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.