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.