proteinuria in males but not in females when there is a prolonged increment in sodium intake.
Belgrade rats carry a disabling mutation in the iron transporter DMT1. Although DMT1 plays a major role in intestinal iron absorption, the transporter is also highly expressed in kidney, where its function remains unknown. The goal of this study was to characterize renal physiology of Belgrade rats. Male Belgrade rats died prematurely with ~50% survival at 20 weeks of age. Necropsy results indicated marked glomerular nephritis and chronic end-stage renal disease. By 15 weeks of age, Belgrade rats displayed altered renal morphology associated with sclerosis and fibrosis. Creatinine clearance was significantly lower compared with heterozygote littermates. Urinary biomarkers of kidney injury, including albumin, fibrinogen and Kim-1, were significantly elevated. Pilot morphological studies suggest that nephrogenesis is delayed in Belgrade rat pups due to their low iron status and fetal growth restriction. Such defects in renal development most likely underlie the compromised renal metabolism observed in adult b/b rats. Belgrade rat kidney non-heme iron levels were not different from controls but urinary iron and transferrin levels were higher. These results further implicate an important role for the transporter in kidney function not only in iron re-absorption but also in glomerular filtration of the serum protein.
The mechanisms underlying the establishment, development, and maintenance of the renal vasculature are poorly understood. Here we propose that the transcription factor "recombination signal binding protein for immunoglobulin kappa J region" (RBP-J) plays a key role in the differentiation of the mural cells of the kidney arteries and arterioles, as well as the mesangial cells of the glomerulus. Deletion of RBP-J in renal stromal cells of the forkhead box D1 (FOXD1) lineage, which differentiate into all mural cells of the kidney arterioles along with mesangial cells and pericytes, resulted in significant kidney abnormalities and mortality by day 30 post-partum. In newborn mutant animals we observed a decrease in the total number of arteries and arterioles, along with thinner vessel walls, and depletion of renin cells. Glomeruli displayed striking abnormalities, including a failure of FOXD1-descendent cells to populate the glomerulus, an absence of mesangial cells, and in some cases complete loss of glomerular interior structure and the development of aneurysms. By P30, the kidney malformations were accentuated by extensive secondary fibrosis and glomerulosclerosis. We conclude that RBP-J is essential for proper formation and maintenance of the kidney vasculature and glomeruli.
High dietary sodium (Na), a feature of the western diet, requires the kidney to excrete ample Na to maintain homeostasis and prevent hypertension. High urinary flow rate, presumably, leads to an increase in fluid shear stress (FSS) and FSS-mediated release of prostaglandin E2 (PGE2) by the cortical collecting duct (CCD) that enhances renal Na excretion. The pathways by which tubular flow biomechanically regulates PGE2 release and, cyclooxygenase-2 (COX-2) expression is limited. We hypothesized that FSS, through stimulation of neutral-sphingomyelinase (N-SM) activity, enhances COX-2 expression to boost Na excretion. To test this, inner medullary CD3 (IMCD3) cells were exposed to FSS in vitro and mice were injected with isotonic saline in vivo to induce high tubular flow. In vitro, FSS induced N-SM activity and COX-2 protein expression in cells while inhibition of N-SM activity repressed FSS-induced COX-2 protein abundance. Moreover, the murine CCD expresses N-SM protein and, when mice are injected with isotonic saline to induce high tubular flow, renal immunodetectable COX-2 is induced. Urinary PGE2 (445±91 pg/ml vs. 205±14 pg/ml; p<0.05) and microdissected CCDs (135.8±21.7 vs. 65.8±11.0 pg/ml/mm CCD; p<0.05) from saline-injected mice generate more PGE2 than sham-injected controls, respectively. Incubation of CCDs with arachidonic acid and, subsequent measurement of secreted PGE2 is a reflection of the PGE2 generating potential of the epithelia. CCDs isolated from polyuric mice doubled their PGE2 generating potential and this was due to induction of COX-2 activity/protein. Thus, high tubular flow and FSS induce COX-2 protein/activity to enhance PGE2 release and, presumably, effectuate Na excretion.
Oxidative stress resulting from unilateral ureteral obstruction (UUO) may be aggravated by increased production of reactive oxygen species (ROS). Previous studies demonstrated increased COX-2 expression in renal medullary interstitial cells (RMIC) in response to UUO. We investigated both in vivo and in vitro the role of ROS in COX-2 induction in rats subjected to UUO and RMIC cells exposed to oxidative and mechanical stress. Rats subjected to 3-day UUO were treated with 2 mechanistically distinct antioxidants, the NADPH-oxidase inhibitor diphenyleneiodonium (DPI) and the complex I inhibitor rotenone (ROT), to interfere with ROS production. We found that UUO-mediated induction of COX-2 in inner medulla was attenuated by both antioxidants. In addition, DPI and ROT reduced tubular damage and oxidative stress after UUO. Moreover, mechanical stretch induce COX-2 and oxidative stress in RMIC. Likewise, RMIC cells exposed to H2O2 as an inducer of oxidative stress showed increased COX-2 expression and activity, both of which were reduced by DPI and ROT. Similarly, ROS production, which was increased following exposure of RMIC cells to H2O2, was also reduced by DPI and ROT. Furthermore, oxidative stress-induced phosphorylation of ERK1/2 and p38 was blocked by both antioxidants, and inhibition of ERK1/2 and p38 attenuated COX-2 induction in RMIC cells. Notably, COX-2 inhibitors further exacerbated the oxidative stress level in H2O2-exposed RMIC cells. We conclude that oxidative stress as a consequence of UUO stimulates the COX-2 expression through activation of multiple MAP kinases and that COX-2 induction may exert a cytoprotective function in RMIC cells.
Kindlin-2 is an adaptor protein that contributes to renal tubulointerstitial fibrosis (TIF). Epithelial-to-mesenchymal transition (EMT) in tubular epithelial cells was regarded as one of the key events in TIF. To determine whether Kindlin-2 is involved in the EMT process, we investigated its regulation on EMT in human kidney TECs and explored the underlying mechanism. In this study, we found that overexpression of Kindlin-2 suppressed epithelial marker E-cadherin and increased the expression of fibronectin and the myofibroblast marker α-SMA. Kindlin-2 significantly activated ERK1/2 and AKT and inhibition of ERK1/2 or AKT reversed Kindlin-2-induced EMT in human kidney TECs. Mechanistically, Kindlin-2 interacted with Ras and Sos-1. Furthermore, overexpression of Kindlin-2 increased Ras activation through recruiting Sos-1. Treatment with a Ras inhibitor markedly repressed Kindlin-2-induced ERK1/2 and AKT activation, leading to restraint of EMT. We further demonstrated that knockdown of Kindlin-2 inhibited EGF-induced Ras-Sos-1 interaction, resulting in reduction of Ras activation and suppression of EMT stimulated by EGF. Importantly, we found that depletion of Kindlin-2 significantly inhibited activation of ERK1/2 and AKT signaling in UUO mice. We conclude that Kindlin-2, through activating Ras and the downstream ERK1/2 and AKT signaling pathways, plays an important role in regulating renal tubular EMT and could be a potential therapeutic target for the treatment of fibrotic kidney diseases.
In the kidney, the sodium-glucose cotransporters SGLT2 and SGLT1 are thought to account for >90% and ~3% of fractional glucose reabsorption (FGR), respectively. Yet, euglycemic humans treated with an SGLT2 inhibitor maintain an FGR of 40-50%, mimicking values in Sglt2 knockout mice. Here we show that oral gavage with a selective SGLT2 inhibitor (SGLT2-I) dose-dependently increased urinary glucose excretion (UGE) in wild-type (WT) mice. The dose-response curve was shifted leftward and the maximum response doubled in Sglt1 knockout (Sglt1-/-) mice. Treatment in diet with the SGLT2-I for 3 weeks maintained 1.5-2 fold higher urine glucose/creatinine ratios in Sglt1-/- vs. WT mice, associated with a temporarily greater reduction in blood glucose in Sglt1-/- vs. WT after 24h (-33 vs -11%). Subsequent inulin clearance studies under anesthesia revealed free plasma concentrations of the SGLT2-I (corresponding to early proximal concentration) close to the reported IC50 for SGLT2 in mice, which were associated with FGR of 64±2% in WT and 17±2% in Sglt1-/-. Additional i.p. application of the SGLT2-I (maximum effective dose in metabolic cages) increased free plasma concentrations ~10 fold and reduced FGR to 44±3% in WT and to -1±3% in Sglt1-/-. Absence of renal glucose reabsorption was confirmed in male and female Sglt1/Sglt2 double knockout mice. In conclusion, SGLT2 and SGLT1 account for renal glucose reabsorption in euglycemia, with 97% and 3% being reabsorbed by SGLT2 and SGLT1, respectively. When SGLT2 is fully inhibited by the SGLT2-I, increase in SGLT1-mediated glucose reabsorption explains why only 50-60% of filtered glucose is excreted.
Our previous work has shown that gene-knockout of the sodium-glucose cotransporter SGLT2 modestly lowered blood glucose in streptozotocin-diabetic mice (BG; from 470 to 300mg/dl) and prevented glomerular hyperfiltration, but did not attenuate albuminuria or renal growth and inflammation. Here we determined effects of the SGLT2 inhibitor empagliflozin (300mg/kg of diet for 15 weeks; corresponding to 60-80mg/kg/d) in type 1 diabetic Akita mice that, opposite to streptozotocin-diabetes, upregulate renal SGLT2 expression. Akita-diabetes, empagliflozin, and Akita+empagliflozin similarly increased renal membrane SGLT2 expression (by 38-56%) and reduced the expression of SGLT1 (by 33-37%) vs. vehicle-treated wild-type controls (WT). The diabetes-induced changes in SGLT2/SGLT1 protein expression are expected to enhance the BG lowering potential of SGLT2 inhibition, and empagliflozin strongly lowered BG in Akita (means of 187-237 vs. 517-535mg/dl in vehicle group; 100-140mg/dl in WT). Empagliflozin modestly reduced GFR in WT (250 vs. 306µl/min), and completely prevented the diabetes-induced increase in GFR (255 vs. 397µl/min). Empagliflozin attenuated increases in kidney weight and urinary albumin/creatinine ratio in Akita in proportion to hyperglycemia. Empagliflozin did not increase urinary glucose/creatinine ratios in Akita, indicating the reduction in filtered glucose balanced the inhibition of glucose reabsorption. Empagliflozin attenuated/prevented the increase in systolic blood pressure, glomerular size, and molecular markers of kidney growth, inflammation, and gluconeogenesis in Akita. We propose that SGLT2 inhibition can lower GFR independent of reducing BG (consistent with the tubular hypothesis of diabetic glomerular hyperfiltration), while attenuation of albuminuria, kidney growth, and inflammation in the early diabetic kidney may mostly be secondary to lower BG.
Remarkable basic and translational advances have elucidated the role of the mammalian target of rapamycin (mTOR) signaling network in the pathogenesis of renal disease. Many of these advances originated from studies of the genetic disease tuberous sclerosis complex (TSC), leading to one of the clearest therapeutic opportunities to target mTOR with rapamycin and its analogs ("Rapalogs"), which effectively inhibit mTOR complex 1 (mTORC1) by an allosteric mechanism. Clinical trials based on these discoveries have provided strongly positive therapeutic results in TSC (4, 14, 15) In June 2013, the NIDDK convened a small panel of physicians and scientists working in the field to identify key unknowns and define possible "next steps" in advancing understanding of TSC- and mTOR-dependent renal phenotypes. TSC-associated renal disease, which affects more than 85% of TSC patients, and was a major topic of discussion, focused on angiomyolipomas and epithelial cysts. The third major topic was the role of mTOR and mTOR inhibition in the pathogenesis and therapy of chronic renal disease. Renal cell carcinoma, while recognized as a manifestation of TSC that occurs in a small fraction of patients, was not the primary focus of this workshop, and thus was omitted from panel discussions and from this report.
Podocyte apoptosis contributes to the pathogenesis of diabetic nephropathy (DN). However, the mechanisms that mediate high glucose (HG)-induced podocyte apoptosis remain poorly understood. Conditionally immortalized mouse podocytes were cultured in HG medium. A chemical inhibitor or a specific short hairpin RNA (shRNA) vector was used to inhibit the activation of the Notch pathway and the PI3K/Akt pathway in HG-treated podocytes. Western blotting and real-time PCR were used to evaluate the levels of Notch, PI3K/Akt and apoptotic pathway signaling. The apoptosis rate of HG-treated podocytes was assessed by TUNEL and Annexin V/PI staining. In HG-treated podocytes, PI3K/Akt pathway activation prevented podocyte apoptosis in the early stage of HG stimulation and Notch pathway-induced podocyte apoptosis in the late stage of HG stimulation. The inhibition of the Notch pathway or the activation of the PI3K/Akt pathway prevented cell apoptosis in HG-treated podocytes. These findings suggest that the Notch and PI3K/Akt pathways may mediate HG-induced podocyte apoptosis.
Although the kidney is believed to play a minor role in bile acid (BA) excretion, chronic renal failure (CRF) has been reported to be associated with increased serum bile acid levels and alterations in BA homeostasis. The mechanisms for elevated BA levels are poorly understood in both clinical and experimental studies. This study was designed to examine the effects of naturally progressing CRF of longer duration on the hepatic and renal mRNA and protein levels of the BA synthesizing enzyme, Cyp7a1 and the BA transporters, Ntcp, Bsep, Mrp3, Ost-α and Ost-β. Sprague Dawley rats were randomized to the CRF group (5/6 nephrectomy) or to the sham-operated control group and were analyzed 8 weeks after surgery. Results obtained in the CRF rats were compared to those obtained in rats that had undergone uninephrectomy (UNX). The CRF group exhibited significantly increased plasma cholesterol and BA concentrations. Hepatic Cyp7a1 mRNA and protein levels were almost identical in the two groups. Hepatic Mrp3, Ost-α and Ost-β expression was increased, suggesting increased basolateral efflux of bile acids into blood. However, no such changes in BA transporter expression were observed in the remnant kidney. In UNX rats similar changes in plasma BA levels and in the expression of BA transporters were found. We hypothesize that the increase in plasma bile acids is an early event in the progression of CRF and is caused by increased efflux across the basolateral hepatocyte membrane.
Both calcium-containing and non calcium-containing phosphate binders can increase gastrointestinal calcium absorption. Previously, we observed that lanthanum carbonate administration to rats with renal failure is not associated with an increased calciuria. Additionally, lanthanum carbonate treatment in dialysis patients has been associated with a less pronounced initial decrease in serum PTH compared to other phosphate binders. For 8 days, male Wistar rats received a diet supplemented with 2% lanthanum carbonate, 2% sevelamer, 2% calcium carbonate or 2% cellulose. Calciuria was found to be increased in animals treated with sevelamer or calcium carbonate, but not with lanthanum carbonate. In animals with renal failure, cumulative calcium excretion showed similar results. Serum ionized calcium levels were increased after 2 days of treatment with sevelamer, while calcium carbonate showed a smaller increase. Lanthanum carbonate did not induce differences. In animals with renal failure, no differences were found between the sevelamer, calcium carbonate and control groups. Lanthanum carbonate, however, induced lower ionized calcium levels within 2 days of treatment. These results were confirmed in normal human volunteers, showing lower net calcium absorption after a single dose of lanthanum carbonate, compared to sevelamer carbonate. In conclusion, these two non calcium-containing phosphate binding agents showed a differential effect on gastrointestinal calcium absorption. These findings may help to improve management of calcium balance in patients with renal failure, including concomitant use of vitamin D.
Renal damage resulting from acute and chronic kidney injury poses an important problem to public health. Currently patients with end stage renal disease rely solely on kidney transplantation or dialysis for survival. Emerging therapies aiming to prevent and reverse kidney damage are thus in urgent need. Although the kidney was initially thought to lack the capacity for self-repair, several studies have indicated that this might not be the case; Progenitor and stem cells appear to play important roles in kidney repair under various pathological conditions. In this review we summarize recent findings on the role of progenitor/stem cells on kidney repair as well as discuss their potential as a therapeutic approach for kidney diseases.
The aim of the study is to explore the role of miR-194 in mediating the effect of high K (HK) intake on ROMK channel. Northern blot analysis showed that miR-194 was expressed in kidney and that HK intake increased while low K intake decreased the expression of miR-194. Real-time PCR analysis further demonstrated that HK intake increased the miR-194 expression in the cortical collecting duct (CCD). HK intake decreased the expression of intersectin 1 ( ITSN1) which enhanced With-No-Lysine Kinase (WNK)-induced endocytosis of ROMK. Expression of miR-194 mimic decreased luciferase reporter gene activity in HEK293 T cells transfected with ITSN-1-3'UTR containing the complementary seed-sequence for miR-194. In contrast, transfection of miR-194 inhibitor increased the luciferase activity. This effect was absent in the cells transfected with mutated 3'UTR of ITSN1 in which the complimentary seed sequence was deleted. Moreover, the inhibition of miR-194 expression increased the protein level of endogenous ITSN1 in HEK293T cells. Expression of miR-194 mimic also decreased the translation of exogenous ITSN1 in the cells transfected with the ITSN1 containing 3'UTR but not with 3'UTR-free ITSN1. Expression of pre-miR-194 increased K currents and ROMK expression in the plasma membrane in ROMK-transfected cells. Coexpression of ITSN1 reversed the stimulatory effect of miR-194 on ROMK channels. This effect was reversed by coexpression of ITSN1. We conclude that miR-194 regulates ROMK channel activity by modulating ITSN1 expression thereby enhancing ITSN1/WNK-dependent endocytosis. It is possible that miR-194 is involved in mediating the effect of a HK intake on ROMK channel activity.
The adult kidney is derived from the interaction between the metanephric blastema and the ureteric bud. The platelet-derived growth factor (PDGF) receptor β is essential for the development of the mature glomerular tuft, as mice deficient for this receptor lack mesangial cells. This study investigated the role of Src tyrosine kinase in PDGF-mediated reactive oxygen species (ROS) generation and migration of metanephric mesenchymal cells (MMCs). Cultured embryonic MMCs from wild type and PDGF receptor-deficient embryos were established. Migration was determined via wound healing assay. Unlike PDGF AA, PDGF BB induced greater migration in MMCs with respect to control. This was abrogated by neutralizing antibody to PDGF BB. Phosphatidylinositol 3-kinase (PI3K) inhibitors suppressed PDGF BB-induced migration. Conversely, mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitors had no effect. Src inhibitors inhibited PDGF-induced cell migration, PI3K activity, and Akt phosphorylation. Adenoviral dominant negative Src (AD DN Src) abrogated PDGF BB-induced Akt phosphorylation. Hydrogen peroxide stimulated cell migration. PDGF BB-induced wound closure was inhibited by the antioxidants N-acetyl-L-cysteine, tiron, and the flavoprotein inhibitor diphenyleneiodonium. These cells express the NADPH oxidase homolog Nox4. Inhibiting Nox4 with antisense oligonucleotides or small interfering RNA (siRNA) suppressed PDGF-induced wound closure. Inhibition of Src with siRNA reduced PDGF BB-induced ROS generation as assessed by 2',7' - dichlorodihydrofluorescein diacetate fluorescence. Furthermore, PDGF BB-stimulated ROS generation and migration were similarly suppressed by Ad DN Src. In MMCs, PDGF BB-induced migration is mediated by PI3K and Src in a redox-dependent manner involving Nox4. Src may be upstream to PI3K and Nox4.
The kidney proximal tubule is a key target in many forms of acute kidney injury (AKI). The multiligand receptor megalin is responsible for the normal proximal tubule uptake of filtered molecules, including nephrotoxins, cytokines, and markers of AKI. By mediating uptake of nephrotoxins megalin plays an essential role in the development of some types of AKI. However, megalin also mediates the tubular uptake of molecules implicated in the protection against AKI, and changes in megalin expression have been demonstrated in AKI in animal models. Thus, modulation of megalin expression in response to AKI may be an important part of the tubule cell adaption to cellular protection and regeneration, and should be further investigated as a potential target of intervention. This review will explore current evidence linking megalin expression and function to the development, diagnosis, progression, and renal protection in AKI.
Abstract Ischemic acute kidney injury (iAKI) in diabetes mellitus is associated with a rapid deterioration of kidney function, more than in non-diabetic subjects. TVP1022, non-MAO inhibitor S-isomer of rasagiline, possesses anti-oxidative and anti-apoptotic activities. The current study examines the effects of TVP1022 and Tempol, on iAKI in diabetic rats. Diabetes was induced by Streptozotocin. iAKI was induced by clamping left renal artery for 30 min, in both diabetic and non-diabetic rats. Right intact kidney served as control. 48 h following ischemia, urinary flow (V), sodium excretion (UNaV) and glomerular filtration rate (GFR) in both ischemic and non-ischemic kidneys were determined. The nephroprotective effects of tempol and TVP1022 were examined in these rats. Hematoxylin and eosin staining, 4HNE immunofluorescent and nitrotyrosine immunohistochemistry were performed on renal tissues of the various experimental groups. Compared with normoglycemic rats, iAKI in diabetic animals caused more profound reductions in V, UNaV, and GFR. Tempol and TVP1022 treatment increased GFR by two and four fold in diabetic ischemic kidney, respectively. Besides hemodynamic perturbations, iAKI markedly increased renal immunoreactive 4HNE and nitrotyrosin staining in both diabetic and non-diabetic rats. Moreover, iAKI increased medullary necrosis, congestion and casts. Noteworthy, these increases were to a larger extent in ischemic diabetic kidney. TVP1022, and to a lesser extent Tempol, decreased nitrotyrosin and 4HNE immunoreactivities, and necrosis and casts formation in the renal medulla. TVP1022 treatment improves renal dysfunction and histological changes in iAKI diabetic model, suggests a role for TVP1022 therapy in kidney injury.
To better understand the role water and urea fluxes play in the urine concentrating mechanism, we determined transepithelial osmotic water (Pf) and urea (Purea) permeabilities in isolated perfused Munich-Wistar rat long-loop descending (DTL) and ascending (ATL) thin limbs. Thin limbs were isolated either from 0.5 to 2.5 mm below the outer medulla (OM) (upper inner medulla; IMupper) or from the terminal 2.5 mm of the IM (IMlower). Segment types were characterized on the basis of structural features and gene expression levels of the water channel aquaporin 1 (AQP1), which was high in DTLupper, absent in DTLlower, and absent in ATLs and the chloride channel ClCK1, which was absent in DTLs and high in ATLs. DTLupper Pf (μm/s) was high (32045 ± 450.28) whereas DTLlower showed very little or no osmotic water permeability (207.8 ± 241.3). Munich-Wistar rat ATLs have previously been shown to exhibit no water permeability. DTLupper Purea (x 10-5 cm/s) was 40.0 ± 7.3 and much higher in DTLlower (203.8 ± 30.3), ATLupper (203.8 ± 35.7) and ATLlower (265.1 ± 49.8). Phloretin (0.25 mM) did not reduce DTLupper Purea, suggesting that Purea is not due to the urea transporter UT-A2, which is expressed in short-loop DTLs and short portions of some inner medullary DTLs close to the OM. In summary, Purea is similar in all segments having no osmotic water permeability but is significantly lower in DTLupper, a segment having high osmotic water permeability. These data are inconsistent with the passive mechanism as originally proposed.
The importance of mPGES1 in regulating renal function has been examined in mPGES1 deficient mice or by evaluating changes in its expression. However, it is unknown whether the prolonged mPGES1 inhibition induces significant changes of renal function when sodium intake is normal or low. This study examines the renal effects elicited by a selective mPGES1 inhibitor (PF-458) during 7 days in conscious chronically instrumented dogs with normal (NSI) or low (LSI) sodium intake. Results obtained in vitro and in vivo studies strongly suggest that PF-458 is a selective mPGES1 inhibitor. The administration of 2.4 mg/Kg/day of PF-458 to dogs with LSI did not induce significant changes in RBF and GFR. A greater dose of PF-458 (9.6 mg/kg/day) reduced RBF (P<0.05) but not GFR in dogs with LSI, and did not induce changes of renal hemodynamic in dogs with NSI. Both doses of PF-458 elicited a decrease (P<0.05) in PGE2 and an increase (P<0.05) in 6 KetoPGF1α. The administration of PF-458 did not induce significant changes in renal excretory function, plasma renin activity, and aldosterone and TXB2 plasma concentrations in dogs with LSI or NSI. The results obtained suggest that mPGES1 is involved in the regulation of RBF when sodium intake is low and that the renal effects elicited by mPGES1 inhibition are modulated by a compensatory increment in PGI2. These results may have some therapeutical implications since it has been shown that prolonged mPGES1 inhibition has lower renal effects than those elicited by NSAIDs or selective COX2 inhibitors.
