C‐peptide reverses hyperglycemia‐induced mitochondrial membrane depolarization and oxidant generation in murine renal microvascular endothelial cells

Renal endothelial cells are a prime target for hyperglycemic damage during diabetes because the glucose transport rate of these cells does not decline quickly enough, leading to intracellular hyperglycemia. As described by Brownlee, this eventually increases the voltage gradient across the mitochondrial membrane until a threshold is reached and oxidants are generated. The goal of this study was to determine the effect of C‐peptide on mitochondrial membrane potential (Δ Ψm ), as well as oxidant production in conditionally immortalized renal microvascular endothelial cells exposed to a high glucose environment. Renal microvascular endothelial cells from H‐2K b ‐tsA58 mice were exposed to high glucose (25 mM) or low glucose (5 mM) with raffinose in the absence or presence of rat C‐peptide II (10 mM) in non‐permissive conditions. After 48‐hours of exposure to the interventions, cells were incubated with JC‐1 to evaluate Δ Ψm , while the production of oxidants was determined using carboxydihydrodichlorofluorescein diacetate (H 2 DCF‐DA). Exposure of cells to 25 mM glucose for 48‐hours resulted in depolarization of Δ Ψm with significant oxidant production. Treatment of cells exposed to 25 mM glucose with rat C‐peptide II for 48‐hours caused significant hyperpolarization of Δ Ψm with no apparent oxidant generation. Together, these data demonstrate that rat C‐peptide II protects against endothelial mitochondrial dysfunction during hyperglycemia.