Peritoneal dysfunction is a major factor leading to treatment failure of peritoneal dialysis (PD). However, the precise mechanism of the peritoneal diffusion changes related to PD remains to be elucidated. To this end, we have established a novel peritoneal diffusion model in vitro, which consists of a three-dimensional culture system using a collagen vitrigel membrane chamber and a fluid-stream generation system. This artificial peritoneal model revealed that high-glucose culture medium and fluid flow stress promoted the epithelial-mesenchymal transition (EMT) process of mesothelial cells, and that endothelial cells inhibited this mesothelial EMT process. Mesothelial cells in the EMT state showed high expression of connective tissue growth factor and low expression of bone morphogenic protein-7, while non-EMT mesothelial cells showed the opposite expression pattern of these two proteins. In addition, these protein expressions were dependent on the presence of endothelial cells in the model. Our model revealed that the endothelial slit function was predominantly dependent on the covering surface area, while the mesothelial layer possessed a specific barrier function against for small solutes independently of the surface area. Notably, a synergic barrier effect of mesothelial cells and endothelial cells was present with low glucose pretreatment, but high glucose pretreatment abolished this synergic effect. These findings suggest that the mesothelial slit function is not only regulated by the high-glucose-induced EMT process, but is also affected by an endothelial paracrine effect. This peritoneal diffusion model could be a promising tool for the development of PD.
Monocyte/macrophage recruitment correlates strongly with the progression of renal impairment in diabetic nephropathy (DN), yet their direct role is not clear. We hypothesize that macrophages contribute to direct podocyte injury and/or an abnormal podocyte niche leading to DN. Experiments were conducted in CD11b-DTR mice treated with diphtheria toxin (DT) to deplete macrophages following streptozotocin (STZ) induced diabetes. Additional experiments were conducted in bone marrow chimeric (CD11b-DTR-> C57BL6/J) mice. Diabetes was associated with an increase in the M1/M2 ratio by 6 weeks following induction of diabetes. Macrophage depletion in diabetic CD11b-DTR mice significantly attenuated albuminuria, kidney macrophage recruitment, glomerular histologic changes and preserved kidney nephrin and podocin expression compared with diabetic CD11b-DTR mice treated with mutant DT. These data were confirmed in chimeric mice indicating a direct role of bone marrow-derived macrophages in DN. In vitro, podocytes grown in high glucose media significantly increased macrophage migration compared to podocytes grown in normal glucose media. In addition, classically activated M1 macrophages; but not M2 macrophages; induced podocyte permeability. These findings provide evidence that macrophages directly contribute to kidney injury in DN; perhaps by altering podocyte integrity through the pro-inflammatory M1 subset of macrophages. Attenuating the deleterious effects of macrophages on podocytes could provide a new therapeutic approach to the treatment of DN.
We investigated the influence of sex and puberty stage on the circadian urine production and levels of antidiuretic hormone (AVP) in healthy children. Thirty-nine volunteers (9 prepuberty boys, 10 prepuberty girls, 10 midpuberty boys and 10 midpuberty girls) were included. All participants underwent a 24-hours circadian in-patient study under standardized conditions regarding sodium and fluid intake. Blood samples were drawn every four hours for measurements of plasma AVP, serum 17-β-estradiol and testosterone and the urine was fractionally collected for measurements of electrolytes, aquaporin 2 (AQP2) and prostaglandin E2 (PGE2). We found a marked night-time decrease in diuresis (from 1.69±0.08 to 0.86±0.06ml/kg/h,p<0.001) caused by a significant night-time increase in solute-free water reabsorption(TcH2O)(Day-Night ratio 0.64±0.07,p<0.001) concurrent with a significant decrease in osmotic excretion (Day-Night ratio 1.23±0.06,p<0.001). Plasma AVP expressed a circadian rhythm (p<0.01) with a night-time increase and peak levels at midnight (0.49±0.05 pg/ml). The circadian plasma AVP rhythm was not influenced by gender (p=0.56) or puberty stage (p=0.73). There was significantly higher night-time TcH2O in pre-puberty children. This concurred with increased night-time urinary AQP2 excretion in pre-puberty children. Urinary PGE2 exhibited circadian rhythm independent of sex or puberty stage. The levels of serum 17-β-estradiol and testosterone were as expected for sex and pubertal stage and no effect on AVP-AQP2- TcH2O axis was observed. This study finds a circadian rhythm of plasma AVP independent of sex and puberty stage, though night-time solute-free water reabsorption was higher and AQP2 excretion more pronounced in pre-puberty children suggesting higher pre-puberty renal AVP sensitivity.
The renal afferent arteriole reacts to an elevation in blood pressure with an increase in muscle tone and a decrease in luminal diameter. This effect, known as the myogenic response, is believed to stabilize glomerular filtration and to protect the glomerulus from systolic blood pressure increases, especially in hypertension. To study the mechanisms underlying the myogenic response, we developed a mathematical model of intracellular Ca2+ signaling in an afferent arteriole smooth muscle cell. The model represents detailed transmembrane ionic transport, intracellular Ca2+ dynamics, the kinetics of myosin light chain phosphorylation, and the mechanical behavior of the cell. It assumes that the myogenic response is initiated by pressure-induced changes in the activity of non-selective cation channels. Our model predicts spontaneous vasomotion at physiological luminal pressures, and KCl- and diltiazem-induced diameter changes comparable to experimental findings. The time-periodic oscillations stem from the dynamic exchange of Ca2+ between the cytosol and the sarcoplasmic reticulum, coupled to the stimulation of Ca2+-activated potassium (KCa) and chloride (ClCa) channels, and the modulation of voltage-activated L-type channels; blocking sarco/endoplasmic reticulum Ca2+ pumps or ryanodyne receptors (RyR), KCa, ClCa or L-type channels abolishes these oscillations. Our results indicate that the profile of the myogenic response is also strongly dependent upon the conductance of ClCa and L-type channels, as well as the activity of plasmalemmal Ca2+ pumps. Furthermore, inhibition of Ca2+-activated K+ channels is not necessary to induce myogenic contraction. Lastly, our model suggests that the kinetic behavior of L-type channels results in myogenic kinetics that are substantially faster during constriction than during dilation, consistent with in vitro observations (Loutzenhiser et al., Circ. Res. 90:1316--1324, 2002).
With better understanding of the molecular mechanisms underpinning chronic kidney disease, the roles of inflammation and fibrosis are becoming increasingly inseparable. The progression of renal disease is characterized by pathomorphologic changes that consist of early inflammatory responses, followed by tubulointerstitial fibrosis, tubular atrophy, glomerular and vascular sclerosis. Currently available therapies that reduce hypertension, proteinuria, hyperglycemia, and interruption of the renin-angiotensin-aldosterone system (RAAS), are at best only partially effective. Hence there remains a need to explore agents targeting non-RAAS pathways. In this review, we discuss the mechanistic aspects in the physiological and pathological role of semicarbazide-sensitive amine oxidase (SSAO), a protein enzyme involved in cellular trafficking and inflammation, with respect to the kidney. We explore the evidence for the use of SSAO inhibitors as potential agents in renal fibrosis to delay the onset and progression of chronic kidney disease.
Oxidative stress and inflammation play important roles in diabetic complications, including diabetic nephropathy. Metallothionein (MT) is induced in proximal tubular epithelial cells as an antioxidant in diabetic kidney; however, the role of MT in renal function remains unclear. We therefore investigated if MT deficiency accelerates diabetic nephropathy through oxidative stress and inflammation. Diabetes was induced by streptozotocin injection in MT-deficient (MT-/-) and MT+/+ mice. Urinary albumin excretion, histological changes, markers for reactive oxygen species (ROS) and kidney inflammation were measured. Murine proximal tubular epithelial (mProx24) cells were used to further elucidate the role of MT under high-glucose conditions. Parameters of diabetic nephropathy and markers of ROS and inflammation were accelerated in diabetic MT-/- mice compared with diabetic MT+/+ mice, despite equivalent levels of hyperglycemia. MT deficiency accelerated interstitial fibrosis and macrophage infiltration into the interstitium in diabetic kidney. Electron microscopy revealed abnormal mitochondrial morphology in proximal tubular epithelial cells in diabetic MT-/- mice. In vitro studies demonstrated that knockdown of MT by small interfering RNA enhanced mitochondrial ROS generation and inflammation-related gene expression in mProx24 cells cultured under high-glucose conditions. The results of this study suggest that MT may play a key role in protecting the kidney against high glucose-induced ROS and subsequent inflammation in diabetic nephropathy.
Focal segmental glomerulosclerosis (FSGS) is a podocyte disease. Among the various histologies of FSGS, active epithelial changes - hyperplasia as typically seen in the collapsing variant - indicates disease progression. Using a podocyte-specific injury model of FSGS carrying a genetic podocyte tag combined with double immunostaining by different sets of podocytes and parietal epithelial cell (PEC) markers (Nestin/Pax8, WT1/Claudin1, and Podocalyxin/Pax2), we investigated the direction of epithelial phenotypic transition and its role in FSGS. FSGS mice showed progressive proteinuria and renal dysfunction often accompanied by epithelial hyperplasia, wherein X-gal-positive podocyte-tagged cells were markedly decreased. The average numbers of double-positive cells in all sets of markers were significantly increased in the FSGS mice compared to the controls. In addition, the average numbers of double-positive cells for X-gal/Pax8, Nestin/Pax8 and Podocalyxin/Pax2 staining in the FSGS mice were comparable, whereas those of WT1/Claudin1 were significantly increased. When we divided glomeruli from FSGS mice into those with FSGS lesions and those without, double-positive cells tended to be more closely associated with glomeruli without FSGS lesions compared to those with FSGS lesions. Moreover, the majority of double-positive cells appeared to be isolated and very rarely associated with FSGS lesions (1/1,997 glomeruli). This study is the first to show the incidence and localization of epithelial cells with phenotypically changing in FSGS using a genetic tag. The results suggest that the major direction of epithelial phenotypic transition in cellular FSGS is from podocytes to PECs, and that these cells were less participated in the active lesions of FSGS.
To investigate the expression of four subtypes of prostaglandin E2 E-prostanoid receptor (EP1-EP4) and the effects of EP3/EP4 on bladder dysfunction in a new neurogenic bladder model induced by experimental autoimmune encephalomyelitis (EAE), the mice model of EAE were induced using a previously established method and then bladder function in mice with different defined levels of neurological impairment were examined, including micturition frequencies and voiding weight. The bladders were then harvested for analysis of EP receptor expression by Western blot. The activities of agonists/antagonists of the EP3 and EP4 receptors as well as PGE2 were also evaluated at different stages of EAE. The results showed that the EAE mice developed profound bladder dysfunction characterized by significantly increased micturition and significantly decreased urine output per micturition. EAE-induced up-regulation of EP3 and EP4 receptors in the bladder was accompanied by bladder dysfunction. However, EAE had no significant effect on the EP1 and EP2 receptors. Moreover, PGE2 and the agonists/antagonists of EP3 and EP4 receptors significantly affected bladder dysfunction in EAE mice. Thus, we indicated that EAE mice are useful for investigating neurogenic bladder. In addition, the EP3 and EP4 receptors play a role in EAE-induced bladder dysfunction, providing us with a new target for treatment of neurogenic bladder.
Elevated serum parathyroid hormone (PTH) is an important complicated phenomenon in patients with chronic kidney disease (CKD). Emerging evidence indicates the involvement of PTH in organ fibrosis, and suppression of PTH by cinacalcet (CINA) ameliorates the progression of fibrotic disorders. However, the underlying mechanisms are largely unknown. The endothelial-to-mesenchymal transition (EndMT) has been shown to be an important mechanism involved in renal fibrosis. The present study aimed to investigate whether CINA treatment attenuated renal EndMT in rats with adenine-induced chronic renal failure (CRF). Compared with the control group, serum PTH was significantly higher in the CRF group and was suppressed after CINA treatment. The serum calcium, phosphorus and calciumxphosphorus product levels were similar in the CRF group and the CINA-treated CRF group. Renal collagen accumulation was significantly increased in the CRF group which was markedly ameliorated by CINA treatment. The expression of the endothelial marker, CD31 was significantly downregulated in rats with CRF, while the expression of the mesenchymal markers, fibroblast specific-protein (FSP1) and α-smooth muscle actin (α-SMA) was markedly upregulated. These changes were inhibited by CINA treatment. The protein levels of these EndMT-related markers were strongly correlated with serum PTH concentrations. Furthermore, in vitro study showed that PTH could significantly increase the expression of FSP1 and α-SMA and decrease CD31 in mRNA and protein levels in a concentration- and time- dependent manner. In conclusion, our study suggested that reducing serum PTH by CINA treatment could attenuate renal fibrosis via suppression of EndMT in the adenine-induced CRF rat model.
We have recently demonstrated that intrarenal dopamine plays an important role in preventing the development of systemic hypertension. Similarly, renal cytochrome P450 (CYP)-epoxygenase-derived arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs) also are anti-hypertensive through inhibiting sodium reabsorption and vasodilation. The potential interaction between renal dopamine and epoxygenase systems was investigated. COMT-/- mice with increased intrarenal dopamine levels and ptAADC-/- with renal dopamine deficiency were treated with low-salt diet or high-salt diet for 2 weeks. Wild type or Cyp2c44-/- mice were treated with gludopa, which selectively increased renal dopamine levels. In low-salt treated mice, urinary EET levels were related to renal dopamine levels, being highest in COMT-/- mice and lowest in ptAADC-/- mice. In high-salt treated mice, total EET and individual EET levels in both kidney and urine were also highest in COMT-/- mice and lowest in ptAADC-/- mice. Selective increases in renal dopamine in response to gludopa administration led to marked increases in both total and all individual EET levels in the kidney without any changes in blood levels. qRT-PCR and Western analysis indicated that gludopa increased renal Cyp2c44 mRNA and protein levels. Gludopa induced marked increases in urine volume and urinary sodium excretion in wild type mice. In contrast, gludopa did not induce significant increases in urine volume or urinary sodium excretion in Cyp2c44-/- mice. These studies demonstrate that renal EET levels are maintained by intrarenal dopamine, and Cyp2c44-derived EETs play an important role in intrarenal dopamine-induced natriuresis and diuresis.
The circadian clock plays an important role in the regulation of physiological processes including renal function and blood pressure. We have previously shown that the circadian protein Per1 positively regulates expression of multiple sodium transport genes in the collecting duct including the alpha subunit of the renal epithelial sodium channel (αENaC). Consistent with this finding, Per1 KO mice exhibit dramatically lower BP than WT mice. We have also recently demonstrated the potential opposing actions of Cry2 on Per1 target genes. Recent work by others has demonstrated that Cry1/2 regulates aldosterone production through increased expression of the adrenal gland-specific rate-limiting enzyme, 3β-HSD. Therefore, we tested the hypothesis that Per1 plays a role in the regulation of aldosterone levels and renal sodium retention. Using RNA silencing and pharmacological blockade of Per1 nuclear entry in the NCI-H295R human adrenal cell line, we showed that Per1 regulated 3β-HSD expression in vitro. These results were confirmed in vivo: mice with reduced levels of Per1 had decreased levels of plasma aldosterone and decreased mRNA expression of 3β-HSD. We postulated that mice with reduced Per1 would have a renal sodium retaining defect. Indeed, metabolic cage studies demonstrated that Per1 heterozygotes excreted more urinary sodium compared to WT mice. Taken together, these data support the hypothesis that Per1 regulates aldosterone levels and that Per1 plays an integral role in the regulation of sodium retention.
The purpose of this study is to determine whether duloxetine (a serotonin-norepinephrine reuptake inhibitor) combined with transcutaneous foot stimulation or WAY100635 (a 5HT1A antagonist) can enhance inhibition of bladder overactivity in cats. Cystometrograms were performed on 8 cats under α-chloralose anesthesia by infusing saline and then 0.25% acetic acid (AA) to induce bladder overactivity. To inhibit bladder overactivity, foot stimulation (5 Hz) was applied via transcutaneous pad electrodes to the right hind foot at 2 and 4 times the threshold (T) intensity for inducing a toe twitch. Duloxetine (0.003-3 mg/kg) was administered intravenously to determine the effect of combination treatment. After the 3 mg/kg dose of duloxetine, WAY100635 (0.5 mg/kg) was given intravenously. AA irritation significantly (P<0.0001) reduced bladder capacity to 42.7±7.4% of saline control capacity. Foot stimulation alone at both 2T and 4T significantly (P<0.0001) inhibited bladder overactivity and increased bladder capacity to 66.7±6.3% and 85.7±6.5% of saline control, respectively. Duloxetine alone dose-dependently inhibited bladder overactivity and completely restored bladder capacity to saline control (109±15.5%) at 3 mg/kg. Although duloxetine combined with foot stimulation did not further enhance the inhibition, WAY100635 (0.5 mg/kg) given after 3 mg/kg duloxetine further increased (P=0.008) bladder capacity to 162.2±22.5% of saline control. Although duloxetine and foot stimulation independently inhibited bladder overactivity, combined treatment did not enhance the inhibition. Duloxetine combined with WAY100635, however, synergistically enhanced bladder inhibition, indicating a potential novel treatment for overactive bladder if duloxetine is combined with a 5HT1A receptor antagonist drug.
Binding of vasopressin to its type-2 receptor in renal collecting ducts induces cAMP signaling, transcription and translocation of aquaporin-2 (AQP2) water channels to the plasma membrane and water reabsorption from the pro-urine. Demeclocycline is currently used to treat hyponatremia in patients with the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Demeclocycline's mechanism of action, which is poorly understood, is studied here. In mouse cortical collecting duct (mpkCCD) cells, which exhibit dDAVP-dependent expression of endogenous AQP2, demeclocycline decreased AQP2 abundance and gene transcription, but not its protein stability. Demeclocycline did not affect V2R localization, but decreased dDAVP-induced cAMP generation and adenylate cyclase 3 and 5/6 abundances. Addition of exogenous cAMP partially corrected the demeclocycline effect. As in patients, demeclocycline increased urine volume, decreased urine osmolality and reverted hyponatremia in an SIADH rat model. AQP2 and adenylate cyclase 5/6 abundances were reduced in the inner medulla, but increased in the cortex and outer medulla, in the absence of any sign of toxicity. In conclusion, our in vitro and in vivo data indicate that demeclocycline mainly attenuates hyponatremia in SIADH by reducing adenylate cyclase 5/6 expression, and consequently cAMP generation, AQP2 gene transcription and AQP2 abundance in the renal inner medulla, coinciding with a reduced vasopressin-escape response in the other collecting duct segments.
Diabetic nephropathy, the most common cause of progressive chronic renal failure and end-stage renal disease, has now reached global proportions. The only means to rescue diabetic patients on dialysis is renal transplantation, a very effective therapy, but severely limited by availability of donor kidneys. Hence, we tested the role of intravenous renal cell transplantation (IRCT) on obese/diabetic ZS female rats with severe ischemic and diabetic nephropathy. Renal ischemia was produced by bilateral renal clamping of the renal arteries at 10 weeks of age, and IRCT with genetically modified normal ZS male tubular cells was given intravenously at 15 and 20 weeks of age. The rats were terminated at 34 weeks of age. IRCT with cells expressing serum amyloid A (SAA) had strong and long-lasting beneficial effects on renal function and structure, including tubules and glomeruli. However, donor cells were found engrafted only in renal tubules 14 weeks after the second infusion. The results indicate that IRCT with SAA+ cells is effective in preventing CKD progression in rats with diabetic and ischemic nephropathy.
The apoptotic or necrotic death of renal tubule epithelial cells is the main pathogenesis of renal ischemia/reperfusion-induced acute kidney injury (AKI). Pyroptosis is a programmed cell death pathway that depends on the activation of the caspase cascade and interleukin (IL)-1 cytokine family members. However, the role of pyroptosis in AKI induced by ischemia/reperfusion remains unclear. In this study, we found that the levels of the pyroptosis-related proteins, including caspase-1, caspase-11, and IL-1β, were significantly increased after 6 h of renal ischemia/reperfusion injury (IRI) and peaked at 12 h following injury. Enhanced pyroptosis was accompanied by elevated renal structural and functional injury. Similarly, hypoxia/reoxygenation injury (HRI) also induced pyroptosis in renal tubule epithelial NRK-52E cells, which was characterized by increased pore formation and elevated lactate dehydrogenase (LDH) release. In addition, the obvious upregulation of endoplasmic reticulum (ER) stress biomarkers, glucose-regulated protein (GRP) 78, and C/EBP homologous protein (CHOP), preceded the incidence of pyroptosis in cells treated with IRI or HRI. Pre-treatment with a low dose of tunicamycin, an inducer of ER stress, relieved IRI-induced pyroptosis and renal tissue injury. Silencing of CHOP by siRNA interference significantly decreased HRI-induced pyroptosis of NRK-52E cells, as evidenced by reduced caspase-11 activity and IL-1β generation. Therefore, we conclude that pyroptosis of renal tubule epithelial cells is a key event during IRI and that CHOP-caspase-11 triggered by overactivated ER stress may be an essential pathway involved in pyroptosis.
The cation cotransporters NKCC1, NKCC2 and NCC are phosphorylated and activated by the kinases SPAK (Ste20-related proline alanine rich kinase) and OXSR1 (oxidative stress responsive kinase) and their targeted disruption in mice causes phenotypes resembling the human disorders Bartter Syndrome and Gitelman Syndrome, reflecting reduced NKCC2 and NCC activity respectively. We previously cloned a kinase-inactive kidney-specific SPAK isoform, KS-SPAK, which lacks the majority of the kinase domain present in full-length SPAK. Another putative inactive SPAK isoform, SPAK2, which only lacks the initial portion of the kinase domain, is also highly expressed in kidney. The functional relevance of inactive SPAK isoforms is unclear. Here, we tested whether of KS-SPAK and SPAK2 differentially affect cation cotransporter activity. While KS-SPAK and SPAK2 both strongly inhibited NKCC1 activity, SPAK2 was a much weaker inhibitor of NKCC2 activity. Removal of the catalytic loop from SPAK2 resulted in an inhibitory effect on NKCC2 similar to that of KS-SPAK. Full-length SPAK is phosphorylated and activated by members of the WNK kinase family. Mutation of a WNK phosphorylation in KS-SPAK did not alter its ability to inhibit NKCC2 activity. In contrast, we found that residues involved in KS-SPAK interactions with cation cotransporters are required for it to inhibit cotransporter activity. Finally, both KS-SPAK and SPAK2 associated with NKCC2, as demonstrated by coimmunoprecipitation. Together, these data identify the structural basis for the differential effects of KS-SPAK and SPAK2 on cation cotransporter activity that may be physiologically important.
The renal proximal tubule (PT) is a major site for maintaining whole-body pH homeostasis and is responsible for reabsorbing ~80% of filtered bicarbonate (HCO3-), the major plasma buffer, into the blood. The PT adapts its rate of HCO3- reabsorption (JHCO3) in response to acute acid-base disturbances. Our laboratory previously showed that single isolated perfused PTs adapt JHCO3 in response to isolated changes in basolateral (i.e., blood-side) [CO2] and [HCO3-], but surprisingly not pH. The response to [CO2] is blocked by the ErbB-family tyrosine kinase inhibitor PD168393. In the present study, we expose enriched rabbit PT suspensions to five acute acid-base disturbances for 5 and 20 min, using a panel of phosphotyrosine (pY)-specific antibodies to determine the influence of each disturbance on pan-pY, ErbB1-specific-pY (four sites), and ErbB2-specific-pY (two sites). We find that each acid-base treatment generates a distinct temporal pY pattern. For example, the summated responses of the individual ErbB1/2-pY sites to each disturbance show that metabolic acidosis (normal [CO2], reduced [HCO3-]) produces a transient summated pY decrease (5 vs. 20 min), whereas metabolic alkalosis produces a transient increase. Respiratory acidosis (normal [HCO3-], elevated [CO2]) has little effect on summated pY at 5 min but produces an elevation at 20 min, whereas respiratory alkalosis produces a reduction at 20 min. Our data show that ErbB1 and ErbB2 in the PT respond to acute acid-base disturbances, consistent with the hypothesis that they are part of the signaling cascade.
Hypertension is a complex trait that is influenced by both heritable and environmental factors. The search for genes accounting for the susceptibility to hypertension has driven parallel efforts in human research and in research using experimental animals in controlled environmental settings. Evidence from rodent models of genetic hypertension and human Mendelian forms of hypertension and hypotension have yielded mechanistic insights into the pathways that are perturbed in blood pressure homeostasis, most of which converge at the level of renal sodium reabsorption. Yet, the bridging of evidence from these very diverse approaches to identify mechanisms underlying hypertension susceptibility and the translation of these findings to human populations and public health remain a challenge. Furthermore, findings from genome-wide association studies still require functional validation in experimental models. In this review, we highlight results and implications from key studies in experimental and clinical hypertension to date.
Regulation of urea transporter UT-A1 in the kidney is important for the urinary concentrating mechanism. We previously reported that activation of cAMP/PKA pathway by forskolin (FSK) leads to UT-A1 ubiquitination, endocytosis, and degradation. In this study, we discovered that the FSK-induced UT-A1 ubiquitination is monoubiquitination as judged by immunoblotting with specific ubiquitin antibodies to the different linkage of ubiquitin chain. The UT-A1 monoubiquitination induced by FSK was processed mainly on the cell plasma membrane. Monoubiquitination facilitates UT-A1 endocytosis and the internalized UT-A1 is accumulated in the early endosome. Inhibition of ubiquitination by E1 ubiquitin activating enzyme inhibitor PYR-41 significantly reduced FSK-induced UT-A1 endocytosis and degradation. Interestingly, FSK- stimulated UT-A1 degradation is through lysosomal protein degradation system. We further found that the PKA phosphorylation sites of UT-A1 at S486 and S499 are required for FSK induced UT-A1 monoubiquitination. The physiological significance was confirmed using rat kidney IMCD suspensions, showing that vasopressin treatment promotes UT-A1 ubiquitination. We concluded that, unlike under the basal condition that UT-A1 is subject to polyubiquitonation and proteasome-mediated protein degradation, activation of UT-A1 by FSK induces UT-A1 monoubiquitination and protein lysosomal degradation.
Caveolin-1 (Cav1) is expressed in the basolateral membrane domain of renal collecting duct (CD) principal cells (PCs), where it is associated with caveolae. To reveal any potential involvement of Cav1 in vasopressin signaling, we used specific mono- and polyclonal antibodies to examine its localization in CD PCs of Brattleboro (BB) rats treated with vasopressin (DDAVP). Compared to controls, immunofluorescence revealed a time-dependent increase in Cav1 expression in the apical membrane domain of PCs, where it overlapped with aquaporin-2 (AQP2). After 24 h of DDAVP treatment, Cav1 was visible as an increased number of small apical spots. The staining gradually became more extensive and after 2 weeks of DDAVP it occupied a majority of the apical membrane domain of many PCs. Cav1 also assumed an apical localization in PCs of DDAVP-treated Sprague-Dawley (SD) and Long-Evans (LE) rats. Similarly, Cav2 appeared at the apical pole of PCs following DDAVP treatment of BB, SD, and LE rats. Immunogold electron microscopy confirmed bipolar Cav1 membrane expression in DDAVP-treated BB rats, while caveolae were only detected on the basolateral membrane. Immunoblotting of BB rat whole kidney homogenates revealed no significant increase in Cav1 levels in DDAVP-treated rats, suggesting that DDAVP induces Cav1 relocalization or modifies its targeting. We conclude that Cav1 and Cav2 trafficking and membrane localization are dramatically altered by the action of DDAVP. Importantly, the absence of apical caveolae indicates that while caveolins may have an as yet undetermined role in vasopressin-regulated signaling processes, this is probably unrelated to AQP2 internalization by caveolae.
We present a lumped-nephron model that explicitly represents the main features of the underlying physiology, incorporating the major hormonal regulatory effects on both tubular and vascular function, and which accurately simulates hormonal regulation of renal salt and water excretion. This is the first model to explicitly couple glomerulovascular and medullary dynamics, and it is much more detailed in structure than existing whole-organ models and renal portions of multi-organ models. In contrast to previous medullary models, which have only considered the antidiuretic state, our model is able to regulate water and sodium excretion over a variety of experimental conditions in good agreement with data from experimental studies of the rat. Since the properties of the vasculature and epithelia are explicitly represented, they can be altered to simulate pathophysiological conditions and pharmacological interventions. The model serves as an appropriate starting point for simulations of physiological, pathophysiological and pharmacological renal conditions, and for exploring the relationship between the extra-renal environment and renal excretory function in physiological and pathophysiological contexts.
A selective 5-hydroxytryptamine (5-HT) 2A receptor antagonist sarpogrelate (SG) blocks serotonin-induced platelet aggregation. It has been used clinically for the treatment of peripheral arterial disease. SG might be able to improve chronic ischemia, which contributes to renal fibrosis progression by maintaining renal microcirculation. This study investigated whether SG suppresses renal fibrosis. C57BL/6 mice fed a 0.2% adenine-containing diet for 6 weeks developed severe tubulointerstitial fibrosis with kidney dysfunction. Subsequent SG treatment (30 mg/kg/day) for 4 weeks improved these changes significantly by increasing peritubular blood flow in the fibrotic area, as evaluated by intravital microscopy and decreasing fibrin deposition. Urinary L-type fatty acid-binding protein, up-regulated by renal hypoxia, was also reduced by SG. Additionally, results showed that mRNA expression of plasminogen activator inhibitor-1 (PAI-1), which is known to promote fibrosis by mediating and enhancing TGF-β1 signaling, was suppressed by SG treatment in the kidney. In vitro experiments using cultured murine proximal tubular epithelial (mProx) cells revealed that incubation with TGF-β1 and 5-HT increased PAI-1 mRNA expression; SG significantly reduced it. In conclusion, SG reduces renal fibrosis not only by the anti-thrombotic effect of maintaining peritubular blood flow, but also by suppressing PAI-1 expression in renal tubular cells.
Unilateral ureteral obstruction (UUO) in the adult mouse is the most widely used model of progressive renal disease: the proximal tubule is the nephron segment most severely affected and atubular glomeruli are formed after only 7 days of UUO. To determine the proximal nephron response to UUO in the maturing kidney, neonatal mice were examined 7 to 28 days following complete UUO under general anesthesia. Proximal tubular mass and maturation were determined by staining with Lotus tetragolonobus lectin. Superoxide was localized by nitroblue tetrazolium and collagen by Sirius red. Cell proliferation, cell death, PAX-2, megalin, α-smooth muscle actin (α-SMA), renin, and fibronectin were identified by immunohistochemistry. During the first 14 days of ipsilateral UUO, despite oxidative stress (4-hydroxynonenal staining), glomerulotubular continuity was maintained and mitochondrial superoxide production persisted. However, from 14 to 28 days, papillary growth was impaired and proximal tubules collapsed with increased apoptosis, autophagy, mitochondrial loss, and formation of atubular glomeruli. Fibronectin, α-SMA and collagen increased in the obstructed kidney. Oxidative stress was present also in the contralateral kidney: renin was decreased, glomerulotubular maturation and papillary growth were delayed, followed by increased cortical and medullary growth. We conclude that neonatal UUO initially delays renal maturation and results in oxidative stress in both kidneys. In contrast to the adult, proximal tubular injury in the neonatal obstructed kidney is delayed at 14 days, followed only later by the formation of atubular glomeruli. Antioxidant therapies directed at proximal tubular mitochondria during early renal maturation may slow progression of congenital obstructive nephropathy.
Cystinuria is an autosomal recessive disease caused by mutations in SLC3A1 (rBAT) and SLC7A9 (b0,+AT). Gene targeting of the catalytic subunit (Slc7a9) in mice leads to excessive excretion of cystine, lysine, arginine and ornithine. Here we studied this non-type I cystinuria mouse model using gene expression analysis, Western blot, clearance and uptake experiments to characterize the renal and intestinal consequences of losing Slc7a9 function. The electrogenic and flux transport studies in the intestine suggested that arginine and ornithine are transported via other routes apart from system b0,+. No remarkable gene expression changes were observed in other amino acid transporters and the peptide transporters in intestine and kidney. Further, the glomerular filtration rate (GFR) was reduced by 30 % in knockout animals compared to wild-type animals. The fractional excretion of arginine was increased as expected (~100 %), but fractional excretions of lysine (~35 %), ornithine (~16 %) and cystine (~11 %) were less affected. Loss of function of b0,+AT reduced transport of cystine and arginine in renal brush border membrane vesicles and completely abolished the exchanger activity of dibasic amino acids with neutral amino acids. In conclusion, loss of Slc7a9 function decreases the glomerular filtration rate and increases the excretion of several amino acids to a lesser extent than expected with no clear regulation at the mRNA and protein level of alternative transporters and no increased renal epithelial uptake. These observations indicate that transporters located in distal segments of the kidney and/or metabolic pathways may partially compensate Slc7a9 loss of function.
Adipocytes secrete a number of bioactive adipokines that activate a variety of cell signaling pathways in central and peripheral tissues. Obesity is associated with altered production of many adipokines which is linked to a number of pathologies. As an increase in body weight is directly associated with an increased risk for developing chronic kidney disease (CKD), there is significant interest in the link between obesity and renal dysfunction. Altered levels of the adipokines leptin, adiponectin, resistin, and visfatin can decrease glomerular filtration and increase albuminuria which are pathophysiological changes typical of CKD. Specifically exposure of the glomerulus to altered adipokine levels can increase its permeability, fuse the podocytes, and cause mesangial cell hypertrophy, all of which alter the glomerular filtration rate. In addition the adipokines leptin and adiponectin can act on the tubular networks. Thus adipokines can act on multiple cell types in the development of renal pathophysiology. Importantly most studies have been performed using in vitro models, with future studies in vivo required to further elucidate the specific roles that adipokines play in the development and progression of CKD.
p53, a pivotal protein in the apoptotic pathway, has been identified as a mediator of transcriptional responses to ischemia-reperfusion (IR) injury. The characteristics and functional significance of p53 response in vivo are largely unknown in IR-induced kidney injury. Therapeutic opportunities of delivering small interfering RNA (siRNA) via venous injection have gained recognition; however, systemic adverse effects of siRNA therapy should be considered. To prevent IR-induced kidney injury, we tested the efficacy of transarterial administration of siRNA targeting p53 (p53 siRNA). Female C57BL/6 mice underwent unilateral renal artery ischemia for 30 min, followed by reperfusion. siRNA experiments utilized short hairpin (sh) RNA plasmid-based approaches. Transfection of shRNA was performed using cationic polymer transfection reagent. Injection of synthetic p53 shRNA into the left renal artery just after ischemia improved tubular injury, apoptosis, and the swelling of mitochondria in cells of the thick ascending limb of Henle (mTALH) at the outer medullary regions. Staining of upregulated p53 was co-localized with the inducible expression of glycogen synthase kinase 3 beta (GSK-3β) at mTALH after IR injury. p53 shRNA inhibited GSK-3β expression and restored β-catenin expression at mTALH. For IR-induced kidney injury, transarterial delivery of p53 siRNA is an effective pharmacological intervention. Targeting siRNA to p53 leads to an attenuation of apoptosis and mitochondrial damage through the downregulation of GSK-3β expression and upregulation of β-catenin. Local delivery of vectors such as p53 siRNA locally through a transaortic catheter is clinically useful in reducing the adverse effect of siRNA-related therapy.
The epithelial sodium channel (ENaC) is comprised of three homologous subunits. Channels composed solely of α and β subunits (αβ channels) exhibit a very high open probability (Po) and reduced sensitivity to amiloride, in contrast to channels composed of α and subunits or of all three subunits (i.e., α and αβ channels). A mutant channel comprised of α and β subunits, and a chimeric subunit where the region immediately preceding (β12 and wrist) and encompassing the second transmembrane domain (TM2) was replaced with the corresponding region of the β subunit (-βTM2) displayed characteristics reminiscent of αβ channels, including a reduced potency of amiloride block and a loss of Na+ self-inhibition (reflecting an increased Po). Substitutions at key pore-lining residues of the -βTM2 chimera enhanced the Na+ self-inhibition response, whereas key subunit substitutions reduced the response. Furthermore, multiple sites within the TM2 domain of the subunit were required to confer high amiloride potency. In summary, we have identified novel pore-lining residues of the subunit of ENaC that are important for proper channel gating and its interaction with amiloride.
The DCT (distal convoluted tubule) is the site of micro-regulation of water reabsorption and ion handling in the kidneys, which is mainly under the control of aldosterone. Aldosterone binds to and activates mineralocorticoid receptors, which ultimately leads to increased sodium reabsorption in the distal part of the nephron. Impairment of mineralocorticoid signal transduction results in resistance to aldosterone and mineralocorticoids, and, therefore, causes disturbances in electrolyte balance. Pseudohypoaldosteronism type II (PHAII) or familial hyperkalemic hypertension (FHHt) is a rare, autosomal dominant syndrome characterized by hypertension, hyperkalemia, metabolic acidosis, elevated or low aldosterone levels, and decreased plasma renin activity. PHAII is caused by mutations in the WNK isoforms (with no lysine kinase), which regulate the Na-Cl and Na-K-Cl cotransporters (NCC and NKCC2, respectively) and renal outer medullary potassium (ROMK) channel in the DCT. This review focuses on new candidate genes such as KLHL3 and Cullin3, which are instrumental to unravel novel signal transductions pathways involving NCC, to better understand the cause of PHAII along with the molecular mechanisms governing the pathophysiology of PHAII and its clinical manifestations.
The kidney plays a key role in the maintenance of the magnesium (Mg2+) homeostasis. Specifically, the distal convoluted tubule (DCT) is instrumental in fine-tuning of the renal Mg2+ handling. In recent years hereditary Mg2+ transport disorders have helped to identify important players in DCT Mg2+ homeostasis. Nevertheless, several proteins involved in DCT-mediated Mg2+ reabsorption remains to be discovered and a full expression profile of this complex nephron segment may facilitate the discovery of new Mg2+-related genes. Here, we report the Mg2+-sensitive expression of the DCT transcriptome. To this end, transgenic mice expressing eGFP under a DCT-specific parvalbumin promoter were subjected to Mg2+-deficient or Mg2+-enriched diets. Subsequently, the Complex Object Parametric Analyzer and Sorter (COPAS) allowed for the first time isolation of eGFP-positive DCT cells. RNA extracts thereof were analyzed by DNA microarrays comparing high vs. low Mg2+ to identify Mg2+ regulatory genes. Based on statistical significance and a fold-change of at least two, 46 genes showed differential expression. Several known magnesiotropic genes, such as Trpm6 and Parvalbumin, were upregulated under low dietary Mg2+. Moreover, new genes were identified that are potentially involved in renal Mg2+ handling. To confirm that the selected candidate genes were regulated by dietary Mg2+ availability, the expression levels of Slc41a3, Pcbd1, Tbc1d4 and Umod were determined by RT-PCR analysis. Indeed, all four genes show significant upregulation in the DCT of mice fed the Mg2+-deficient diet. By elucidating the Mg2+-sensitive DCT transcriptome new candidate genes in renal Mg2+ handling have been identified.
The sequence of events by which primary hyperoxaluria type 1 (PH1) causes renal failure is unclear. We hypothesize that proximal tubule (PT) is vulnerable because oxalate secretion raises calcium oxalate (CaOx) supersaturation (SS) there, leading to crystal formation and cellular injury. We studied cortical and papillary biopsies from 2 PH1 patients with preserved renal function, and 7 native kidneys removed from 4 patients at the time of transplant, after short-term (2) or longer-term (2) dialysis. In these patients, and another 5 PH1 patients without renal failure, we calculated oxalate secretion, and estimated PT CaOx SS. Plasma oxalate was elevated in all PH1 patients, and inverse to creatinine clearance. Renal secretion of oxalate was present in all PH1, but rare in controls. PT CaOx SS was > 1 in all non-pyridoxine responsive PH1 prior to transplant, most marked in patients who developed ESRD. PT from PH1 with preserved renal function had birefringent crystals, confirming the presence of CaOx SS, but had no evidence of cortical inflammation or scarring by histopathology or hyaluronan staining. PH1 with short ESRD showed CaOx deposition and hyaluronan staining particularly at the corticomedullary junction in distal PT while cortical collecting ducts were spared. Longer ESRD showed wide-spread cortical CaOx, and in both groups papillary tissue had marked intratubular CaOx deposits and fibrosis. CaOx SS in PT causes CaOx crystal formation, and CaOx deposition in distal PT appears to be associated with ESRD. Minimizing PT CaOx SS may be important for preserving renal function in PH1.
The KCl cotransporters KCC3 and KCC4 are expressed at the basolateral membrane of proximal convoluted tubule cells, and KCC4 is present in the basolateral membrane of the thick ascending loop of Henle's limb and the α-intercalated cells of the collecting duct. Little is known, however, about the physiological roles of these transporters in the kidney. We evaluated the KCC3 and KCC4 mRNA and protein expression levels and intrarenal distribution in male Wistar rats or C57 mice under five experimental conditions: hyperglycemia after a single dose of streptozotocin, a low salt diet, metabolic acidosis induced by ammonium chloride in drinking water, and low or high K+ diet. Both KCC3 mRNA and protein expression was increased during hyperglycemia in the renal cortex and at the basolateral membrane of proximal tubule cells but not a low salt diet or acidosis. In contrast, KCC4 protein expression was increased by a low sodium diet in whole kidney and by metabolic acidosis in the renal outer medulla, specifically at the basolateral membrane of α-intercalated cells. The increased protein expression of KCC4 by a low salt diet was also observed in WNK4 knockout mice, suggesting that up-regulation of KCC4 in these circumstances is not WNK4 dependent. No change in KCC3 or KCC4 protein expression was observed under low or high K+ diets. Our data are consistent with a role for KCC3 in the proximal tubule glucose reabsorption mechanism and for KCC4 in salt reabsorption of the TAL and acid secretion of the collecting duct.
A number of studies have shown that rats with congestive heart failure (CHF) have increased protein levels of the vasopressin-regulated water channel AQP2 even during conditions with unchanged circulating levels of vasopressin (AVP), suggesting an increase in the sensitivity of the AVP type-2 (V2) receptor in experimental CHF. The present study was aimed at investigating AVP signaling in rats with moderate CHF (left ventricular end diastolic pressure >10 mmHg; normal plasma AVP levels) induced by ligation of the left anterior descending coronary artery. Sham-operated rats were used as controls. Western blotting analyses revealed an increased abundance of AQP2 in renal cortex (+ 33±9 % of Sham; p<0.05) and in inner medulla (IM) (+54±15% of Sham; p<0.05) in CHF rats when compared to sham operated controls. Dose response studies on isolated collecting ducts (CD's) showed an increased accumulation of cAMP in response to AVP in CHF rats when compared to controls. V2-receptor surface binding studies in isolated IMCD's showed a marked and comparable AVP-induced V2-receptor internalization in response to AVP in both CHF and control rats. As expected V2-receptor surface binding remained low after AVP challenge in control rats. In contrast to this, V2-receptor surface binding returned to pre-AVP levels within 30 minutes in the CHF rats indicating an obtained recycling ability of the V2-receptor in CHF. Together the results indicate the presence of an increased AVP sensitivity in the CD's from CHF rats, associated with an acquired recycling ability of the V2-receptor.
Transforming growth factor (TGF)-β has been associated with podocyte injury; we have examined its effect on podocyte bioenergetics. We studied transformed mouse podocytes, exposed to TGF-β1, using a label-free assay system, Seahorse XF24, which measures oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). Both basal OCR and ATP generation-coupled OCR were significantly higher in podocytes exposed to 0.3-10 ng/mL of TGF-β1 for 24, 48, and 72 hours. TGF-β1 (3 ng/mL) increased oxidative capacity 75%, and 96% relative to control after 48 and 72 hours, respectively. ATP content was increased 19% and 30% relative to control after 48 and 72 hours exposure, respectively. Under conditions of maximal mitochondrial function, TGF-β1 increased palmitate-driven OCR by 49%. Thus, TGF-β1 increases mitochondrial oxygen consumption and ATP generation in the presence of diverse energy substrates. TGF-β1 did not increase cell number or mitochondrial DNA copy number, but did increase mitochondrial membrane potential (MMP), which could explain the OCR increase. Reactive oxygen species (ROS) increased by 32% after TGF-β1 exposure for 48 hours. TGF-β1 activated the mammalian target of rapamycin (mTOR) pathway, and rapamycin reduced the TGF-β1-stimulated increases in OCR, ECAR, ATP generation, cellular metabolic activity, and protein generation. Our data suggest that TGF-β1, acting in part via mTOR, increases mitochondrial MMP and OCR, resulting in increased ROS generation, and that this may contribute to podocyte injury.
In this study, we examined the possibility that 5-HT1A receptors may underlie sexually dimorphic mechanisms affecting regulation of urethral functions in anesthetized rats. Simultaneous recordings of the intravesical pressure under isovolumetric conditions (isovolumetric IVP), external urethral sphincter-electromyography (EUS-EMG), and urethral perfusion pressure (UPP) were used to examine the effects of a 5-HT1A receptor agonist (8-OH-DPAT) and antagonist (WAY-100635) on bladder and urethral functions. This research also evaluated the effects of 8-OH-DPAT and α-bungarotoxin (a neuromuscular blockade agent) on urethral continence using leak point pressure (LPP) testing, and the distribution of 5-HT1A receptors in the lower urinary tract was assessed by immunohistochemistry (IHC). The serotonergic mechanism that controls the urinary bladder and EUS-EMG activity showed no significant sexual difference, but urethral activity in UPP and LPP values exhibited some sexual differences. 8-OH-DPAT enhanced urethral pressure during continence in rats of both sexes, but the drug elevated the pressure during voiding in males and reduced it in females. The distribution of 5-HT1A receptors in the spinal cord also showed some sexual differences. The present study contributes to our understanding of the role of 5-HT1A receptors in physiological and IHC properties of urethral smooth muscle in rats of different sexes. These findings may be a basis for the future development of pharmacotherapies for stress urinary incontinence in men.
Dendritic cells (DC) play critical roles in immune-mediated kidney diseases. Little is known, however, about DC subsets in human chronic kidney disease, with previous studies restricted to a limited set of pathologies and to using immunohistochemical methods. In this study, we developed novel protocols for extracting renal DC subsets from diseased human kidneys and identified, enumerated and phenotyped them by multi-colour flow cytometry. We detected significantly greater numbers of total DC, CD141hi and CD1c+ myeloid DC (mDC) subsets in diseased biopsies with interstitial fibrosis than diseased biopsies without fibrosis or healthy tissue. In contrast, plasmacytoid DC numbers were significantly higher in the fibrotic group compared to healthy tissue only. Numbers of all DC subsets correlated with loss of kidney function, recorded as estimated glomerular filtration rate. CD141hi DC expressed CLEC9A, whilst the majority of CD1c+ DC lacked expression of CD1a and DC-SIGN, suggesting these mDC subsets may be circulating CD141hi and CD1c+ blood DC infiltrating kidney tissue. Our analysis revealed CLEC9A+ and CD1c+ cells were restricted to the tubulointerstitium. Notably, DC expression of the costimulatory and maturation molecule CD86 was significantly increased in both diseased cohorts compared to healthy tissue. TGF-β levels in dissociated tissue supernatants were significantly elevated in diseased biopsies with fibrosis compared to non-fibrotic biopsies, with mDC identified as a major source of this pro-fibrotic cytokine. Collectively, our data indicate that activated mDC subsets, likely recruited into the tubulointerstitium, are positioned to play a role in the development of fibrosis and thus, progression to chronic kidney disease.
Obesity-related kidney disease occurs as a result of complex interactions between metabolic and hemodynamic effects. Changes in microvascular perfusion may play a major role in kidney disease; however, these changes are difficult to assess in vivo. Here, we used perfusion ultrasound imaging to evaluate cortical blood flow in a mouse model of high fat induced kidney disease. C57BL/6J mice were randomized to a standard diet (STD) or a high-fat diet (HFD), for 30 weeks, then treated either with Losartan or a placebo for an additional 6 weeks. Non-invasive ultrasound perfusion imaging of the kidney was performed during infusion of a microbubble contrast agent. Blood flow within the microvasculature of the renal cortex and medulla was derived from imaging data. An increase in the time required to achieve full cortical perfusion was observed for HFD mice relative to STD. This was reversed following treatment with losartan. These data were concurrent with an increased glomerular filtration rate in HFD mice compared to STD or HFD-losartan-treated mice. Losartan treatment also abrogated fibro-inflammatory disease, assessed by markers at the protein and messenger level. Finally, a reduction in capillary density was found in HFD mice, and this was reversed upon Losartan treatment. This suggests that alterations in vascular density may be responsible for the elevated perfusion time observed by imaging. These data demonstrate that ultrasound contrast imaging is a robust and sensitive method for evaluating changes in renal microvascular perfusion, and that cortical perfusion time may be a useful parameter for evaluating obesity-related renal disease.
Deletions of claudin-2 (Cldn2) and aquaporin1 (AQP1) reduce proximal fluid reabsorption (PFR) by about 30% and 50% respectively. Experiments were done to replicate these observations and to determine in AQP1/claudin-2 double knockout mice (DKO) if the effects of deletions of these established water pores are additive. PFR was determined in inactin/ketamine anesthetized mice by free flow micropuncture using single nephron I125-iothalamate (io) clearance. Animal means of PFR (% of GFR) derived from TF/Pio ratios in 12 mice in each of 4 groups (WT, Cldn2-/-, AQP1-/-, and DKO) were 45.8 ± 0.85 (51 tubules), 35.4 ± 1 (54 tubules; p<0.01 vs. WT), 36.8 ± 1 (63 tubules; p<0.05 vs. WT), and 33.9 ± 1.4 (69 tubules; p<0.01 vs. WT). Kidney and single nephron GFRs (SNGFR) were significantly reduced in all mutant strains . The direct relationship between PFR and SNGFR was maintained in mutant mice, but the slope of this relationship was reduced in the absence of Cldn2 and/or AQP1. Transtubular osmotic pressure differences were not different between WT and Cldn2-/- mice, but markedly increased in DKO. In conclusion, the deletion of Cldn2, AQP1, or of both Cldn2 and AQP1 reduces PFR by 22.7%, 19.6%, and 26% respectively. Our data are consistent with an up to 25% paracellular contribution to PFR. The reduced osmotic water permeability caused by absence of AQP1 augments luminal hypotonicity. Aided by a fall in filtered load the capacity of non AQP1-dependent transcellular reabsorption is sufficient to maintain PFR without AQP1 and claudin-2 at 75% of control.
Quantitative real time-PCR was used to test whether cavernous nerve injury leads to a decrease in major pelvic ganglia (MPG) neuronal nicotinic acetylcholine receptor (nAChR) subunit and postsynaptic density-93 (PSD-93) transcript levels. Subunits α3, β4 and α7, commonly expressed in MPG, were selected for analysis. After 72 hours in explant culture, MPG transcript levels for α3, β4, α7, and PSD-93 were depressed significantly. Three days following cavernous nerve axotomy or crush in vivo, transcript levels for α3, β4 and PSD-93, but not for α7, were significantly depressed. Three days after dissecting the cavernous nerve free of underlying tissue and applying a 5 mm lateral stretch (manipulation), the transcript levels for α3 and PSD-93 also were decreased significantly. Seven days following all 3 surgical procedures, α3 transcript levels remained depressed, but PSD-93 transcript levels were still decreased only following axotomy or nerve crush. At 30 days post surgery, transcript levels for the nAChR subunits and PSD-93 had recovered. Acetylcholine-induced currents were significantly smaller in MPG neurons dissociated from 3-day explant cultured ganglia than from those recorded in neurons dissociated from acutely isolated ganglia; this observation provides direct evidence that a decrease in nAChR function was coincident with a decrease in nAChR subunit transcript levels. We conclude that a down regulation of nAChR subunit and PSD-93 expression following cavernous nerve injury, or even manipulation, could interrupt synaptic transmission within the MPG and thus contribute to the loss of neural control of urogenital organs following pelvic surgeries.
Introduction: Inhibition of prostate smooth muscle contraction is an important strategy for medical treatment of lower urinary tract symptoms (LUTS). Besides α1-adrenoceptors, prostate smooth muscle contraction is induced by activation of thromboxane (TXA2) receptors (TXA2-R). Here, we examined the effects of the TXA2-R antagonist, picotamide, on contraction of human prostate tissue. Methods: Prostate tissues were obtained from radical prostatectomy. Effects of picotamide (300 μM), L-665,240 (3 μM), and seratrodast (3 μM) on U46619-, electric field stimulation- (EFS-), phenylephrine-, and noradrenaline-induced contractions were studied in organ baths. Expression of TXA2-R and TXA2 synthase (TXS) was examined by fluorescence stainings. Results: Picotamide, seratrodast, and L-655,240 inhibited concentration-dependent contractions induced by the TXA2 analogue, U46619. Picotamide, but not seratrodast or L-655,240 inhibited frequency-dependent contractions induced by EFS. Picotamide inhibited concentration-dependent contractions induced by noradrenaline, or by the selective α1-adrenoceptor agonist, phenylephrine. In prostate strips, where only submaximal contraction by a low dose of phenylephrine was induced, application of U46619 raised tone to maximum phenylephrine-induced tension. Immunoreactivity for TXA2-R and TXS was observed in the stroma, and in epithelial cells of glands. Colocalization of both immunoreactivites was observed with the smooth muscle markers calponin and α-smooth muscle actin, with the epithelial marker pan-cytokeratin, and with prostate-specific antigen in the stroma and glands. Conclusions: The receptor antagonist picotamide inhibits α1-adrenergic, TXA2-mediated, and EFS-induced contractions in the human prostate. To the best of our knowledge, this is the first antagonist able to inhibit two different contraction systems in the prostate.
Hibernators periodically undergo profound physiological changes including dramatic reductions in metabolic, heart and respiratory rates and core body temperature. This review discusses the effect of hypoperfusion and hypothermia observed during hibernation on glomerular filtration and renal plasma flow, as well as specific adaptations in renal architecture, vasculature, the renin-angiotensin system and upregulation of possible protective mechanisms during the extreme conditions endured by hibernating mammals. Understanding the mechanisms of protection against organ injury during hibernation may provide insights into potential therapies for organ injury during cold storage and re-implantation during transplantation.
Aldosterone is a major regulator of Na+ absorption and acts by activating the mineralocorticoid receptor to stimulate the epithelial Na+ channel (ENaC). MR-/- mice exhibited pseudohypoaldosteronism type 1 (hyponatremia, hyperkalemia, salt wasting, and high levels of aldosterone) and died around P10. However, if and how MR regulates ENaC transcription remain incompletely understood. Our earlier work demonstrated that aldosterone activates αENaC transcription by reducing expression of Dot1a and Af9 and by impairing Dot1a-Af9 interaction. Most recently, we reported identification of a major Af9 binding site in the αENaC promoter and upregulation of αENaC mRNA expression in mouse kidneys lacking Dot1a. Despite these findings, the putative antagonism between the MR/aldosterone and Dot1a-Af9 complexes has never been addressed. The molecular defects leading to PHA-1 in MR-/- mice remain elusive. Here, we report that MR competes with Dot1a to bind Af9. MR/aldosterone and Dot1a-Af9 complexes mutually counterbalance ENaC mRNA expression in IMCD3 cells. Real-time RT-qPCR revealed that 5-day-old MR-/- vs. MR+/+ mice had significantly lower αENaC mRNA levels. This change was associated with an increased Af9 binding and H3 K79 hypermethylation in the αENaC promoter. Therefore, this study identified MR as a novel binding partner and regulator of Af9 and a novel mechanism coupling MR-mediated activation with relief of Dot1a-Af9-mediated repression via MR-Af9 interaction. Impaired ENaC expression due to failure to inhibit Dot1a-Af9 may play an important role in the early stages of PHA-1 (prior to P8) in MR-/- mice.
Na-Cl cotransporter (NCC) in the distal tubules in kidney is known to be excreted in urine. However, its clinical significance has not been established because of the lack of quantitative data on urinary NCC. We developed highly sensitive enzyme-linked immunosorbent assays (ELISAs) for urinary total NCC (tNCC) and its active form, phosphorylated NCC (pNCC). We first measured the excretion of tNCC and pT55-NCC in urinary exosomes in pseudohypoaldosteronism type II (PHAII) patients since PHAII is caused by NCC activation. Highly increased excretion of tNCC and pNCC was observed in PHAII patients. In contrast, the levels of tNCC and pNCC in the urine of patients with Gitelman's syndrome were not detectable or very low, indicating that both assays could specifically detect the changes in urinary NCC excretion caused by the changes of NCC activity in the kidney. Then, to test whether these assays could be feasible for a more general patient population, we measured tNCC and pNCC in the urine of outpatients with different clinical backgrounds. Although urinary protein levels greater than 30 mg/dl interfered with our ELISA, we could measure urinary pNCC in all patients without proteinuria. Thus, we established highly sensitive and quantitative assays for urinary NCC, which could be valuable tools for estimating NCC activity in vivo.
CD44 family members are cell surface glycoproteins, which are expressed on tubular epithelial cells (TEC) solely upon kidney injury and are involved in renal fibrosis development. Renal interstitial fibrosis is the final manifestation of chronic kidney diseases and is regulated by a complex network of cytokines, including the pro-fibrotic factor TGF-β1 and the two anti-fibrotic cytokines BMP-7 and HGF. The present study investigates the potential role of CD44 standard (CD44s) and CD44v3-v10 (CD44v3) isoforms as modulators of the balance between TGF-β1 and HGF/BMP-7. CD44s is the shortest and most common isoform. CD44v3-v10 (CD44v3) has heparan sulfate moieties, which enable the binding to HGF/BMP-7, and hence, might exert reno-protective effects. Using transgenic mice overexpressing either CD44s or CD44v3 specifically on proximal TEC, we found that in vitro the overexpression of CD44v3 on primary TEC renders cells less susceptible to TGF-β1 pro-fibrotic actions and more sensitive to BMP-7 and HGF in comparison to TEC overexpressing CD44s. One day after unilateral ureteric obstruction, obstructed kidneys from CD44v3 transgenic mice showed less tubular damage and myofibroblasts accumulation, which was associated with decreased TGF-β1 signaling and increased BMP-7 synthesis and signaling compared to kidneys from WT and CD44s transgenic mice. These data suggest that CD44v3 plays a renoprotective role in early stage of chronic obstructive nephropathy.
Idiopathic uric acid nephrolithiasis is characterized by elevated urinary net acid excretion and insufficient buffering by ammonium, resulting in excessively acidic urine and titration of the relatively soluble urate anion to insoluble uric acid. Patients with type 2 diabetes have similar changes in urinary pH, net acid excretion and ammonium in 24 hour urine collections at baseline, even after controlling for dietary factors, and are at increased risk for uric acid nephrolithiasis. However, not all patients with type 2 diabetes develop kidney stones, suggesting that uric acid stone formers may have additional urinary defects, perhaps not apparent at baseline. We performed a metabolic study of 14 patients with idiopathic uric acid nephrolithiasis, 13 with type 2 diabetes, and 8 healthy control subjects of similar body mass index. Following equilibration on a fixed diet for 5 days, subjects were given a single oral acid load (50 mEq ammonium chloride), and urine was collected hourly for 4 hours. Uric acid stone formers had lower ammonium excretory response to acute acid loading compared with diabetic and non-diabetic non-stone formers, suggesting that an ammonium excretory defect unique to uric acid stone formers was unmasked by the acid challenge. The Zucker diabetic fatty rat also did not show impaired urinary ammonium excretion in response to acute acid challenge. Blunted renal ammonium excretory response to dietary acid loads may contribute to the pathogenesis of idiopathic uric acid nephrolithiasis.
Thiazolidinediones (TZDs) which are synthetic PPAR agonists are highly effective for treatment of type 2 diabetes. However, the side effect of fluid retention has significantly limited their application. Most of the previous studies addressing TZD-induced fluid retention employed healthy animals. The underlying mechanism of this phenomenon is still incompletely understood, particularly in the setting of disease state. The present study was undertaken to examine rosiglitazone (RGZ)-induced fluid retention in db/db mice and to further investigate the underlying mechanism. In response to RGZ treatment, db/db mice exhibited an accelerated plasma volume expansion as assessed by hematocrit (Hct) and fluorescent nanoparticles, in parallel with a greater increase in body weight, as compared with lean controls. In response to RGZ-induced fluid retention, urinary Na+ excretion and urine volume were significantly increased in lean mice. In contrast, the natriuretic and diuretic responses were significantly blunted in db/db mice. RGZ db/db mice exhibited a parallel decrease in plasma Na+ concentration and plasma osmolality, contrasting to unchanged levels in lean controls. Imunoblotting analysis showed downregulation of renal AQP2 expression in response to RGZ treatment in lean mice but not in db/db mice. Renal AQP3 protein expression was unaffected by RGZ treatment in lean mice but was elevated in db/db mice. In contrast, the expression of NHE3 and NKCC2 was unchanged in either mouse strain. Together these results suggest that compared with the lean controls, db/db mice exhibited accelerated plasma volume expansion that was in part due to the inappropriate response of renal water transporters.
The NBCn1 Na+/HCO3- cotransporter catalyze the electroneutral movement of 1 Na+:1 HCO3- into kidney cells. We characterized the intracellular pH (pHi) regulation in human embryonic kidney cells (HEK) subjected to NH4Cl prepulse acid loading, and we examined the NBCn1 expression and function in HEK cells subjected to 24-h elevated pCO2 (10-15%). After acid loading, in the presence of HCO3-, ~50% of the pHi recovery phase was blocked by the Na+/H+ exchanger (NHE) inhibitors EIPA (10-50 μM) and amiloride (1 mM), and was fully cancelled by 30 μM EIPA under nominally HCO3--free conditions. In addition, in the presence of HCO3-, pHi recovery after acid loading was completely blocked when Na+ was omitted in the buffer. pHi recovery after acidification in HEK cells was repeated in the presence of the NBC-inhibitor S0859, and the pHi recovery was inhibit by S0859 in a dose-dependent manner (Ki= 30 μM, full inhibition at 60 μM), which confirmed NBC Na+/HCO3- cotransporter activation. NBCn1 expression increased threefold after 24-h exposure of cultured HEK cells to 10% CO2 and sevenfold after exposure to 15% CO2, examined by immunoblots. Finally, exposure of HEK cells to high CO2 significantly increased the HCO3--dependent recovery of pHi after acid loading. We conclude that HEK cells expressed the NBCn1 Na+/HCO3- cotransporter as the only HCO3--dependent mechanism responsible for cellular alkaline loading. NBCn1, which expresses in different kidney cell types, was upregulated by 24-h high CO2 exposure of HEK cells, and this upregulation was accompanied by increased NBCn1-mediated HCO3- transport.
Diabetes is associated with impaired vascular reactivity and development of diabetic nephropathy. In a rat model of streptozotocin-induced diabetic nephropathy the effects of systemic nitric oxide (NO) synthesis inhibition on intrarenal diffusion and oxygenation were determined by magnetic resonance diffusion tensor imaging (DTI) and blood oxygen level dependency imaging (BOLD), respectively. Eight weeks after diabetes induction 21 rats (n=7 per group) - untreated controls, diabetes (DM), diabetes with uninephrectomy (DM UNX) - were examined. DTI and BOLD sequences were acquired before and after NO synthesis inhibition with N-nitro-L-arginine-methyl-ester (L-NAME). In the same rats mean arterial pressure (MAP) and vascular conductance were determined, with and without the influence of L-NAME. In control animals, NO synthesis inhibition was associated with a significant increase of MAP of 33.8±4.3 mmHg (p<0.001) and a decrease of vascular conductance of -17.8±2.0 µl/(min*100mmHg) (p<0.001). These changes were attenuated in both diabetes groups with no significant difference between pre and post L-NAME measurements in DM UNX animals. Similarly, L-NAME challenge induced a significant reduction of renal T2* at MRI in control animals, indicating reduced renal oxygenation after L-NAME injection compared to baseline. DM UNX animals did not show a significant T2*-reduction after NO synthesis inhibition in the renal cortex and attenuated T2*-reduction in the outer medulla. MRI parameters of tissue diffusion were not affected by L-NAME in all groups. In conclusion, BOLD imaging proved valuable to non-invasively measure renal vascular reactivity upon NO synthesis inhibition in control animals and to detect impaired vascular reactivity in animals with diabetic nephropathy.
Thick ascending limbs (TAL) reabsorb 30% of the filtered NaCl load. Na enters the cells via apical Na/K/2Cl cotransporters and Na/H exchangers and exits via basolateral Na pumps. Chronic angiotensin II (Ang II) infusion increases net TAL Na transport and Na apical entry; however little is known about its effects on the basolateral Na pump. We hypothesized that in rat TALs Na pump activity is enhanced by Ang II-infusion, a model of Ang II-induced hypertension. Rats were infused with 200 ng/kg/min Ang II or vehicle for 7 days and TAL suspensions were obtained. We studied plasma membrane Na pump activity by measuring changes in: 1) intracellular Na (Nai) induced by ouabain; and 2) ouabain-sensitive oxygen consumption (QO2). We found that the ouabain-sensitive rise in Nai in TALs from Ang II-infused rats was 12.8 ± 0.4 Arbitrary Fluorescent Units (AFU)/mg/min compared to only 9.9 ± 1.1 AFU /mg/min in controls (p < 0.024). Ouabain-sensitive oxygen consumption was 17 ± 5% (p < 0.043) greater in tubules from Ang II-treated than vehicle rats. Ang II infusion did not alter total Na pump expression, the number of Na pumps in the plasma membrane or the affinity for Na. When furosemide (1.1 mg/Kg/day) was co-infused with Ang II, no increase in plasma membrane Na pump activity was observed. We concluded that in Ang II-induced hypertension Na pump activity is increased in the plasma membrane of TALs, and that this increase is caused by the chronically enhanced Na entry occurring in this model.
Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512-530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys523 to Gln, Val524 to Leu, Ser526 to Thr, Pro527 to Cys, and Lys529 to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (-/-) animals did not develop, but heterozygous (+/-) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation (EFS) by the detrusor smooth muscle (DSM) from the CaD mutant was high compared to that of the wild type (WT). CaD mutant mice revealed non-voiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase, and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the C-terminal region of CaD.
During hypovolemia and hyperkalemia, the kidneys defend homeostasis by sodium retention and potassium secretion, respectively. Aldosterone mediates both effects, but it is unclear how the same hormone can evoke such different responses. To address this, we mimicked hypovolemia and hyperkalemia in four groups of rats with a control diet, low sodium diet, high potassium diet or a combined diet. The low sodium and combined diets increased plasma and kidney angiotensin II. The low sodium and high potassium diets increased plasma aldosterone to a similar degree (3-fold), while the combined diet increased aldosterone to a greater extent (10-fold). Despite similar sodium intake and higher aldosterone, the high potassium and combined diets caused a greater natriuresis than the control and low sodium diets, respectively (p<0.001 for both). This potassium-induced natriuresis was accompanied by a decreased abundance but not phosphorylation of the sodium chloride cotransporter (NCC). In contrast, the epithelial sodium channel (ENaC) increased in parallel with aldosterone showing the highest expression with the combined diet. The high potassium and combined diets also increased WNK4, but decreased Nedd4-2 in the kidney. Total and phosphorylated SPAK were also increased but were retained in the cytoplasm of distal convoluted tubule cells. In summary, high dietary potassium overrides the effects of angiotensin II and aldosterone on NCC to deliver sufficient sodium to ENaC for potassium secretion. Potassium may inhibit NCC through WNK4 and help activate ENaC through Nedd4-2.
Urinary exosomes are small vesicles secreted into urine from all renal epithelial cell types and known to contain proteins that are involved in renal secretion and reabsorption. Among these proteins, urinary exosomal aquaporin-2 (AQP2) has been suggested to be useful for diagnosis of renal disease. However, the mechanisms underlying the excretion of urinary exosomal AQP2 are largely unknown. In this study, we examined the mechanisms of urinary exosomal AQP2 excretion in vivo, using diuretics including furosemide (FS), an inhibitor of the sodium-potassium-chloride symporter, acetazolamide (ACTZ), an inhibitor of carbonic anhydrase, OPC-31260 (OPC), a vasopressin type 2 receptor antagonist, and NaHCO3, a urinary alkalizing agent. Samples of urine from rats were collected for 2 h just after treatment with each diuretic, and urinary exosomes were isolated by ultracentrifugation. Urinary exosomal AQP2 excretion was dramatically increased by treatment with FS accompanied by urine acidification, or with ACTZ accompanied by urine alkalization. Immunohistochemistry showed that apical localization of AQP2 was clearly evident and plasma vasopressin level was increased after each treatment. Although treatment with OPC alone had no significant effect, co-administration of OPC completely inhibited the FS-induced, and partially reduced the ACTZ-induced responses, respectively. Treatment with NaHCO3 increased the excretion of urinary exosomal AQP2 accompanied by urine alkalization. This increased response was partially inhibited by co-administration of OPC. These data suggest that an increased plasma level of vasopressin promoted the excretion of urinary exosomal AQP2 and that urine alkalinization also increased it independently of vasopressin.
The Gi-coupled adenine receptor (AdeR), binds adenine with high affinity, and potentially reduces cellular cAMP levels. Since cAMP is an important second messenger in renal transport of water and solutes, we localized AdeR in the rat kidney. Real-time RT-PCR showed higher relative expression of AdeR mRNA in the cortex (CTX) and outer medulla (OM) as compared to inner medulla (IM). Immunoblots using a peptide-derived and affinity-purified rabbit polyclonal antibody specific for an 18-amino acid C-terminal sequence of rat AdeR which we generated, detected two bands between ~30-40 kDa (MW of native protein 37 kDa) in CTX, OM and IM. These bands were ablated by pre-adsorption of the antibody with the immunizing peptide. Immunofluorescence labeling showed expression of AdeR protein in all regions of the kidney. Immunoperoxidase revealed strong labeling of AdeR protein in the cortical vasculature including the glomerular arterioles, and less intense labeling in the cells of the collecting duct (CD) system. Confocal immunofluorescence imaging co-localized AdeR with AQP2 protein to the apical plasma membrane in the CD. Functionally, adenine (10 µM) significantly decreased (P < 0.01) dDAVP (10 nM)-induced cAMP production in ex vivo preparations of inner medullary collecting ducts, which was reversed by PSB-08162 (20 µM; P < 0.01)), a selective antagonist of AdeR. Thus, we demonstrated the expression of AdeR in renal vasculature and collecting ducts, and its functional relevance. This study may open a new avenue for exploration of autocrine/paracrine regulation of renal vascular and tubular functions by the nucleobase adenine in health and disease.
The chemokine receptor CCR2 is central for migration of monocytes into inflamed tissues. The novel CCR2 antagonist CCX140-B, which is currently in two separate Phase 2 clinical trials in diabetic nephropathy, was recently shown to reduce hemoglobin A1c and fasting blood glucose levels in Type 2 diabetics. In this report, we describe the effects of this compound on glycemic and renal function parameters in diabetic mice. Since CCX140-B has low affinity for mouse CCR2, transgenic human CCR2 knock-in mice were generated and rendered diabetic either with a high-fat diet (DIO) or by deletion of the leptin receptor gene (db/db). CCX140-B treatment in both models resulted in decreased albuminuria, which was associated with decreased glomerular hypertrophy and increased podocyte density. Moreover, treatment of DIO mice with CCX140-B resulted in decreased levels of fasting blood glucose and insulin, normalization of HOMA-IR values, and decreased numbers of adipose tissue inflammatory macrophages. Unlike other CCR2 antagonists, CCX140-B had no effect on plasma levels of the CCR2 ligand CCL2 or on the numbers of blood monocytes. These results support the ongoing evaluation of this molecule in diabetic subjects with impaired renal function.
Organ cross-talk exists in many diseases of the human and animal models of human diseases. Recent study had demonstrated that inflammatory mediators can cause acute kidney injury and neutrophil infiltration in a mouse model of DSS-colitis. However, the chemokines and their receptors that may mediate distant organ effects in colitis are unknown. We hypothesized that KC/IL-8 receptor CXCR2 mediates DSS-colitis induced acute kidney injury. Consistent with our hypothesis, WT mice developed severe colitis with DSS treatment which was associated with inflammatory cytokine and chemokine expression and neutrophil infiltration in the colon. DSS-colitis in WT was accompanied by acute kidney injury and enhanced expression of inflammatory cytokines in the kidney. However, CXCR2 knockout mice were protected against DSS-colitis as well as acute kidney injury. Moreover, the expression of cytokines and chemokines and neutrophil infiltration was blunted in CXCR2 KO mice in the colon and kidney. Administration of recombinant KC exacerbated DSS-colitis induced acute kidney injury. Our results suggest that KC/IL-8 and its receptor CXCR2 are critical and major mediators of organ cross talk in DSS colitis and neutralization of CXCR2 will help to reduce the incidence of acute kidney injury due to ulcerative colitis and Chron's disease in humans.
This study was to determine whether prostacyclin (prostaglandin I2; PGI2) evokes mouse renal vasoconstriction, if so, the underlying mechanism(s) and how its synthesis via cysclooxygenase-1 (COX-1) influences local vasomotor reaction. Experiments were performed on vessels from C57BL/6 mice and/or those with COX-1 deficiency (COX-1-/-). Results showed that in renal arteries, PGI2 evoked contraction more potently than in carotid arteries, where COX-1 is suggested to mediate prominent endothelium-dependent contraction. A similar result was observed with the thromboxane-prostanoid (TP) receptor agonist U46619. However, in renal arteries TP receptor antagonism, which inhibited the contraction, did not result in any relaxation in response to PGI2. Moreover, we noted that the endothelial muscarinic receptor agonist ACh evoked an increase in the production of the PGI2 metabolite 6-keto-PGF1α, which was prevented by endothelial denudation or COX-1-/-. Interestingly, COX-1-/- was further found to abolish a force development that was sensitive to TP receptor antagonism and result in enhanced relaxation evoked by ACh following NO synthase inhibition. Also, in renal arteries the COX substrate arachidonic acid evoked a vasoconstrictor response, which was again abolished by COX-1-/-. Meanwhile, non-selective COX inhibition did not show any effect in vessels from COX-1-/- mice. Thus, in mouse renal arteries high expression of TP receptors together with little functional involvement from the vasodilator PGI2 receptors results in a potent vasoconstrictor effect evoked by PGI2. Also, our data imply that endogenous COX-1-mediated PGI2 synthesis leads to vasoconstrictor activity and this could be an integral part of endothelium-derived mechanisms in regulating local renal vascular function.
Recent data suggests possible net transport of urinary constituents across mammalian urinary tract epithelia (urothelia). To evaluate the effect of animal hydration status on such transport we instilled urine collected during two day water-deprivation, water-loading or ad-libitum water intake into isolated in-situ bladder(s) of groups of rats undergoing one of the same three hydration states. After 1 hour bladder dwell we retrieved the urine and measured differences in volume and solute concentration between instilled and retrieved urine. We previously reported results regarding changes in urine volume and net urea and creatinine transport and herein report the results of net urinary sodium, potassium, and chloride transport in the same animals. During water-loading conditions, urinary concentrations of Na, K, and Cl rose 4.9 mEq/L (30.7%), 2.6 mEq/L (16.5%), and 6.0 mEq/L (26.8%) respectively - indicating urothelial secretion into urine. During ad-libitum water intake urinary K and Cl concentrations fell 33.6 mEq/L (14.8%) and 28.4 mEq/L (12%) respectively (Na did not change) and during water deprivation urine Na, K and Cl concentrations fell dramatically by 53.2 mEq/L (18.6%), 159.4 mEq/L (34.6%) and 133.7 mEq/L (33.8%) - reflecting urothelial reabsorption of each ion. For each ionic species two factors independently influenced transport - the instilled urinary ion concentration, and the animal hydration state. These results demonstrate significant regulated ion transport across mammalian urothelia, support the notion that lower urinary tract modifies final urine, and suggest that the lower urinary tract may play a role in local and whole animal solute homeostasis.
It is recognized that dopamine promotes natriuresis by inhibiting multiple transporting systems in the proximal tubule. In contrast, less is known about molecular targets of dopamine actions on the water-electrolyte transport in the cortical collecting duct (CCD). Epithelial cells in the CCD are exposed to dopamine which is synthesized locally or secreted from sympathetic nerve endings. Basolateral K+ channels in the distal renal tubule are critical for potassium recycling and controlling basolateral membrane potential to establish driving force for Na+ reabsorption. We demonstrate that Kir4.1 and Kir5.1 are highly expressed in the mouse kidney cortex and are localized to the basolateral membrane of the CCDs. Using patch clamp electrophysiology in freshly isolated CCDs, we detected highly abundant 40 pS and scarce 20 pS single channel conductances most likely representing Kir4.1/5.1 and Kir4.1 channels, respectively. Dopamine reversibly decreased open probability of both channels having a relatively greater action on Kir4.1/5.1 heterodimer. This effect was mediated by D2-like but not D1-like dopamine receptors. Protein kinase C blockade abolished inhibition of the basolateral K+ channels by dopamine. Importantly, dopamine significantly decreased the amplitude of Kir4.1/5.1 and Kir4.1 unitary currents. Consistently, dopamine induced an acute depolarization of basolateral membrane potential as was directly monitored using current clamp mode in isolated CCD. Therefore, we demonstrate that dopamine inhibits basolateral Kir4.1/5.1 and Kir4.1 channels in CCD cells via stimulation of D2-like receptors and subsequently PKC. This leads to depolarization of basolateral membrane and a decreased driving force for sodium reabsorption in the distal renal tubule.
Background GDF15 is emerging as valuable biomarker in cardiovascular disease and diabetic kidney disease. Also, GDF15 represents an early response gene induced after tissue injury and studies performed in GDF15 knockout mice suggest that GDF15 plays a protective role after injury. In the current study, we investigated the role of GDF15 in the development of diabetic kidney damage in type 1 and type 2 models of diabetes. Methods and results Renal damage was assessed in GDF15 knockout (ko) mice and wild type (wt) mice in streptozotocin type 1 and db/db type 2 diabetic models. Genetic deletion of GDF15 augmented tubular and interstitial damage in both models of diabetes, despite similar diabetic states in ko and wt mice. Increased tubular damage in ko animals was associated with increased glucosuria and polyuria in both type 1 and type 2 models of diabetes. In both models of diabetes, ko mice showed increased interstitial damage as indicated by increased α-SMA staining and collagen type 1 expression. In contrast, glomerular damage was similarly elevated in diabetic ko and wt mice. In type 1 diabetes, GDF15 ko mice demonstrated increased expression of inflammatory markers. In type 2 diabetes, elevated levels of plasma creatinine indicated impaired kidney function in ko mice. Conclusion GDF15 protects the renal interstitium and tubular compartment in experimental type 1 and 2 diabetes without affecting glomerular damage.
Both sodium reabsorption in the thick ascending limb of the loop of Henle (TAL) and macula densa salt-sensing crucially depend on the function of the Na/K/2Cl cotransporter NKCC2. The NKCC2 gene gives rise to at least three different full-length NKCC2 isoforms derived from differential splicing. In the present study, we addressed the influence of dietary salt intake on the differential splicing of NKCC2. Mice were subjected to diets with low, standard, and high salt content for 7 days, and NKCC2 isoform mRNA abundance was determined. With decreasing salt intake, we found a reduced abundance of the low-affinity isoform NKCC2A and an increase in the high-affinity isoform NKCC2B in the renal cortex and the outer stripe of the outer medulla. This shift from NKCC2A to NKCC2B during a low-salt diet could be mimicked by furosemide in vivo and in cultured kidney slices. Furthermore, the changes in NKCC2 isoform abundance during a salt-restricted diet were partly mediated by the actions of angiotensin II on AT1 receptors, as determined using chronic angiotensin II infusion. In contrast to changes in oral salt intake, water restriction (48 hrs) and water loading (8% sucrose solution) increased and suppressed the expression of all NKCC2 isoforms, without changing the distribution pattern of the single isoforms. In summary, the differential splicing of NKCC2 pre-mRNA is modulated by dietary salt intake, which may be mediated by changes in intracellular ion composition. Differential splicing of NKCC2 appears to contribute to the kidney's adaptive capacity to cope with changes in reabsorptive needs.
Hypertension is a risk factor for chronic kidney disease, particularly when associated with impaired renal autoregulation and thereby increased intraglomerular pressure (Pgc). Elevated Pgc can be modeled in vitro by exposing glomerular mesangial cells to mechanical strain. We previously showed that RhoA mediates strain-induced matrix production. Here we show that RhoA activation is dependent on an intact microtubule network. Upregulation of the profibrotic cytokine CTGF by mechanical strain is dependent on RhoA activation and inhibited by microtubule disruption. We tested the effects of the microtubule depolymerizing agent colchicine in 5/6 nephrectomized rats, a model of chronic kidney disease driven by elevated intraglomerular pressure. Colchicine inhibited glomerular RhoA activation and attenuated both glomerular sclerosis and interstitial fibrosis without affecting systemic blood pressure. Upregulation of the matrix proteins collagen I and fibronectin, as well as CTGF, were attenuated by colchicine. Activity of the profibrotic cytokine TGFβ, as assessed by Smad3 phosphorylation, was also inhibited by colchicine. Microtubule disruption significantly decreased renal infiltration of lymphocytes and macrophages. Our studies thus indicate that colchicine modifies hypertensive renal fibrosis. Its protective effects are likely mediated by inhibition of RhoA signaling and renal infiltration of inflammatory cells. Already well established in clinical practice for other indications, prevention of hypertension-associated renal fibrosis may represent a new potential use for colchicine.
Background: miRNAs are frequently dysregulated in the development of renal fibrosis. Exosomes are small membrane vesicles that could be isolated from urine secreted from all nephron segments. Here we sought to observe for the first time whether miRNA in urine exosome could serve as a potential biomarker of renal fibrosis. Methods: Urine samples were collected from 32 chronic kidney disease (CKD) patients who underwent kidney biopsy and 7 controls. Exosome was isolated and confirmed by immunogold staining of exosome marker. Members of miR-29, miR-200 and RNU6B as endogenous control were detected by RT quantitative PCR. Results: Electronic microscopy verified a typical shape of exosome with average size of 65.1nm and was labelled with anti-CD9 and anti-AQP2 antibody. Members of miR-29 and miR-200 are readily measured with reduced levels compared with controls (p<0.05), and can robustly distinguish CKD from controls (area under the curve (AUC) varied from 0.902 to 1 by ROC analysis).miR-29c correlated with both eGFR (r=0.362, p<0.05) and degree of tubulointerstitial fibrosis (r=-0.359, p<0.05) for CKD patients. Moreover, miRNAs in exosome was decreased in mild fibrosis group compared with moderated to severe group. miR-29a and miR-29c could predict degree of tubulointerstitial fibrosis with AUC of 0.883 and 0.738 (p<0.05). The sensitivity and specificity for distinguishing mild from moderate to severe fibrosis was 93.8%, 81.3% with the use of miR-29a, and 68.8%, 81.3% for miR-29c. Conclusions: Overall, miR-29c in urinary exosome correlates with both renal function and degree of histological fibrosis, suggesting it as novel, non-invasive marker for renal fibrosis.
Hypoxia-inducible factors (HIFs) are transcription factors consisting of an oxygen-sensitive α-subunit binding to a stable β-subunit. HIFs regulate multiple signaling pathways that could contribute to fibrogenesis, supporting their potential role in hypoxia-mediated renal fibrosis. We previously reported that HIF-1 is upregulated and required for TGF-β induction of collagen in renal tubular cells. Here, we performed in vitro and in vivo studies of potential glomerular crosstalk between transforming growth factor (TGF)-β and normoxic HIF signaling. HIF-α has two major isoforms, HIF-1α and HIF-2α, with different target gene sets. In cultured human mesangial cells (HMC), TGF-β1 treatment increased both HIF-1α and HIF-2α expression in normoxia. TGF-β1 did not increase HIF-1α/2α mRNA levels nor decrease the rate of protein degradation, suggesting that it enhances HIF-1α/2α expression through translation. TGF-β receptor (ALK5) kinase activity was required for increased, TGF-β-stimulated HIF-α expression in response to TGF-β, and inhibiting PI3-kinase markedly decreased HIF-α expression. Blocking HIF-1α/2α expression using siRNA decreased basal and TGF-β1-stimulated type-I collagen expression, while overexpressing non-degradable HIF-α increased the collagen response, with HIF-2α being significantly more effective than HIF-1α. In Adriamycin (ADR)-induced mouse glomerulosclerosis, HIF-2α target genes were upregulated in sclerosing glomeruli. Taken together, our data demonstrate potential signaling interaction between TGF-β and HIFs to promote renal fibrogenesis in normoxia and suggest that the HIF-2α isoform is more important during glomerulosclerosis.
Diabetic nephropathy (DN) is a leading cause of end-stage renal disease (ESRD). The inhibitors of renin-angiotensin-aldosterone system (RAAS) can alleviate some of the symptoms of DN but fail to stop the progression to ESRD. Our previous studies demonstrate renoprotective action of nitro-oleic acid (OA-NO2) in several rodent models of renal disease. Here we examined the therapeutic potential and the underlying mechanism of combination of Losartan and OA-NO2 in db/db mice. OA-NO2 was infused at 5 mg/kg/day via osmotic mini-pump and Losartan was incorporated into diet at 10 mg/kg/day, each administered alone or in combination for 2 weeks. Diabetic db/db mice developed progressive albuminuria and glomerulosclerosis, accompanied by podocytes loss, increased indices of renal fibrosis, oxidative stress, and inflammation. Treatment of the diabetic mice with OA-NO2 or Losartan alone moderately ameliorated kidney injury; however, the combined treatment remarkably reduced albuminuria, restored glomerular filtration barrier structure, and attenuated glomerulosclerosis, accompanied with significant suppression of renal oxidative stress and inflammation. These data demonstrate that combination of Losartan and OA-NO2 effectively reverses renal injury in DN.
In this study, we assessed the acute effects of angiotensin II on the albumin glomerular sieving coefficient (GSC) using intravital microscopy. The experiments were performed on Munich Wistar Froemter (MWF) rats. Alexa-Fluor-594 albumin was injected intravenously, and the fluorescence intensity in the glomerular capillaries and Bowman's space was determined to calculate the albumin GSC. The GSC was measured before and during the constant infusion of angiotensin II (10 ng/min/kg BW). Baseline mean arterial pressure (MAP) was 99±5 mm Hg and stabilized at 137±5 mm Hg during angiotensin II infusion. The baseline GSC averaged 0.00044±4.8*10-5 and increased by 286±44% after angiotensin II infusion (p<.0001). The proximal tubular Alexa-Fluor-594 albumin uptake was enhanced during angiotensin II infusion (518% of the baseline value during angiotensin II vs. 218% in controls; p<.0001). No change in GSC was observed when the AT1 antagonist losartan was injected before the start of angiotensin II infusion. The AT2 antagonist PD123319 increased the baseline GSC from 0.00052±3.6*10-5 to 0.00074±8.2*10-5 (p=.02) without altering the MAP. During angiotensin II infusion with losartan, PD123319 increased the albumin GSC from 0.00037±5.8*10-5 to 0.00115±.00015 (p=.001). When the renal perfusion pressure was mechanically controlled, the GSC increased from 0.0007±.00019 to 0.0025±.00063 during angiotensin II infusion (p=.047), similar to what was observed when the renal perfusion pressure was allowed to increase. In summary, AT1 activation acutely increases the albumin GSC. This effect appears to be largely independent of changes in the renal perfusion pressure. The AT2 receptor partially attenuates the proteinuric effects of the AT1 receptor.
The inner medullary collecting duct (IMCD) is the nephron segment with the highest production of endothelin-1 (ET-1) and the greatest expression of ET-1 receptors that function to adjust Na+ and water balance. We have reported that male rats have reduced natriuresis in response to direct intramedullary infusion of ET-1 compared to females. Our aim was to determine whether alterations of ET-1 receptor expression and downstream intracellular Ca2+ signaling within the IMCD could account for these sex differences. IMCDs from male and female rats were isolated for radioligand binding or microdissected for intracellular Ca2+ ([Ca2+]i) measurement by fluorescence imaging of fura 2-AM. IMCD from male and female rats had similar ETB expression (655±201 vs. 567±39 fmol/mg protein, respectively) while male rats had significantly higher ETA expression (436±162 vs. 47±29 fmol/mg protein respectively; p<0.05). The [Ca2+]i response to ET-1 was significantly greater in IMCDs from male compared to female rats (288±52 vs. 118±32 AUC, nM x 3 min, respectively; p<0.05). In IMCDs from males, the [Ca2+]i response to ET-1 was significantly blunted by the ETA antagonist BQ-123, but not the ETB antagonist BQ-788 (control: 137±27, BQ-123: 53±11, and BQ-788: 84±25 AUC, nM x 3 min, respectively; p<0.05), consistent with greater ETA receptor function in males. These data demonstrate a sex difference in ETA receptor expression that results in differences in ET-1 Ca2+ signaling in IMCD. As activation of ETA receptors is thought to oppose ETB receptor activation, enhanced ETA function in males could limit the natriuretic effects of ETB receptor activation.
Relaxin, a pregnancy hormone, has anti-apoptotic and anti-inflammatory properties. The aim of this study was to determine the effects of relaxin on ischemia/reperfusion (IR)-induced acute kidney injury. Male rats underwent unilateral nephrectomy and contralateral renal IR (45 min of renal pedicle clamping). Rats were divided into three groups: 1) sham group, 2) IR group, and 3) IR-RLX group (rats treated with relaxin before ischemia). In this group, relaxin was infused at 500 ng/hr via subcutaneous osmotic minipump for 24 h beginning 2 h before renal ischemia. At 24 h after reperfusion, renal function was assessed and kidneys were removed for analysis. There was no significant difference in blood pressure among the three groups. IR increased plasma levels of creatinine and urea nitrogen, and relaxin provided protection against the increases in these two parameters. Relaxin significantly decreased plasma TNF-α levels and renal TNF receptor 1 mRNA expression, compared to the IR group. Semi-quantitative assessment of the histological lesions showed marked structural damage in IR rats compared with the IR-RLX rats. RLX significantly reduced apoptotic cell counts compared to the IR group. Overexpression of caspase-3 observed in the IR kidneys was reduced in the IR-RLX group. The results demonstrated that relaxin provided protection against IR-induced renal injury by reducing apoptosis and inflammation.
1, 25-dihydroxycholechalciferol (calcitriol) and 19-nor-1, 25-dihydroxyvitamin D2 (paricalcitol) are vitamin-D receptor (VDR) agonists. Previous data suggest VDR agonists may actually increase renin-angiotensin activity, and this has always been assumed to be mediated by hypercalcemia. We hypothesized that calcitriol and paricalcitol would increase plasma renin activity (PRA) independently of plasma Ca2+ via hypercalciuria-mediated polyuria, hypovolemia and subsequent increased β-adrenergic sympathetic activity. We found that both calcitriol and paricalcitol increased PRA 3-fold, p<0.01). Calcitriol caused hypercalcemia, but paricalcitol did not. Both calcitriol and paricalcitol caused hypercalciuria (9 and 7-fold vs control, p<0.01) and polyuria (increasing 2.6 and 2.2-fold vs control, p<0.01). Paricalcitol increased renal calcium-sensing receptor (CaSR) expression, suggesting a potential cause of paricalcitol-mediated hypercalciuria and polyuria. Volume replacement completely normalized calcitriol-stimulated PRA and lowered plasma epinephrine by 43% (p<0.05). β-adrenergic blockade also normalized calcitriol-stimulated PRA. Cyclooxygenase-2 inhibition had no effect on calcitriol-stimulated PRA. Our data demonstrate that vitamin D increases PRA independently of plasma Ca2+ via hypercalciuria, polyuria, hypovolemia and increased β-adrenergic activity.
In the present study, we examine the hypothesis that the nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays a protective role in the development of angiotensin II (AngII) induced hypertension and renal injury by minimizing oxidative stress and the inflammation induced by tumor necrosis factor-alpha (TNF-α). Systemic blood pressure (SBP) and renal injury responses to chronic infusions of AngII (via implanted mini-pumps) were evaluated for 2 weeks in wild type (WT) and in eNOS knockout mice (KO) co-treated with or without a superoxide (O2-) scavenger, tempol (400 mg/L in the drinking water) or a TNF-α receptor blocker, etanercept (5 mg/kg/day i.p.). In study 1, when AngII was given at a dose of 25 ng/min, it increased mean SBP in WT ( 36± 3 mmHg; n=7) and this effect was attenuated in mice pretreated with tempol ( 24±3 mmHg; n=6). In KO mice (n=9), this dose of AngII resulted in severe renal injury associated with high mortality. To avoid this high mortality in KO, study 2 was conducted with a lower dose of AngII (10 ng/min) that increased SBP slightly in WT ( 17 ± 7 mmHg; n=6) but exaggeratedly in KO ( 48 ± 12 mmHg, n=6) associated with severe renal injury. Co-treatment with either tempol (n=6) or etanercept (n=6) ameliorated the hypertensive as well as the renal injury responses in KO compared to WT. These data demonstrate a protective role for eNOS activity in preventing renal inflammatory injury and hypertension induced by chronic increases in AngII.
Numerous proinflammatory cytokines have been implicated in the reorganization of lower urinary tract function following cyclophosphamide (CYP)-induced cystitis. The present study investigated the functional profile of three pleiotropic transforming growth factor-beta (TGF-β) isoforms and receptor (TβR) variants in the normal and inflamed (CYP-induced cystitis) rat urinary bladder. Our findings indicate that TGF-β (1, 2 and 3) and TβR (1, 2 and 3) transcript and protein expression were regulated to varying degrees in the urothelium or detrusor smooth muscle following intermediate (48 h; 150 mg/kg, i.p.) or chronic (75 mg/kg, i.p.; once every three days for ten days), but not acute (4 h; 150 mg/kg, i.p.), CYP-induced cystitis. Conscious, open outlet cystometry was performed to determine whether aberrant TGF-β signaling contributes to urinary bladder dysfunction following intermediate (48 h) CYP-induced cystitis. TβR-1 inhibition with SB505124 (5 µM) significantly (p ≤ 0.001) decreased voiding frequency and increased bladder capacity (2.5-fold), void volume (2.6-fold) and intercontraction intervals (2.5-fold) in CYP-treated (48 h) rats. Taken together, these results provide evidence for: (i) the involvement of TGF-β in lower urinary tract neuroplasticity following urinary bladder inflammation, (ii) a functional role of TGF-β signaling in the afferent limb of the micturition reflex and (iii) urinary bladder TβR-1 as a viable target to reduce voiding frequency with cystitis.
Focal segmental glomerulosclerosis (FSGS) and collapsing glomerulopathy are common causes of nephrotic syndrome. Variants in over 20 genes have been associated with these podocyte diseases, including genes critical for mitochondrial function. One such gene, PDSS2, is required for synthesis of the decaprenyl tail of coenzyme Q10 in humans. The mouse gene Pdss2 is mutated in the kd/kd mouse model of collapsing glomerulopathy. We examined the hypothesis that human PDSS2 polymorphisms are associated with podocyte diseases. We genotyped 377 cases with primary FSGS or collapsing glomerulopathy, together with 900 controls, for nine single nucleotide polymorphisms (SNPs) in the PDSS2 gene in a case-control study. Subjects included 247 African American (AA) and 130 European American (EA) cases, and 641 AA and 259 EA controls. Among EAs, a pair of proxy SNPs was significantly associated with podocyte disease, and patients homozygous for one PDSS2 haplotype had a strongly increased risk for podocyte disease. By contrast, the distribution of PDSS2 genotypes and haplotypes were similar in AA cases and controls. Thus, a PDSS2 haplotype, which has a frequency of 13% in the EA control population and a homozygote frequency of 1.2%, is associated with a significantly increased risk for FSGS and collapsing glomerulopathy in EA. Lymphoblastoid cell lines (LCLs) from FSGS patients had significantly less coenzyme Q10 than cell lines from controls; unexpectedly this finding was independent of PDSS2 haplotype. These results suggest that FSGS patients have coenzyme Q10 deficiency, and that this deficiency is manifested in patient-derived LCLs.
The inbred genetic hypercalciuric stone-forming (GHS) rats exhibit many features of human idiopathic hypercalciuria and have elevated levels of vitamin D receptors (VDR) in calcium (Ca) transporting organs. On a normal Ca diet, 1,25(OH)2D3 (1,25D) increases urine (U) Ca to a greater extent in GHS than in controls (SD). The additional UCa may result from an increase in intestinal Ca absorption and/or bone resorption. To determine the source, we asked whether 1,25D would increase UCa in GHS fed a low Ca (0.02%) diet. With 1,25D, UCa in SD increased from 1.2±0.1 to 9.3±0.9 mg/d and increased more in GHS from 4.7±0.3 to 21.5±0.9 mg/d (p<0.001). In GHS rats on LCD with or without 1,25D, UCa far exceeded daily Ca intake (2.6 mg/d). While the greater excess in UCa in GHS rats must be derived from bone mineral, there may also be a 1,25D-mediated decrease in renal tubular Ca reabsorption. RNA expression of the components of renal Ca transport indicated that 1,25D administration results in a suppression of klotho, an activator of the renal Ca reabsorption channel TRPV5, in both SD and GHS rats. This fall in klotho would decrease tubular reabsorption of the 1,25D-induced bone Ca release. Thus the greater increase in UCa with 1,25D in GHS fed LCD strongly suggests that the additional UCa results from an increase in bone resorption, likely due to the increased number of vitamin D receptors in the GHS rat bone cells, with a possible component of decreased renal tubular calcium reabsorption.
We have demonstrated that mesenchymal cells from spontaneously hypertensive rats genetically express complement 3 (C3). Mature tubular epithelial cells can undergo epithelial-to-mesenchymal transition (EMT) that is linked to the pathogenesis of renal fibrosis and injury. In this study, we investigated the contribution of C3 in EMT and in the renal renin-angiotensin (RA) systems associated with hypertension. C3a induced EMT in mouse TCMK-1 epithelial cells, which displayed increased expression of renin and Krüppel-like factor 5 (KLF5) and nuclear localization of liver X receptor α (LXRα). C3 and renin were strongly stained in the degenerated nephrotubulus and co-localized with LXRα and prorenin receptor in unilateral ureteral obstruction (UUO) kidneys from wild type mice. In C3-deficient mice, hydronephrus and EMT were suppressed with no expression of renin and C3. After UUO, systolic blood pressure was increased in wild type but not in C3-deficient mice. In wild type mice, intrarenal angiotensin II (Ang II) levels were markedly higher in UUO kidneys than normal kidneys, and decreased with aliskiren. There were no increases in intrarenal Ang II levels after UUO in C3-deficient mice. Thus C3 induces EMT and dedifferentiation of epithelial cells, which produce renin through induction of LXRα. We firstly indicate that C3 may be a primary factor to activate the renal RA systems to induce hypertension.
Aldosterone increases tubular sodium absorption largely by increasing epithelial Na+ channel (ENaC) α transcription in collecting duct principal cells. How aldosterone reprograms basal αENaC transcription to high-level activity in the collecting duct is incompletely understood. Promoter methylation, a covalent, but reversible epigenetic process, has been implicated in the control of gene expression in health and disease. We investigated the role of promoter methylation/demethyation in epigenetic control of basal and aldosterone-stimulated αENaC transcription in mIMCD3 collecting duct cells. Bisulfite treatment and sequencing analysis after treatment of the cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR) identified clusters of methylated cytosines (5mC) in a CpG island near the transcription start site of the αENaC promoter. 5-Aza-CdR treatment or siRNA-mediated knockdown of DNMT3b or methyl-CpG binding domain protein (MBD)-4 de-repressed basal αENaCtranscription, indicating that promoter methylation suppresses basal αENaC transcription. Aldosterone triggered a time-dependent decrease in 5mC and DNMT3b, and a concurrent enrichment in 5-hydroxymethylcytosine (5hmC) and ten-eleven translocation (Tet) 2 at the αENaC promoter, consistent with active demethylation. 5-Aza-CdR mimicked aldosterone by enhancing Sp1 binding to the αENaC promoter. We conclude that DNMT3b- and MBD4-dependent methylation of the αENaC promoter limits basal αENaC transcription, in part by limiting Sp1 binding and trans-activation. Aldosterone stimulates dispersal of DNMT3b and recruitment of Tet2 to demethylate the αENaC promoter to induce αENaC transcription. These results disclose a novel epigenetic mechanism for control of basal and aldosterone-induced αENaC transcription that adds to previously described epigenetic controls exerted by histone modifications.
The aims of this study were to 1) determine whether renal localization of aliskiren and its anti-hypertensive and renoprotective effects persist after stopping administration of the drug, and 2) define the renal localization of aliskiren by light microscopy autoradiography. Hypertensive double transgenic rats (dTGR) overexpressing genes for human renin and angiotensinogen were treated with aliskiren (3 mg/kg/day sc; osmotic minipumps) or enalapril (18mg/L in drinking water). After 2 weeks treatment dTGR were assigned to either continued treatment with aliskiren ("continued"), or to cessation of their respective treatment ("stopped") for three weeks washout. One week treatment with aliskiren and enalapril reduced BP and albuminuria vs baseline. After cessation of either treatment, blood pressure had returned to pre-treatment levels and albuminuria remained relatively low for 1 week, but rose thereafter similarly in both groups. In contrast, renal mRNA for TGF-β and renal collagen IV was reduced by aliskiren (continued and stopped groups), but not after cessation of enalapril. Similar patterns were found for collagen IV protein expression. Even 3 weeks after stopping aliskiren treatment, renal levels of the drug exceeded its IC50, whereas enalaprilat was not detected. To localize aliskiren accumulation, Wistar rats were treated with [3H]-aliskiren for 7 days. Autoradiography demonstrated specific labeling in glomeruli, arterioles, and afferent arterioles as well as in the distal nephron. Labeling could still be observed even after 7 days washout. These results suggest that the renophilic properties of aliskiren are different from enalapril and could have contributed to the renoprotective mechanism of this renin inhibitor.
Renal ischemia reperfusion injury (IRI) is a major factor responsible for acute renal failure (ARF). An intermediate in heme synthesis, 5-aminolevulinic acid (5-ALA), is fundamental in aerobic energy metabolism. Hemeoxygenase-1 (HO-1) cleaves heme to form biliverdin, carbon monoxide (CO) and iron (Fe2+), which is used with 5-ALA. In the present study, we investigated the role of 5-ALA in attenuating acute renal ischemic-reperfusion injury (IRI) using a mouse model. Male Balb/c mice received 30 mg/kg of 5-ALA with Fe2+ 48, 24 and 2 hours prior to IRI, and were subsequently subjected to bilateral renal pedicle occlusion for 45 minutes. The endogenous CO concentration of the kidneys from the mice administered 5-ALA/Fe2+ increased significantly, and the peak concentrations of serum creatinine and BUN decreased. The 5-ALA/Fe2+ treatments significantly decreased the tubular damage and the number of apoptotic cells. The IRI-induced renal TBARS level was also significantly decreased in the 5-ALA/Fe2+ group. Furthermore, the mRNA expression of HO-1, TNF-α and IFN- was significantly increased following IRI. The levels of HO-1 was increased and TNF-α and IFN- were decreased in the 5-ALA/Fe2+-pretreated renal parenchyma after IRI. F4/80 staining showed reduced macrophage infiltration, and TUNEL staining revealed that there were fewer interstitial apoptotic cells. These findings suggest that 5-ALA/Fe2+ can protect the kidneys against IRI by reducing macrophage infiltration and decreasing the renal cell apoptosis via the generation of CO.
Cilia, membrane-enclosed organelles protruding from the apical side of the cells, can be divided into two classes, motile and primary cilia. During the past decades, motile cilia have been intensively studied. However, it was not until 1990s that people began to realize the importance of primary cilia as cellular specific sensors, particularly in kidney tubular epithelial cells. Furthermore, accumulating evidence indicates that primary cilia may be involved in the regulation of cell proliferation, differentiation, apoptosis, and planar cell polarity. Many signaling pathways, such as Wnt, Notch, and Hedgehog, have been located to the primary cilia. Thus, primary cilia have been regarded as a hub that integrates signals from the extracellular environment. More importantly, dysfunction of this organelle may contribute to the pathogenesis of a large spectrum of human genetic diseases named ciliopathies. The significance of primary cilia in acquired human diseases such as hypertension and diabetes has gradually drawn attention. Interestingly, recent reports disclosed that cilia length varies during kidney injury, and shortening of cilia enhances the sensitivity of epithelial cells to injury cues. This review briefly summarizes the current status of cilia research and explores the potential mechanisms of cilia length changes during kidney injury as well as provides some thoughts in order to allure more insightful ideas and promotes the further study of primary cilia in the context of kidney injury.
IL-18 is an important mediator of obstruction-induced renal fibrosis and renal tubular epithelial cell (TEC) injury. IL-18's pro-inflammatory properties have been attributed, in part, to NFB activation and the stimulation of cytokine gene expression, however, STAT3 has increasingly been shown to mediate renal fibrotic injury. We therefore hypothesized that IL-18 mediates pro-fibrotic TEC injury via STAT3 activation. Male C57BL6 wild-type mice and transgenic mice for human IL-18-binding protein were subjected to unilateral ureteral obstruction or sham operation. The kidneys were harvested 1 or 2 weeks after operation, and analyzed for active STAT3 (p-STAT3) expression (western blot, immunohistochemistry) and suppressor of cytokine signaling 3 (SOCS3) expression. In a separate arm, renal tubular cells (HK-2) were directly stimulated with IL-18 for 2 days with or without the STAT3 inhibitor, S3I-201 (50μM). Cell lysates were then analyzed for p-STAT3 and SOCS3 expression, pro-fibrotic cellular changes (collagen and α–SMA expression), and tubular cell apoptosis. p-STAT3 and SOCS3 expression increased significantly in response to obstruction, however, a significant reduction in p-STAT3 and SOCS3 expression occurred following 1 week, but not 2 weeks, of obstruction in the presence of IL-18 neutralization. In vitro results similarly demonstrate increased p-STAT3, SOCS3, α-SMA, and collagen III expression, and increased collagen production and TEC apoptosis in response to IL-18 stimulation, but the response was significantly diminished in the presence of STAT3 inhibition. These results demonstrate that IL-18-induces pro-fibrotic cellular changes and collagen production in TECs via STAT3 activation.
In peritoneal dialysis (PD) therapy, physical stresses such as exposure to peritoneal dialysate, catheter trauma and peritonitis induce peritoneal injuries which can prevent continued long-term PD therapy. Therefore, protection of the peritoneum is an important target to enable long-term PD therapy in patients with end-stage renal disease. We previously showed that neutralizations of membrane complement regulators (CRegs), Crry and CD59, in rat peritoneum provokes development of acute peritoneal injuries due to uncontrolled complement activation. C5a is a key effecter molecule of the complement system, released during acute inflammation. Control of C5a has been proposed as a strategy to suppress inflammatory reactions and, because peritoneal injuries are accompanied by inflammation, we hypothesized that C5a targeted therapy might be an effective way to suppress peritoneal injuries. In the present study, we have used the established acute peritonitis model induced by neutralization of CRegs to investigate effects on acute peritoneal injuries of inhibiting C5a. Intravenous administration of an anti-C5a complementary peptide (AcPepA) up to 4 h after induction of injury significantly and dose-dependently prevented accumulation of inflammatory cells and reduced tissue damage in the model, accompanied by decreased C3b deposition. Here we showed that C5a contributed to the development of peritoneal injuries. Our results suggest that C5a is a target to prevent or treat peritoneal injuries in PD patients on prolonged therapy or with infectious complications.
Albuminuria is strongly associated with progressive kidney tubulo-interstitial damage and chronic kidney disease (CKD) progression. In proteinuric nephropathies, albumin reabsorption by the proximal tubule is saturated and the distal nephron is exposed to high concentrations of luminal albumin that may produce adverse effects. Since proximal tubular cells exposed to albuminuria exhibit a pro-inflammatory and pro-fibrotic response, we assessed the effect of albuminuria in the collecting duct (CD). Using kidney sections and isolated cortical CDs (CCD) from puromycin-aminonucleoside-induced nephrotic rats (PAN rats) exhibiting proteinuria, immunofluorescence microscopy revealed internalized albumin in CD cells. In these proteinuric rats, increased expression levels of cytokines and pro-fibrotic signaling markers were detected in isolated CCDs and bands of inflammatory fibrosis could be observed around CDs. Albumin endocytosis was confirmed by FITC-albumin uptake in cultured murine CCD (mCCDcl1) cells. Exposure of mCCDcl1 cells to albumin induced NF-B activation assessed by luciferase reporter gene assay, nuclear translocation of NF-B p65 subunit and increased NF-B target gene expression. Moreover, albuminuria-like conditions results in TGFβ1 over-expression and the up-regulation of pro-fibrotic signaling markers such as Snail or Vimentin via an autocrine mechanism. In mCCDcl1 cells NGAL/lipocalin-2/24p3 receptor (24p3R) mediates albumin endocytosis as well as activation of NF-B and TGFβ1 signaling pathways. Therefore, CD may play a key role in initiation and/or progression of inflammation and fibrosis in response to proteinuria.
Renal hemodynamic studies tested the hypothesis that CD38 and superoxide anion (O2.-) participate in vasoconstriction produced by activation of thromboxane prostanoid (TP) receptors in the mouse kidney. CD38 is the major mammalian ADP-ribosyl cyclase contributing to vasomotor tone through generation of cyclic ADP-ribose, a second messenger that activates ryanodine receptors to release Ca2+ from the sarcoplasmic reticulum in vascular smooth muscle cells. We evaluated whether the stable thromboxane mimetic U-46619 causes less pronounced renal vasoconstriction in CD38 deficient mice and the involvement of O2.- in U-46619-induced renal vasoconstriction. Our results indicate that U-46619 activation of TP receptors causes renal vasoconstriction in part by activating cADP-ribose signaling in renal resistance arterioles. Based on maximal renal blood flow and renal vascular resistance responses to bolus injections of U-46619, CD38 contributes 30-40% of the TP receptor-induced vasoconstriction. We also found that the anti-oxidant superoxide dismutase mimetic tempol attenuated the magnitude of vasoconstriction by U-46619 in both groups of mice, suggesting mediation by O2.-. The degree of tempol blockage of U-46619-induced renal vasoconstriction was greater in wild-type mice, attenuating renal vasoconstriction by 40% as compared to 30% in CD38 null mice. In other studies U-46619 rapidly stimulated O2.- production (DHE fluorescence) in isolated mouse afferent arterioles, an effect abolished by tempol. These observations provide the first in vivo demonstration of CD38 and O2.- involvement in the vasoconstrictor effects of TP receptor activation in the kidney and in vitro evidence for TP receptor stimulation of O2.- production by the afferent arteriole.
Pigment epithelium derived factor (PEDF) is a multifunctional protein with antiangiogenic, anti-oxidative and anti-inflammatory properties. PEDF is involved in the pathogenesis of diabetic retinopathy, but its direct role in the kidneys remains unclear. We hypothesize that a PEDF fragment (P78-PEDF) confers kidney protection in diabetic nephropathy (DN). The localization of the full length PEDF protein were determined in DBA mice following multiple low doses of streptozotocin. Using immunohistochemistry, PEDF was localized in the kidney vasculature, interstitial space, glomeruli, tubules and renal medulla. Kidney PEDF protein and mRNA expression were reduced significantly in diabetic mice. Continuous infusion of P78-PEDF for 6 wks resulted in protection from DN as indicated by reduced albuminuria and blood urea nitrogen, increased nephrin expression, decreased kidney macrophage recruitment and inflammatory cytokines, and reduced histological changes compared with vehicle-treated diabetic mice. In vitro, P78-PEDF blocked the increase in podocyte permeability to albumin and disruption of the actin cytoskeleton induced by puromycin aminonucleoside (PAN) treatment. These findings highlight the importance of P78-PEDF peptide as a potential therapeutic modality in early phase diabetic renal injury.
Nephrogenic diabetes insipidus (NDI) is the most common renal side effect in patients undergoing lithium therapy for bipolar affective disorders. Approximately 2 million US patients take lithium of whom ~50% will have altered renal function and develop NDI (2),(33). Lithium-induced NDI is a defect in the urinary concentrating mechanism. Lithium therapy also leads to proliferation and abundant renal cysts (microcysts), commonly in the collecting ducts of the cortico-medullary region. The mTOR pathway integrates nutrient and mitogen signals to control cell proliferation and cell growth (size) via the mTOR Complex 1 (mTORC1). To address our hypothesis that mTOR activation may be responsible for lithium-induced proliferation of collecting ducts we fed mice lithium chronically, and assessed mTORC1 signaling in the renal medulla. We demonstrate that mTOR signaling is activated in the renal collecting ducts of lithium-treated mice; lithium increased the phosphorylation of rS6 (Ser-240/Ser244), p-TSC2 (Thr1462) and p-mTOR (Ser 2448). Consistent with our hypothesis, treatment with rapamycin, an allosteric inhibitor of mTOR, reversed lithium-induced proliferation of medullary collecting duct cells and reduced levels of p-rS6 and p-mTOR. Medullary levels of p-GSK3β were increased in the renal medullae of lithium-treated mice and remained elevated following rapamycin treatment. However mTOR inhibition did not improve lithium-induced nephrogenic diabetes insipidus, and did not restore the expression of collecting duct proteins AQP2 or UT-A1.
Bone marrow-derived mesenchymal stem cells (BMSCs) transplantation can repair acute kidney injury (AKI), but with limited effect. We test the hypothesis that CXCR4 overexpression improves BMSCs repair ability and that this is related to increased release of cytokines. Hypoxia/re-oxygenation pretreated renal tubular epithelial cells (HR-RTECs) were used. BMSCs, null-BMSCs and CXCR4-BMSCs were co-cultured with HR-RTECs. The number of migrating BMSCs was counted. Proliferating cell nuclear antigen (PCNA) expression, cell death and expressions of cleaved Caspase-3 and Bcl-2 in the co-cultured HR-RTECs were measured. Cytokeratin 18 (CK18) expression and cytokine secretions for the BMSCs cultured with HR-RTEC supernatant were detected. BMSC homing, renal function, proliferation and cell death of tubular cells were assayed in the AKI mouse model. CXCR4-BMSCs showed a remarkable expression of CXCR4. Stromal cell-derived factor-1 (SDF-1) in the HR-RTEC supernatant was increased. Migration of BMSCs was CXCR4-dependent. Proportions of CK18+ cells in BMSCs, null-BMSCs and CXCR4-BMSCs showed no difference. However, CXCR4 overexpression in BMSCs stimulated secretion of BMP-7, HGF, and IL-10. The neutralizing anti-CXCR4 antibody AMD3100 abolished this. In the co-cultured HR-RTECs, proportion of PCNA+ cells and Bcl-2 expression were enhanced; however, proportion of Annexin V+ cells and expression of cleaved Caspase-3 were reduced. The in vivo study showed increased homing of CXCR4-BMSCs in kidneys, associated with improved renal function, reduced ATN scoring, accelerated mitogenic response of tubular cells, and reduced tubular cell death. The enhanced homing and paracrine actions of BMSCs with CXCR4 overexpression suggest beneficial effects of such cells in BMSC-based therapy of AKI.
A cellular compartment was added to our previous mathematical model of steady-state acid-base and fluid-electrolyte chemistry to gain further understanding and aid diagnosis of complex disorders involving cellular involvement in critically-ill patients. An important hypothesis to be validated was that the thermodynamic, standard free-energy of cellular H+ and Na+ pumps remained constant under all conditions. In addition, a hydrostatic-osmotic pressure balance was assumed to describe fluid exchange between plasma and interstitial fluid, including incorporation of compliance curves of vascular and interstitial spaces. The description of the cellular compartment was validated by close comparison of measured and model-predicted cellular pH and electrolyte changes in vitro and in vivo. The new description of plasma-interstitial fluid exchange was validated using measured changes in fluid volumes after isoosmotic and hyperosmotic fluid infusions of NaCl and NaHCO3. The validated model was used to explain the role of cells in the mechanism of saline or dilutional acidosis and acid-base effects of acidic or basic fluid infusions and the acid-base disorder due to potassium depletion. A module was created that would allow users, who do not possess the software, to determine, for free, the results of fluid infusions and urinary losses of water and solutes to the whole body.
Renal anemia has been recognized as a characteristic complication of chronic kidney disease (CKD). Although many factors are involved in renal anemia, the predominant cause of renal anemia is a relative deficiency in erythropoietin (EPO) production. To date, exogenous recombinant human EPO (rhEPO) has been used widely as a powerful drug for treating patients with renal anemia. Despite its clinical effectiveness, a potential risk for increased mortality has been suggested in patients who receive rhEPO, in addition to the economic burden of rhEPO administration. The induction of endogenous EPO is another therapeutic approach that might have advantages over rhEPO administration. However, the physiological and pathophysiological regulation of EPO is not fully understood, and this lack of understanding has hindered the development of an endogenous EPO inducer. In this review, we will discuss the current treatment for renal anemia and its drawbacks, provide an overview of EPO regulation in healthy and diseased conditions, and propose future directions for therapeutic trials that more directly target the underlying pathophysiology of renal anemia.
During migration, passerine birds typically complete a series of multi-hour flights, each followed by a period of stopover. During flight, rates of respiratory water loss are high, yet these birds show no signs of dehydration after flights. During stopover, birds become hyper-phagic to replenish fat reserves, often consuming food with high water content, such as fruit. Thus, migratory birds seem to face an osmoregulatory challenge; they must reduce water losses during flight, but retain the ability to excrete large quantities of water while maintaining osmotic balance at stopover. Our goal was to measure glomerular filtration rate (GFR) and fractional water reabsorption (FWR) of a migratory bird in free flight, at rest, and during feeding to assess the role of the kidney in maintaining water balance during migration. We used FITC-Inulin and one- and two-phase exponential decay models to first validate a technique and then measure GFR in the Swainson's thrush, a small (~30 g) songbird. Single-phase exponential decay models and the modified slope intercept method overestimated GFR by 26% compared to two-phase exponential decay models. We found no differences in GFR among fed, resting and flying birds, but fractional water reabsorption was significantly higher in resting and flying birds relative to feeding birds. There was no effect of the rate of respiratory water loss on GFR or FWR in flight. These data support the idea that birds in flight do not dramatically alter GFR, but rely on increased fractional water reabsorption to minimize excretory water losses.
Epithelial cells are continuously exposed to mechanical forces including shear stress and stretch, though the impact these forces have on tight junction (TJ) organization and function are poorly understood. Umbrella cells form the outermost layer of the stratified uroepithelium and undergo large cell shape and surface area changes during the bladder cycle. Here we investigated the effects of bladder filling and voiding on the umbrella cell TJ. We found that bladder filling promoted a significant increase in the length of the TJ ring, which was quickly reversed within 5 min of voiding. Interestingly, when isolated uroepithelial tissue was mounted in Ussing chambers and exposed to physiological stretch, we observed a tenfold drop in both transepithelial electrical resistance (TER) and the umbrella cell junctional resistance. The effects of stretch on TER were reversible and dependent on the applied force. Furthermore, the integrity of the umbrella cell TJ was maintained in the stretched uroepithelium, as suggested by the limited permeability of biotin, fluorescein, and ruthenium red. Finally, we found that depletion of extracellular Ca2+ by EGTA completely disrupted the TER of unstretched, but not of stretched uroepithelium. Taken together our studies indicate that the umbrella cell TJ undergoes major structural and functional reorganization during the bladder cycle. The impact of these changes on bladder function is discussed.
The role of bone marrow marrow-derived cells after kidney endothelial injury is controversial. In this study, we investigated if and to what extent extrarenal cells incorporate into kidney endothelium after acute as well as during chronic endothelial injury. Fischer F-344wt (wild type) rat kidney grafts were transplanted into R26-hPAP (human Placental Alkaline Phosphatase) transgenic Fischer F-344 recipient rats to allow identification of extrarenal cells by specific antibody staining. A severe model of renal thrombotic microangiopathy was induced via graft perfusion with anti-glomerular endothelial cell (GEN) antibody and resulted in eradication of 85 % of the glomerular and 69 % of the peritubular endothelium (GEN group). At week 4 after injury, renal endothelial healing as well as recovery of the kidney function was seen. Endothelial chimerism was evaluated by double staining for hPAP and endothelial markers RECA-1 or JG-12. Just 0.25 % of the glomerular and 0.1 % of the peritubular endothelium was recipient-derived. In a second experiment, chronic endothelial injury was induced by combination of kidney transplantation with 5/6 nephrectomy (5/6 Nx group). After 14 weeks, only 0.86 % of the peritubular and 0.05 % of the glomerular endothelium was of recipient origin. In summary, despite demonstration of extensive damage and loss as well as excellent regeneration, just a minority of extrarenal cells were incorporated into kidney endothelium in rat models of acute and chronic renal endothelial cell injury. Our results highlight that kidney endothelial regeneration after specific and severe injury is almost exclusively of renal origin.
Renin is synthesized and released from juxtaglomerular (JG) cells. Adenosine inhibits renin release via an adenosine A1 receptor (A1R), calcium-mediated pathway. How this occurs is unknown. In cardiomyocytes, adenosine increases intracellular calcium via transient receptor potential canonical (TRPC) channels. We hypothesized adenosine inhibits renin release via A1R activation, opening TRPC channels. However, higher concentrations of adenosine may stimulate renin release through A2R activation. Using primary cultures of isolated mouse JG cells, Immunolabeling demonstrated renin and A1R in JG cells, but not A2R subtypes, though RT-PCR indicated mRNA of both A2AR and A2BR. Incubating JG cells with increasing concentrations of adenosine decreased renin release. Different concentrations of the adenosine receptor agonist NECA didn't change renin. Activating A1R with 0.5µM of CHA decreased basal renin release from 0.22±0.05 to 0.14±0.03 µg AngI/ml/mg prot (p<0.03), and higher concentrations also inhibited renin. Reducing extracellular calcium with EGTA increased renin release (0.35±0.08 µgAngI/ml/mg prot (p<0.01), and blocked renin inhibition by CHA (0.28±0.06 ugAngI/ml/mg prot p<0. 005 vs. CHA alone). The intracellular calcium chelator BAPTA-AM increased renin release by 55%, and blocked the inhibitory effect of CHA. Repeating these experiments in JG cells from A1R knockout mice using CHA or NECA demonstrated no effect on renin release. However, RT-PCR showed mRNA from TRPC 3 and 6 in isolated JG cells. Adding the TRPC blocker SKF-96365 reversed CHA-mediated inhibition of renin release. Thus, A1R activation results in a calcium-dependent inhibition of renin release via TRPC-mediated calcium entry, but A2 receptors do not regulate renin release.
Epithelial Na+ channel (ENaC) activity, which determines the rate of renal Na+ reabsorption, can be regulated by G-protein coupled receptors. Regulation of ENaC by Gα mediated downstream effectors has been studied extensively, but the effect of Gβ dimers on ENaC is unclear. A6 cells endogenously contain high levels of Gβ1 but low levels of Gβ3 ,Gβ4 and Gβ5 were detected by Q-PCR. We tested G2 combined individually with Gβ1 through Gβ5 expressed in A6 cells, after which we recorded single ENaC activity. Among the five β and 2 combinations, β12 strongly inhibits ENaC activity by reducing both ENaC channel number (N) and open probability (Po) compared to control cells. In contrast, the other four β isoforms combined with 2 have no significant effect on ENaC activity. By using various inhibitors to probe Gβ12 effects on ENaC regulation, we found that Gβ12-mediated ENaC inhibition involved activation of phospholipase C-β and its enzymatic products that induce protein kinase C and ERK1/2 signaling pathways.
Nadph oxidase 4 is an important cellular source of reactive oxygen species (ROS) generation in the kidney. Novel anti-oxidant drugs, such as Nox4-inhibitor compounds, are being developed. There is however, very little experimental evidence for the biological role and regulation of Nadph oxidase isoforms in the kidney. Herein, we show that Fulvene-5 is an effective inhibitor of Nox-generated ROS and report the role of Nox isoforms in activating epithelial sodium channels (ENaC) in A6 distal nephron cells via oxidant signaling and cell stretch activation. Using single channel patch clamp analysis, we report that Fulvene-5 blocked the increase in ENaC activity that is typically observed with H2O2 treatment of A6 cells: average ENaC NPo values decreased from a baseline level of 1.04±0.18 (mean±SE) to 0.25±0.08 following Fulvene-5 treatment. H2O2 treatment failed to increase ENaC activity in the presence of Fulvene-5. Moreover, Fulvene-5 treatment of A6 cells blocked the osmotic-cell stretch response of A6 cells; indicating that stretch activation of Nox-derived ROS plays an important role in ENaC regulation. Together, these findings indicate that Fulvene-5, and perhaps other classes of antioxidant inhibitors, may represent a novel class of compounds useful for the treatment of pathological disorders stemming from inappropriate ion channel activity, such as hypertention.
Oxygenation defects may contribute to renal disease progression but the chronology of events is difficult to define in vivo without recourse to invasive methodologies. BOLD MRI provides an attractive alternative but the R2* signal is physiologically complex. Post-acquisition data analysis often relies on manual selection of region(s) of interest. This approach excludes from analysis significant quantities of biological information and is subject to selection bias. We present a semi-automated, anatomically unbiased approach to compartmentalize voxels into two quantitatively related clusters. In control F344 rats, low R2* clustering was located predominantly within the cortex and higher R2* clustering within the medulla (70.96±1.48 versus 79.00±1.50; 3 scans per rat; n=6; P<0.01) consistent anatomically with a cortico-medullary oxygen gradient. An intravenous bolus of acetylcholine caused a transient reduction of the R2* signal in both clustered segments (P<0.01). This was nitric oxide dependent and temporally distinct from the hemodynamic effects of acetylcholine. Rats were then chronically infused with angiotensin II (60ng/min) and rescanned three days later. Clustering demonstrated a disruption of the cortico-medullary gradient, producing less distinctly segmented mean R2* clusters (71.30±2.00; versus 72.48±1.27; n=6; NS). The acetylcholine-induced attenuation of the R2* signal was abolished by chronic angiotensin II infusion, consistent with reduced nitric oxide bioavailability. This global map of oxygenation, defined by clustering individual voxels on the basis of quantitative nearness might be more robust in defining deficits in renal oxygenation than the absolute magnitude of R2* in small, manually selected regions of interest defined exclusively by anatomical nearness
Patients with idiopathic hypercalciuria (IH) have decreased renal calcium reabsorption, most marked in the post-prandial state, but mechanisms are unknown. We compared 29 subjects with IH and 17 normal subjects (N) each fed identical meals. Urine and blood samples were collected fasting and after meals. Levels of 3 candidate signallers - serum calcium (Sca), insulin (I), and plasma parathyroid hormone (PTH) did not differ between IH and N either fasting or fed, but all changed with feeding, and the change in Sca was greater in IH than in N. Regression analysis of fractional excretion of calcium (FECa) was significant for PTH and Sca in IH but not N. Using multivariable analysis, Sca entered the model while PTH and I did not. To avoid internal correlation we decomposed FECa into its independent terms: adjusted urine calcium (UCa) and UFCa molarity. Analyses using adjusted Uca and unadjusted Uca parallel those using FECa, showing a dominant effect of Sca with no effect of PTH or I. The effect of Sca may be mediated via VDR-stimulated increased abundance of CaSR, which is supported by the fact PTH levels also seem more responsive to serum Ca in IH than in N. Although our data support an effect of Sca on FECa and urine Ca which is more marked in IH than in N, it can account for only a modest fraction of the meal effect, perhaps 10 - 20%, suggesting additional mediators are also responsible for the exaggerated post-prandial hypercalciuria seen in IH.
Obesity is prevalent worldwide and is a major risk factor for many diseases including renal complications. Thrombospondin 1 (TSP1), a multifunctional extracellular matrix protein, plays an important role in diabetic kidney diseases. However, whether TSP1 plays a role in obesity-related kidney disease is unknown. In the present studies, the role of TSP1 in obesity-induced renal dysfunction was determined by using a diet-induced obese mouse model. The results demonstrated that TSP1 was significantly up-regulated in the kidney from obese mice. The increased TSP1 was localized in the glomerular mesangium as well as in the tubular system from obese wild type mice. Obese wild type mice developed renal hypertrophy and albuminuria, which was associated with increased kidney macrophage infiltration, augmented kidney inflammation, and activated TGF-β signaling and renal fibrosis. In contrast, obese TSP1 deficient mice did not develop these kidney damages. Furthermore, in vitro studies demonstrated that leptin treatment stimulated the expression of TSP1, TGF-β 1, fibronectin and collagen type IV in mesangial cells isolated from wild type mice. These leptin stimulated effects were abolished in TSP1 deficient mesangial cells. Taken together, these data suggest that TSP1 is an important mediator for obesity or hyperleptinemia induced kidney dysfunction.
As the molecular revolution continues to inform a deeper understanding of disease mechanisms and pathways, there exist unprecedented opportunities for translating discoveries at the bench into novel therapies for improving human health. Despite the availability of several different classes of antihypertensive medications, only about half of the 67 million Americans with hypertension manage their blood pressure appropriately. A broader selection of structurally diverse antihypertensive drugs acting through different mechanisms of action would provide clinicians with greater flexibility in developing effective treatment regimens for an increasingly diverse and aging patient population. An emerging body of physiological, genetic, and pharmacological evidence has implicated several renal ion-transport proteins, or regulators thereof, as novel, yet clinically unexploited, diuretic targets. These include the Renal Outer Medullary potassium channel, ROMK (Kir1.1), Kir4.1/5.1 potassium channels, ClC-Ka/b chloride channels, UTA/B urea transporters, the chloride/bicarbonate exchanger pendrin, and the STE20/SPS1-related proline/alanine-rich kinase SPAK. The molecular pharmacology of these putative targets is poorly developed or lacking altogether; however, recent efforts by a few academic and pharmaceutical laboratories have begun to lessen this critical barrier. Here, we review the evidence in support of the aforementioned proteins as novel diuretic targets and highlight examples where progress toward developing the small-molecule pharmacology has been made.
The vacuolar H+-ATPase (V-ATPase) in intercalated cells contributes to luminal acidification in the kidney collecting duct and non-volatile acid excretion. We previously showed that the A subunit in the cytoplasmic V1 sector of the V-ATPase (ATP6V1A) is phosphorylated by the metabolic sensor AMP-activated protein kinase (AMPK) in vitro and in kidney cells. Here we demonstrate that treatment of rabbit isolated perfused collecting ducts with the AMPK activator AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) inhibited V-ATPase-dependent H+ secretion from intercalated cells after an acid load. We have identified by mass spectrometry that Ser-384 is a major AMPK phosphorylation site in the V-ATPase A subunit, a result confirmed by comparing AMPK-dependent phosphate labeling of wild-type A-subunit (WT-A) with that of a Ser-384-to-Ala A subunit mutant (S384A-A) in vitro and in intact HEK-293 cells. As compared with WT-A-expressing HEK-293 cells, S384A-A-expressing cells exhibited greater steady-state acidification of HCO3--containing media. Moreover, AICAR treatment of Clone C rabbit intercalated cells expressing WT-A subunit reduced V-ATPase-dependent extracellular acidification, an effect that was blocked in cells expressing the phosphorylation-deficient S384A-A mutant. Finally, expression of the S384A-A mutant prevented cytoplasmic redistribution of the V-ATPase by AICAR in Clone C cells. In summary, direct phosphorylation of the A subunit at Ser-384 by AMPK represents a novel regulatory mechanism of the V-ATPase in kidney intercalated cells. Regulation of the V-ATPase by AMPK may couple V-ATPase activity to cellular metabolic status with potential relevance to ischemic injury in the kidney and other tissues.
Renalase is a kidney secreted catecholamines-degrading enzyme whose expression and activity are down-regulated by increased dietary phosphate. A renalase knockout (KO) mouse model was used to explore the mechanisms mediating renalase's effect on phosphate excretion. Compared to wild type (WT) mice maintained on a regular diet, KO mice show decreased serum PO4- (KO=5.3±0.2 vs WT=6.0±0.1, n= 6, p<0.04), and increased urinary PO4- excretion (urine PO4-/creatinine: KO=7.7±0.3 vs WT=6.1±0.3, n=6, p<0.02). However, both WT and KO mice respond similarly to PO4- restriction by increasing renal COMT-1 activity and markedly decreasing PO4- excretion, which excludes an intrinsic renal defect in the KO. Renal sodium-phosphate co-transporter, Npt2a, and sodium proton exchanger, NHE3 expression, and MAO-A and B activity did not differ between WT and KO. Only catechol-O-methyl transferase (COMT) expression and activity were significantly increased in KO mice. In spite of that, urinary dopamine increased by 2 fold, while urinary L-DOPA excretion decreased by 2 fold, indicating an up-regulation of renal DA synthesis. These data indicate that renalase deficiency is associated with increased renal DA synthesis, stimulated PO4- excretion, and moderately severe hypophosphatemia. The signal to increase renal DA synthesis is strong as it overcomes a compensatory increase in COMT activity.
Signal regulatory protein α (SIRPα) is a transmembrane protein that contains tyrosine phosphorylation sites in its cytoplasmic region; two tyrosine phosphatases, SHP-1 and SHP-2, bind to these sites in a phosphorylation-dependent manner and transduce multiple intracellular signals. Recently, SIRPα was identified as one of the major tyrosine-phosphorylated proteins in the glomeruli and found to be expressed in podocytes. In the present study, we examined the role of SIRPα expression in podocytes using knock-in mice (C57BL/6 background) expressing mutant SIRPα that lacks a cytoplasmic region (SIRPα-mutant mice). Light microscopic examination revealed no apparent morphological abnormalities in the kidneys of the SIRPα-mutant mice. On the other hand, electron microscopic examination revealed abnormal podocytes with irregular major processes and wider and flattened foot processes in the SIRPα-mutant mice as compared to their wild-type counterparts. Significantly impaired renal functions and slight albuminuria were demonstrated in the SIRPα-mutant mice. In addition, adriamycin injection induced massive albuminuria together with focal glomerulosclerosis in the SIRPα-mutant mice, while their wild-type counterparts were resistant to adriamycin-induced nephropathy. These data demonstrate that SIRPα is involved in the regulation of podocyte structure and function as a filtration barrier under both physiological and pathological conditions.
Unaccustomed strenuous physical exertion in hot environments can result in heat stroke and acute kidney injury (AKI). Both exercise-induced muscle damage and AKI are associated with the release of interleukin-6 but whether muscle damage causes AKI in the heat is unknown. We hypothesized that muscle damaging exercise, prior to exercise in the heat would increase kidney stress. Ten healthy, euhydrated males underwent a randomized, crossover trial involving both a 60 minute downhill muscle-damaging run (EIMD), and an exercise intensity-matched non-muscle damaging flat run (CON), in random order separated by two weeks. Both treatments were followed by heat stress elicited by a 40 minute run at 33oC. Urine and blood were sampled at baseline, after treatment, and after running in the heat. By design, EIMD induced higher plasma creatine kinase and interleukin-6 than CON. EIMD elevated kidney injury biomarkers (e.g. urinary NGAL after running in the heat: EIMD-CON, mean diff [95%CI]: 12 [5, 19] ng/ml) and reduced kidney function (e.g. plasma creatinine after running in the heat: EIMD-CON, mean diff [95%CI]: 0.2 [0.1, 0.3] mg/dl). Plasma interleukin-6 was positively correlated with plasma NGAL (r = 0.9, P = 0.001). Moreover, following EIMD, 5 of 10 participants met AKIN criteria for AKI. Thus, for the first time we demonstrate that muscle damaging exercise prior to running in the heat results in a greater inflammatory state and kidney stress compared to non-muscle damaging exercise. Muscle damage should therefore be considered a risk factor for AKI when performing exercise in hot environments.
The role of the 5-HT3 receptors in pudendal neuromodulation of bladder activity and its interaction with opioid receptors were investigated in anesthetized cats. The bladder was distended with either saline to induce normal bladder activity or with 0.25% acetic acid (AA) to induce bladder overactivity. Pudendal afferent nerves were activated by 5 Hz stimulation at multiples of the threshold (T) intensity for inducing anal twitching. AA irritation significantly reduced bladder capacity to 16.5±3.3% of saline control capacity, while pudendal nerve stimulation (PNS) at 1.5-2T and 3-4T restored the capacity to 82.0±12% (P=0.0001) and 98.6±15% (P<0.0001), respectively. Cumulative doses (1-3mg/kg, i.v.) of ondansetron, a 5-HT3 receptor antagonist, eliminated low intensity (1.5-2T) PNS inhibition and reduced high intensity (3-4T) PNS inhibition of bladder overactivity. During saline distention, PNS at 1.5-2T and 3-4T significantly increased bladder capacity to 173.2±26.4% (P=0.036) and 193.2±22.5% (P=0.008), respectively, of saline control capacity, but ondansetron (0.003-3mg/kg, i.v.) did not alter PNS inhibition. Ondansetron (0.1-3 mg/kg) also significantly (P<0.05) increased control bladder capacity (50-200%) during either AA irritation or saline distention. In both conditions the effect of low and high intensity PNS were not significantly different. After ondansetron (3 mg/kg) treatment, naloxone (1 mg/kg, i.v.) significantly (P<0.05) decreased control bladder capacity (40-70%) during either AA irritation or saline distention, but failed to affect PNS inhibition. This study revealed that activation of 5-HT3 receptors has a role in PNS inhibition of bladder overactivity. It also indicated that 5-HT3 receptor antagonists might be useful for treating overactive bladder symptoms.
Chronic Ang II infusion in rodents is widely used as an experimental model of hypertension, yet very limited data are available describing the resulting BP - RBF relationships in conscious rats. Accordingly, male Sprague-Dawley rats (n=19) were instrumented for chronic measurements of BP (radiotelemetry) and RBF (Transonic). One week later, 2-3 separate 2 hr recordings of BP and RBF were obtained in conscious rats at 24 hr intervals in addition to separate 24 hr BP recordings. Rats were then administered either Ang II (n=11, 125 ng/kg/min) or phenylephrine (PE, n=8, 50 mg/kg/day) as a control, Ang II-independent, pressor agent. Three days later the BP-RBF and 24 hr BP recordings were repeated over several days. Despite similar increases in BP, PE led to significantly greater BP lability at the heart beat and very low frequency bandwidths. Conversely, Ang II, but not PE, caused significant renal vasoconstriction (a 62% increase in renal vascular resistance and a 21% decrease in RBF) and increased variability in BP-RBF relationships. Transfer function analysis of BP (input) and RBF (output) were consistent with a significant potentiation of the renal myogenic mechanism during Ang II administration, likely contributing, in part, to the exaggerated reductions in RBF during periods of BP elevations. We conclude that relatively equipressor doses of Ang II and PE lead to greatly different ambient BP profiles and effects on the renal vasculature when assessed in conscious rats. These data may have important implications regarding the pathogenesis of hypertension-induced injury in these models of hypertension.
The cellular morphology of the collecting duct is altered by chronic lithium treatment. We have previously shown that lithium increases the fraction of type-A intercalated cells and lowers the fraction of principal cells along the collecting duct. Moreover, type-A intercalated cells acquire a long-row distribution pattern along the tubules. In the present study, we show that these morphological changes reverse progressively after discontinuation of lithium, and finally disappear after 19 days from lithium suspension. In this time frame we have identified for the first time, in vivo, a novel cellular type positive for both intercalated and principal cells functional markers, as recognized by co-labelling with H+-ATPase/AQP4 and AE-1/AQP2 and Foxi1/AQP4. This cell type is mainly present after 6 days of lithium washout and it disappears in parallel with the long-row pattern of the type-A intercalated cells. It usually localizes either in the middle or at the edge of the long-row pattern. Its ultra-structure resembles the intercalated cells as shown both by differential interference contrast and by electron microscopy. The time course of appearance, the localization along the collecting duct and the ultra-structure suggest that the cells double labelled for principal and intercalated cells markers could represent a transition element driving the conversion of intercalated cells into principal cells.
Increasing evidence nowadays is showing that obesity by itself, independent of other co-morbidities like diabetes and hypertension, is associated with renal structural damage and functional changes. Intentional weight loss demonstrates beneficial reduction in proteinuria and albuminuria in patients with mild to moderate chronic kidney disease, particularly those whose renal damage is directly induced by obesity. The safety of some weight loss interventions, particularly the use of high-protein diets and/or medications, is questionable in this population due to the lack of well-designed randomized controlled studies reporting on their efficacy or harm. Bariatric surgery showed the most promising results with regards to ameliorating glomerular hyperfiltration and albuminuria albeit with a modest risk of increased perioperative complications with advanced stages of chronic kidney disease.
Cisplatin is widely used to treat malignancies. However, its major limitation is the development of dose-dependent nephrotoxicity. The precise mechanisms of cisplatin-induced kidney damage remain unclear, and the renoprotective agents during cisplatin treatment are still lacking. Here we demonstrated that the expression and activity of cGMP-dependent protein kinase-I (PKG-I) was reduced in cisplatin treated renal tubular cells in vitro as well as in the kidney tissues from cisplatin treated mice in vivo. Increasing PKG activity by both pharmacological and genetic approaches attenuated cisplatin-induced kidney cell apoptosis in vitro. This was accompanied by decreased Bax/Bcl2 ratio, caspase 3 activity, and cytochrome c release. Cisplatin-induced mitochondria membrane potential loss in the tubular cells was also prevented by increased PKG activity. All of these data suggest a protective effect of PKG on mitochondria function in renal tubular cells. Importantly, increasing PKG activity pharmacologically or genetically diminished cisplatin-induced tubular damage and preserved renal function during cisplatin treatment in vivo. Mitochondria structural and functional damage in the kidney from cisplatin treated mice was inhibited by increased PKG activity. In addition, increasing PKG activity enhanced ciaplatin induced cell death in several cancer cell lines. Taken together, these results suggest that increasing PKG activity may be a novel option for renoprotection during cisplatin-based chemotherapy.
Every collecting duct (CD) of the rat inner medulla is uniformly surrounded by about four abutting ascending vasa recta (AVR) running parallel to it. One or two ascending thin limbs (ATLs) lie between and parallel to each abutting AVR pair, opposite the CD. These structures form boundaries of axially running interstitial compartments. Viewed in transverse sections, these compartments appear as four interstitial nodal spaces (INSs) positioned symmetrically around each CD. The axially running compartments are segmented by interstitial cells spaced at regular intervals. The pairing of ATLs and CDs bounded by an abundant supply of AVR carrying reabsorbed water, NaCl and urea make a strong argument that mixing of NaCl and urea within the INSs, and countercurrent flows play a critical role in generating the inner medullary osmotic gradient. This study fully supports that hypothesis. We quantified interactions of all structures comprising INSs along the corticopapillary axis for 2 rodent species, the Munich-Wistar rat and the kangaroo rat. Results show remarkable similarities in configurations of INSs, suggesting that the structural arrangement of INSs is a highly conserved architecture that plays a fundamental role in renal function. The number density of INSs along the corticopapillary axis directly correlates with a loop population that declines exponentially with distance below the outer medullary-inner medullary boundary. Axial configurations are consistent with discrete association between near-bend loop segments and INSs, and with upper loop segments lying distant from INSs.
20-Hydroxyeicosatetraenoic acid (20-HETE) is a cytochrome P450-derived arachidonic acid metabolite that has been shown to increase smooth muscle contractions and proliferation, stimulate endothelial dysfunction and activation and promote hypertension. We examined if 20-HETE contributes to microvascular remodeling in hypertension. In Sprague-Dawley rats, administration of the 20-HETE biosynthesis inhibitor, HET0016, or the 20-HETE antagonist, 20-HEDE prevented 5α-dihydrotestosterone (DHT)-induced increases in blood pressure as well as abrogated DHT-induced increases in media-to-lumen ratio (M/L), media thickness and collagen IV deposition in renal interlobar arteries. Reserpine prevented blood pressure elevation in DHT-treated rats but did not affect microvascular remodeling (M/L, media thickness and collagen deposition); under these conditions, treatment with 20-HETE antagonist attenuated microvascular remodeling, suggesting that 20-HETE contributes to DHT-induced vascular remodeling independent of blood pressure elevation. In Cyp4a14(-/-) mice, which display androgen-driven and 20-HETE-dependent hypertension, treatment with 20-HETE antagonist abolished remodeling of renal resistance arteries measured as media thickness (24±1 vs. 15±1μm) and M/L (0.29±0.03 vs. 0.17±0.01). Moreover, in the Cyp4a12 transgenic mice in which the expression of Cyp4a12-20-HETE synthase is driven by a tetracyclin-sensitive promoter, treatment with doxycycline resulted in blood pressure elevation (140±4 vs. 92±5 mmHg) and a significant increase in remodeling of renal resistance arteries (media thickness, 23±1 vs. 16±1 μm; M/L, 0.39±0.04 vs. 0.23±0.02); these increases were abrogated by co-treatment with 20-6,15-HEDE. This study demonstrated that 20-HETE is a key regulator of microvascular remodeling in hypertension; its effect is independent of blood pressure elevation and androgen levels.
The rat kidney ablation and infarction (A/I) model of subtotal or 5/6th nephrectomy is the most commonly studied model of non-diabetic chronic kidney disease (CKD). The A/I kidney at one-week exhibits reductions in kidney function as determined by glomerular filtration rate (GFR), and diminished metabolic efficiency as determined by oxygen consumption per sodium transport (QO2/TNa). As renoprotective adenosine monophosphate-activated protein kinase (AMPK) activity is affected by metabolic changes and cellular stress, we evaluated AMPK activity in this model system. We show that these early pathophysiologic changes are accompanied by a paradoxical decrease in AMPK activity. Over time these kidney parameters progressively worsen with extensive kidney structural, functional, metabolic and fibrotic changes observed at four weeks post A/I. We show that induction of AMPK activity with either metformin or AICAR increases AMPK activity in this model, and also corrects kidney metabolic inefficiency, improves kidney function and ameliorates kidney fibrosis and structural alterations. We conclude that AMPK activity is reduced in the subtotal nephrectomy model of non-diabetic CKD; that altered regulation of AMPK is coincident with the progression of disease parameters; and that restoration of AMPK activity can suppress the progressive loss of function characteristic of this model. We propose that induction of AMPK activity may prove an effective therapeutic target for the treatment of non-diabetic CKD.
Cold storage of kidneys before transplantation is problematic because of limited survival time of the allografts. In this study, Zinc-N-acetylcysteine (ZnNAC) was shown to be a potent endonuclease inhibitor and antioxidant, and it was tested as a potential additive to a cold storage solution for kidney preservation. Exposure of normal rat kidney NRK-52E cells to ZnNAC resulted in zinc delivery to the cells determined by TFL-Zn fluorophore and partial protection of the cells against injury by cold storage in the University of Wisconsin solution (UWS) as measured by propidium iodide assay. Ex vivo, rat kidneys demonstrated time- and temperature-dependent DNA fragmentation assessed by TUNEL assay, indicating irreversible cell death. The DNA fragmentation was faster in medulla than in cortex, and tubules were affected more than glomeruli. Perfusion of rat kidneys with cold ZnNAC solution in UWS significantly inhibited cell death both in the cortex and medulla at concentrations 0.3-30 mM as compared to UWS alone, with the maximum effect at 1-10 mM ZnNAC. Cold storage of the kidney significantly increased quantities of cleaved caspase-3 and endonuclease G in the tissue which was abolished by 10 mM ZnNAC indicating its ability to suppress both caspase-dependent and -independent cell death. Therefore, supplementation of UWS with ZnNAC can decrease DNA fragmentation and protect kidney allograft from cell death due to cold storage.
Endothelial cells (ECs) are highly susceptible to hypoxia and easily affected upon ischemia/reperfusion (I/R) during renal transplantation. Pericytes and angiopoeitins play important roles in modulating EC function. In the present study, we investigate the effect of renal I/R on dynamics of angiopoietin expression and its association with pericytes and fibrosis development. Male Lewis rats were subjected to unilateral renal ischemia for 45 minutes followed by removal of the contralateral kidney. Rats were sacrificed at different time points after reperfusion. Endothelial integrity (RECA-1),pericytes (PDGFRβ), Angiopoietin-2 (Ang-2)/Angiopoietin-1 (Ang-1) expression and interstitial collagen deposition (Sirius Red and α-SMA) were assessed using immunohistochemistry and RT-PCR. Our study shows an increase in protein expression of Ang-2 starting at 5 hours and remaining elevated up to 72 hours, with consequently higher Ang-2/Ang-1 ratio after renal I/R (p<0.05 at 48 hours). This was accompanied by an increase in protein expression of the pericytic marker PDGFRβ and a loss of ECs (both at 72 hours after I/R, p<0.05). Nine weeks after I/R, when renal function was restored, we observed normalization of the Ang-2/Ang-1 ratio and PDGFRβ expression and increase in cortical ECs, which was accompanied by fibrosis. Renal I/R induces a dysbalance of Ang-2/Ang-1 accompanied by proliferation of pericytes, EC loss and development of fibrosis. The Ang-2/Ang-1 balance was reversed to baseline at 9 weeks after renal I/R, which coincided with restoration of cortical ECs and pericytes. Our findings suggest that angiopoietins and pericytes play an important role in renal microvascular remodeling and development of fibrosis.
The inhibitor of apoptosis protein survivin is a bifunctional molecule which regulates cellular division and survival. We have previously shown that survivin protein can be found at high concentrations in the adult kidney, particularly in the proximal tubules. Here, survivin is localized primarily at the apical membrane, a pattern which may indicate absorption of the protein. Several proteins in primary urine are internalised by megalin, an endocytosis receptor, which is in principle found in the same localization as survivin. Immunolabeling for survivin in different species confirmed survivin signal localising to the apical membrane of the proximal tubule. Immuno-electron microscopy also showed apical localisation of survivin in human kidneys. Furthermore, in polarised human primary tubular cells endogenous as well as external recombinant survivin stored in the apical region of the cells. Co-staining of survivin and megalin by immunohistochemistry and immuno-electron microscopy confirmed colocalization. Finally, by surface plasmon resonance we were able to demonstrate that survivin binds megalin and cubilin and that megalin knockout mice loose survivin through the urine. Survivin accumulates at the apical membrane of the renal tubule by reuptake, which is achieved by the endocytic receptor megalin, collaborating with cubilin. For this to occur, survivin will have to circulate in blood and be filtered into the primary urine. It is not known at this stage what the functional role of tubular survivin is. However, a small number of experimental and clinical reports implicate that renal survivin is important for functional integrity of the kidney.
Podocyte damage and accumulation of advanced glycation end-products (AGEs) are characteristic of diabetic nephropathy (DN). The pathophysiology of AGE-challenged podocytes is closely related to the induction of cell cycle inhibitor p27Kip1 and to the inhibition of neuropilin 1 (NRP1). We previously demonstrated that treatment with erythropoietin is associated with protective effects for podocytes in vitro. db/db mice with overt DN aged 15-16 weeks were treated with either placebo, or epoetin-β, or CERA (continuous erythropoietin receptor activator) for 2 weeks. db/db mice compared with non-diabetic db/m controls revealed the expected increases in body weight, blood glucose, albumin-to-creatinine ratio (ACR) and AGE-accumulation. Whereas no differences in body weight, hyperglycemia and AGEs were observed among the diabetic groups receiving epoetin-β resp, The ACR were significantly lower in db/db mice treated with epoetin-β or CERA. Furthermore, kidney weights in db/db mice were increased compared with the db/m controls indicating renal hypertrophy, whereas the increase in renal weight in epoetin-β- or CERA-treated db/db was significantly lower than in the placebo-treated controls. Induction of p27Kip1 and suppression of NRP1 were significantly reduced in the epoetin-β resp. CERA treatment group. Furthermore, erythropoietin treatment diminished the diabetes-induced podocyte loss. Together, independently from hematopoetic effects, epoetin-β or CERA treatment was associated with protective changes in DN, especially that the NRP1 and p27Kip1 expressions as well as the number of podocytes returned to normal level. Our data show for the first time that medication of overt DN with erythropoietin for a short time can ameliorate albuminuria and podocyte loss.
The aim of this study was to assess the potential of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to predict and evaluate functional outcome after renal artery revascularization for renal artery stenosis (RAS). Single-kidney glomerular filtration rate (SK-GFR) was measured in 15 patients with atherosclerotic RAS with DCE-MRI and radioisotopes at baseline, and 4 months after revascularization. DCE-MRI also produced a measurement of blood flow, blood volume, extraction fraction, tubular transit time and functional volume. Stented kidneys (n=22) were divided into three response groups on the basis of the changes in radioisotope SK-GFR: improved (n=5), stable (n=13), deteriorated (n=4). A good agreement was found between SK-GFR values from DCE-MRI and radioisotopes. Before intervention, kidneys that improved had lower extraction fraction, higher blood volume, longer tubular transit time and lower SK-GFR. After intervention, improved kidneys had increased functional volume and deteriorated kidneys had reduced functional volume and extraction fraction. Revascularization improved blood flow and blood volume in all groups. This pilot study leads to the hypothesis that well-vascularized kidneys with reduced extraction fractions are most likely to benefit from revascularization. More generally, DCE-MRI has the potential to replace radioisotope measurement of SK-GFR, and may improve patient management by providing additional information on tissue perfusion.
Podocyte injury, a major contributor to the pathogenesis of diabetic nephropathy, is caused at least in part by the excessive generation of reactive oxygen species (ROS). Overproduction of superoxide by the NADPH oxidase isoform Nox4 plays an important role in podocyte injury. The plant extract silymarin is attributed antioxidant and antiproteinuric effects in humans and in animal models of diabetic nephropathy. We investigated the effect of silybin, the active constituent of silymarin, in cultures of mouse podocytes and in the OVE26 mouse, a model of type 1 diabetes mellitus and diabetic nephropathy. Exposure of podocytes to high glucose (HG) increased 60% the intracellular superoxide production, 90% the NADPH oxidase activity, 100% the Nox4 expression and 150% the number of apoptotic cells, effects that were completely blocked by 10 μM silybin. These in vitro observations were confirmed by similar in vivo findings. The kidney cortex of vehicle-treated control OVE26 mice displayed greater Nox4 expression and twice as much superoxide production than cortex of silybin-treated mice. The glomeruli of control OVE26 mice displayed 35% podocyte drop out which was not present in the silybin-treated mice. Finally, the OVE26 mice experienced 54% more pronounced albuminuria than the silybin-treated animals. In conclusion, this study demonstrates a protective effect of silybin against HG-induced podocyte injury and extends this finding to an animal model of diabetic nephropathy.
Renal glutamine synthetase catalyzes the reaction of NH4+ with glutamate, forming glutamine and decreasing ammonia available for net acid excretion. The purpose of the current study was to determine glutamine synthetase's specific cellular expression in mouse kidney and its regulation by hypokalemia, a common cause of altered renal ammonia metabolism. Glutamine synthetase mRNA and protein were present in the renal cortex and in both the outer and the inner stripe of outer medulla (OMo and OMi). Immunohistochemistry showed glutamine synthetase expression throughout the entire proximal tubule and in non-proximal tubule cells. Double-immunolabel with cell-specific markers demonstrated glutamine synthetase expression in Type A intercalated cells, non-A, non-B intercalated cells and distal convoluted tubule cells, but not in principal cells, Type B intercalated cells or connecting segment cells. Hypokalemia induced by feeding a nominally K+-free diet for twelve days decreased glutamine synthetase expression throughout the entire proximal tubule and in the distal convoluted tubule and simultaneously increased glutamine synthetase expression in Type A intercalated cells in both the cortical and outer medullary collecting duct. We conclude that glutamine synthetase is widely and specifically expressed in renal epithelial cells, and that the regulation of expression differs in specific cell populations. Glutamine synthetase is likely to mediate an important role in renal ammonia metabolism.
This study determined role of PKC-α and associated inducible heat shock protein70 (iHSP70) in the repair of mitochondrial function in renal proximal tubular cells (RPTC) following oxidant injury. Wild-type PKC-α (wtPKC-α) and inactive PKC-α (dnPKC-α) mutant were overexpressed in primary cultures of RPTC and iHSP70 levels and RPTC regeneration were assessed following treatment with oxidant, tert-butylhydroperoxide (TBHP). TBHP exposure increased ROS production and induced RPTC death that was prevented by ferrostatin and necrostatin-1, but not cyclosporin A. Overexpressing wtPKC-α maintained mitochondrial levels of active PKC-α, reduced cell death, and accelerated proliferation without altering ROS production in TBHP-injured RPTC. In contrast, dnPKC-α blocked proliferation and monolayer regeneration. Co-immunoprecipitation and proteomic analysis demonstrated association between inactive, but not active, PKC-α and iHSP70 in mitochondria. Mitochondrial iHSP70 levels increased as levels of active PKC-α decreased after injury. Overexpressing dnPKC-α augmented whereas overexpressing wtPKC-α abrogated oxidant-induced increases in mitochondrial iHSP70 levels. iHSP70 overexpression: 1) maintained mitochondrial levels of phosphorylated-PKC-α, 2) improved recovery of state 3 respiration and ATP content, 3) decreased RPTC death (an effect abrogated by cyclosporin A), and 4) accelerated proliferation following oxidant injury. In contrast, iHSP70 inhibition blocked recovery of ATP content and exacerbated RPTC death. Inhibition of PKC-α in RPTC overexpressing iHSP70 blocked protective effects of iHSP70. We conclude that active PKC-α maintains mitochondrial function and decreases cell death following oxidant injury. iHSP70 is recruited to mitochondria in response to PKC-α dephosphorylation, associates with and re-activates inactive PKC-α, which promotes recovery of mitochondrial function, decreases RPTC death, and improves regeneration.
Plasma urate levels are higher in humans than rodents (240-360 µM vs. ~30 µM) because humans lack the liver enzyme uricase. High uricemia in humans may protect against oxidative stress but hyperuricemia also associates with the metabolic syndrome and urate and uric acid can crystallize to cause gout and renal dysfunctions. Thus, hyperuricemic animal models to study urate-induced pathologies are needed. We recently generated mice with liver-specific ablation of glut9, a urate transporter providing access of urate to uricase (LG9KO mice). LG9KO mice had moderately high uricemia (~120 µM). To further increase their uricemia, here we gavaged LG9KO mice for 3 days with inosine, a urate precursor; this treatment was applied in both chow and high fat-fed mice. In chow-fed LG9KO mice, uricemia peaked at 300 µM 2h after the first gavage and normalized 24h after the last gavage. In contrast, in high fat-fed LG9KO mice, uricemia further rose to 500µM. Plasma creatinine strongly increased, indicating acute renal failure. Kidneys showed tubule dilation, macrophage infiltration, and urate and uric acid crystals, associated with a more acidic urine. Six weeks after inosine gavage, plasma urate and creatinine had normalized. However, renal inflammation, fibrosis and organ remodeling had developed despite the disappearance of urate and uric acid crystals. Thus, hyperuricemia and high fat diet feeding combined to induce acute renal failure. Furthermore, a sterile inflammation caused by the initial crystal-induced lesions developed despite the disappearance of urate and uric acid crystals.
The epithelial Na+ channel (ENaC) in the distal nephron constitutes the rate-limiting step for renal sodium eabsorption. Aldosterone increases tubular sodium absorption in large part by increasing αENaC transcription in collecting duct cells. We previously reported that Af9 binds to +78/+92 of αENaC and recruits Dot1a to repress basal and aldosterone-sensitive αENaC transcription in mIMCD3 cells. Despite this epigenetic repression, basal αENaC transcription is still evident and physiologically necessary, indicating basal operation of positive regulators. In the present study, we identified Sp1 as one such regulator. Gel shift and antibody competition assays using a +208/+240 probe revealed DNA-Sp1-containing complexes in mIMCD3 cells. Mutation of the +222/+229 element abrogated Sp1 binding in vitro and in promoter-reporter constructs stably expressed in mIMCD3 cells. Compared to the wild type promoter, an αENaC promoter-luciferase construct with +222/+229 mutations exhibited much lower activity and impaired trans-activation in Sp1 overexpression experiments. Conversely, Sp1 knockdown inhibited endogenous αENaC mRNA and the activity of the wild type αENaC promoter but not the mutated construct. Aldosterone triggered Sp1 recruitment to the αENaC promoter, which was required for maximal induction of αENaC promoter activity and was blocked by spironolactone. Sequential chromatin immunoprecipitation assays and functional tests of +78/+92 and +222/+229 αENaC promoter mutants indicated that while Sp1, Dot1a, and Af9 co-occupy the αENaC promoter, the Sp1 effects are functionally independent from Dot1a and Af9. In summary, Sp1 binding to a cis-element at +222/+229 represents the first identified constitutive driver of αENaC transcription, and contributes to maximal aldosterone trans-activation of αENaC.
Meprin metalloproteases are highly expressed at the luminal interface of the intestine and kidney and in certain leukocytes. Meprins cleave a variety of substrates in vitro including extracellular matrix proteins, adherens junction proteins and cytokines, and have been implicated in a number of inflammatory diseases. The linkage between results in vitro and pathogenesis however, has not been elucidated. The current study aimed to determine whether meprins are determinative factors in disrupting the barrier function of the epithelium. Active meprin A or meprin B applied to the Madin-Darby canine kidney (MDCK) cell monolayers increased permeability to fluorescein isothiocyanate (FITC)-dextran and disrupted immunostaining of the tight junction protein occludin but not claudin-4. Meprin A, but not meprin B, cleaved occludin in MDCK monolayers. Studies with recombinant occludin demonstrated meprin A cleaves the protein between Gly100 and Ser101 on the first extracellular loop. In vivo experiments demonstrated that meprin A infused into the mouse bladder increased the epithelium permeability to sodium fluorescein. Furthermore, monocytes from meprin knockout (KO) mice on the C57BL/6 background were less able to migrate through an MDCK monolayer than monocytes from wild-type (WT) counterparts. These results demonstrate the capability of meprin A to disrupt epithelial barriers and implicate occludin as one of the important targets of meprin A that may modulate inflammation.
Metabolic acidosis is a relatively common pathological condition that is defined as a decrease in blood pH and bicarbonate concentration. The renal proximal convoluted tubule responds to this condition by increasing the extraction of plasma glutamine and activating ammoniagenesis and gluconeogenesis. The combined processes increase the excretion of acid and produce bicarbonate ions that are added to the blood to partially restore acid-base homeostasis. Only a few cytosolic proteins, such as phosphoenolpyruvate carboxykinase, have been determined to play a role in the renal response to metabolic acidosis. Therefore, further analysis was performed to better characterize the response of the cytosolic proteome. Proximal convoluted tubule cells were isolated from rat kidney cortex at various times after onset of acidosis and fractionated to separate the soluble cytosolic proteins from the remainder of the cellular components. The cytosolic proteins were analyzed using two-dimensional liquid chromatography and tandem mass spectrometry. Spectral counting along with average MS/MS total ion current were used to quantify temporal changes in relative protein abundance. In all, 461 proteins were confidently identified, of which 24 exhibited statistically significant changes in abundance. To validate these techniques, several of the observed abundance changes were confirmed by western blotting. Data from the cytosolic fractions were then combined with previous proteomic data and pathway analyses were performed to identify the primary pathways that are activated or inhibited in the proximal convoluted tubule during the onset of metabolic acidosis.
We examined the effects of increased expression of proximal tubule PPARα in a mouse model of renal fibrosis. After 5 days of unilateral ureteral obstruction (UUO) PPARα expression was significantly reduced in kidney tissue of wild type mice but this downregulation was attenuated in proximal tubules of PPARα Tg mice. When compared with wild type mice subjected to UUO, PPARα Tg mice had reduced mRNA and protein expression of proximal tubule TGFβ1, with reduced production of extracellular matrix proteins including collagen 1, fibronectin, α-SMA, and reduced tubulo-interstitial fibrosis. UUO-mediated increased expression of microRNA 21 (miR21) in kidney tissue was also reduced in PPARα Tg mice. Over-expression of PPARα in cultured proximal tubular cells by adenoviral transduction reduced aristolochic acid(AA)-mediated increased production of TGFβ, demonstrating PPARα signaling reduces epithelial TGFβ production. Flow cytometry studies of dissociated whole kidneys demonstrated reduced macrophage infiltration to kidney tissue in PPARα Tg mice after UUO. Increased expression of pro-inflammatory cytokines including IL1-β, IL-6, and TNF-α in wild type mice was also significantly reduced in kidney tissue of PPARα Tg mice. In contrast, the expression of anti-inflammatory cytokines IL-10 and Arginase-1 was significantly increased in kidney tissue of PPARα Tg mice when compared with wild type mice subjected to UUO. Our studies demonstrate several mechanisms by which preserved expression of proximal tubule PPARα reduces tubulo-interstitial fibrosis and inflammation associated with obstructive uropathy.
Renal ischemia/reperfusion (IR) is associated with activation of the coagulation system and accumulation of blood clotting factors in the kidney. The aim of the present study was to examine the functional impact of Fibrinogen on renal inflammation, damage and repair in the context of IR injury. In this study, we found that IR was associated with a significant increase in renal deposition of circulating Fibrinogen. In parallel, IR-stress induced de novo expression of Fibrinogen in tubular epithelial cells as reflected by RT-PCR, immunofluorescence and in situ hybridization. In vitro, Fibrinogen expression was induced by Oncostatin-M and Hyper-IL-6 in primary tubular epithelial cells and Fibrinogen containing medium had an inhibitory effect on tubular epithelial cell adhesion and migration. Fibrinogen+/- mice showed similar survival as wild-type mice but better preservation in early postischemic renal function. In Fibrinogen-/- mice renal function and survival were significantly worse than in Fibrinogen+/- mice. Renal transplant experiments revealed reduced expression of tubular damage markers and attenuated proinflammatory cytokine expression but increased inflammatory cell infiltrates and TGF-β expression in Fibrinogen-/- isografts. These data point to heterogeneous effects of Fibrinogen in renal IR injury. While a complete lack of Fibrinogen may be detrimental, partial reduction of Fibrinogen in heterozygous mice can improve renal function and overall outcome.
Salt reabsorption is the major energy-requiring process in the kidney and AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism. Mice with a targeted deletion of the β1 subunit of AMPK (AMPK β1-/- mice) had significantly increased urinary sodium excretion on a normal salt diet. This was associated with reduced expression of the β subunit of the epithelial sodium channel (ENaC) and increased sub-apical tubular expression of the kidney-specific Na-K-2Cl co-transporter NKCC2. AMPK β1-/- mice fed a salt-deficient diet were able to conserve sodium but renin secretion increased 180% compared with controls. Cox-2 mRNA also increased in the kidney cortex, indicating greater signaling through the macula densa tubular salt-sensing pathway. To determine whether the increase in renin secretion was due to a change in regulation of fatty acid metabolism by AMPK, mice with a mutation of the inhibitory AMPK phosphosite in acetyl-CoA carboxylase 1 (ACC1-KIS79A mice) were examined. ACC1-KIS79A mice on a normal-salt diet had no increase in salt loss or renin secretion, and expression of NKCC2, NCC and βENaC were similar to those in control mice. When placed on a salt-deficient diet, however, renin secretion and cortical expression of Cox-2 mRNA increased significantly in the ACC1-KIS79A mice compared to controls. In summary, our data suggest that renin synthesis and secretion are regulated by AMPK and coupled to metabolism by phosphorylation of ACC1.
The classical role of the Na,K-ATPase is that of a primary active transporter that utilizes cell energy to establish and maintain transmembrane Na+ and K+ gradients to preserve cell osmotic stability, support cell excitability and drive secondary active transport. Recent studies have revealed that Na,K-ATPase located within cholesterol-containing lipid rafts serves as a receptor for cardiotonic steroids, including ouabain. Traditionally, ouabain was viewed as a toxin produced only in plants and it was used in relatively high concentrations to experimentally block the pumping action of the Na/K-ATPase. However, the new and unexpected role of the Na,K-ATPase as a signal transducer revealed a novel facet for ouabain in the regulation of a myriad of cell functions, including cell proliferation, hypertrophy, apoptosis, mobility and metabolism. The seminal discovery that ouabain is endogenously produced in mammals and circulates in plasma has fueled the interest in this endogenous molecule as a potentially important hormone in normal physiology and disease. In this article, we review the role of the Na/K-ATPase as an ion transporter in the kidney, the experimental evidence for ouabain as a circulating hormone, the function of the Na,K-ATPase as a signal transducer that mediates ouabain's effects and novel results for ouabain-induced Na,K-ATPase signaling in cystogenesis of autosomal dominant polycystic kidney disease (ADPKD).
We developed a mathematical model of calcium (Ca2+) transport along the rat nephron to investigate the factors that promote hypercalciuria. The model is an extension of the flat medullary model of Hervy and Thomas (Am J Physiol Renal Physiol 284: F65-F81, 2003). It explicitly represents all the nephron segments beyond the proximal tubule, and distinguishes between superficial and deep nephrons. It solves dynamic conservation equations to determine NaCl, urea, and Ca2+ concentration profiles in tubules, vasa recta, and interstitium. Calcium is known to be reabsorbed passively in the thick ascending limb, and actively in the distal convoluted (DCT) and connecting (CNT) tubules. Our model predicts that the passive diffusion of Ca2+ from the vasa recta and loops of Henle generates a significant axial Ca2+ concentration gradient in the medullary interstitium. In the base case, the urinary Ca2+ concentration and fractional excretion are predicted as 2.7 mM and 0.32 % respectively. Urinary Ca2+ excretion is found to be strongly modulated by water and NaCl reabsorption along the nephron. Our simulations also suggest that Ca2+ molar flow and concentration profiles differ significantly between superficial and deep nephrons, such that the latter deliver less Ca2+ to the collecting duct. Finally, our results suggest that the DCT and CNT can act to counteract upstream variations in Ca2+ transport, but not always sufficiently to prevent hypercalciuria.
Hypertension is a leading cause of morbidity and mortality worldwide and disordered sodium balance has long been implicated in its pathogenesis. Aldosterone is perhaps the key regulator of sodium balance and thus blood pressure. The sodium chloride cotransporter (NCC) in the distal convoluted tubule of the kidney is a major site of sodium reabsorption and plays a key role in blood pressure regulation. Chronic exposure to aldosterone increases NCC protein expression and function. However, more acute effects of aldosterone on NCC are unknown. In our salt-abundant modern society where chronic salt deprivation is rare, understanding the acute effects of aldosterone is critical. Here, we examined the acute effects (12-36 hours) of aldosterone on NCC in rodent kidney and in a mouse distal convoluted tubule cell line. Studies demonstrated that aldosterone acutely stimulated NCC activity and phosphorylation without affecting total NCC abundance or surface expression. STE20/SPS-1 related proline/alanine rich kinase (SPAK) phosphorylation also increased and gene silencing of SPAK eliminated the effect of aldosterone on NCC activity. Aldosterone administration via minipump in adrenalectomized rodents confirmed an increase in NCC phosphorylation without a change in NCC total protein. These data indicate that acute aldosterone-induced SPAK-dependent phosphorylation of NCC increases individual transporter activity.
Adriamycin (ADR) administration in susceptible rodents such as BALB/c mouse strain produces injury to the glomerulus mimicking human focal glomerular sclerosis (FSGS). The goal of the present study was to use this model to investigate antiproteinuric action of nitro-oleic acid (OA-NO2), a nitric oxide-derived endogenous lipid product, which has exhibited multiple attractive signaling properties particularly in the kidney. BABL/c mice were pretreated for 2 days with OA-NO2 at 5 mg/kg/day via osmotic mini-pump, followed by a single injection of vehicle or adriamycin (10 mg/kg) via tail vein. Albuminuria and renal function were analyzed at 1 wk post ADR treatment. ADR mice developed prominent albuminuria, hypoalbuminemia, hyperlipidemia and severe ascites. In contrast, the symptoms of nephrotic syndrome were greatly improved by OA-NO2 treatment. In parallel, plasma creatinine and plasma urea nitrogen were elevated in ADR group and the severity was less in the ADR+OA-NO2 group. OA-NO2 attenuates ADR-induced glomerulosclerosis, podocyte loss, and tubulointerstitial fibrosis. Indices of oxidative stress including plasma and urinary TBARS and renal expression of NAD(P)H oxidase p47phox and gp91phox, and inflammation including renal expression of TNF-α, IL-1β and MCP-1 in response to ADR were all similarly suppressed. Together, these findings suggest that OA-NO2 exerts renoprotective action against ADR nephropathy likely via its anti-inflammatory and anti-oxidant properties.
Recent studies from our laboratory showed that aldosterone plays a central role in the development of functional and structural injury induced by cyclosporine. These findings, however, do not allow us to establish if mineralocorticoid receptor antagonism could also be a helpful strategy to prevent chronic tacrolimus nephrotoxicity. This study was designed to evaluate if aldosterone participates in the renal injury induced by tacrolimus. For this purpose, four groups of male Wistar rats fed a low-sodium diet were studied: a vehicle group (V), spironolactone-treated rats (Sp 20 mg/kg), tacrolimus-treated rats (2 mg/kg) and rats treated simultaneously with tacrolimus+Sp for 28 days. At the end of the experimental period, mean arterial pressure , glomerular filtration rate (GFR) and renal blood flow (RBF) were measured. Tubulo-interstitial fibrosis (TIF), arteriolopathy percentage and arteriolar thickening were also quantified. Protein levels of TGF-β, phosho-Smad-3, Rho kinase (Rhok), Twist, and α-SMA were determined. In addition, endothelin and its receptor mRNA levels were quantified. Tacrolimus produced chronic nephrotoxicity characterized by significant decreases in GFR and RBF and significant increases in TIF, arteriolopathy, and arteriolar thickening. These alterations were associated with greater TGF-β protein and activity, as evidenced by a greater phosphorylation of Smad-3, which together with Rhok and Twist elevation promoted a greater epithelial-to-mesenchymal transition (EMT). In contrast, MR antagonism prevented TIF and reduced arteriolopathy, hypoperfusion and hypofiltration. The renoprotection conferred by Sp was associated with restoration of TGF-β and prevention of EMT.