Activated Protein C Controls p66shc: A Novel Defense Mechanism in Diabetic Nephropathy

Previously it has been shown that activated protein C (APC) protects against diabetic nephropathy (DN). APC's cytoprotective effect in DN may be related to its in vitro ability to downregulate reactive oxygen species (ROS) and maintain mitochondrial membrane potential in glucose stressed cells. Cellular targets, through which APC, an extracellular serine protease, regulates mitochondrial function, are unknown. Since mice lacking the ROS-inducing mitochondrial protein p66shc are protected from DN we hypothesized that APC's cytoprotective effect may be mechanistically linked to p66shc.

Immortalized mouse podocytes and murine endothelial cells were stressed in vitro with glucose (30 mM). In mice persistent hyperglycaemia was induced by streptozotocin injections. Markers of DN were analysed in wild-type mice, mice expressing a thrombomodulin (TM) mutant lacking cofactor activity towards protein C activation (TMPro/Pro), mice expressing high levels of APC (hPChigh), and p66−/− x TMPro/Pro double mutant mice. Immunoblotting, RT-PCR, cellular subfractionation, immunohistochemistry and immunofluorescensce were employed for analyses.

8-OH-Desoxyguanosine, a marker for ROS, is induced in diabetic mice. Nitrotyrosine, a marker for ROS-dependent damage, localizes mainly to podocytes. This is paralleled by p66shc-upregulation in glomerular podocytes. These effects are aggravated in diabetic TMPro/Pro, ameliorated in aPChigh, and reversed in p66shc x TMPro/Pro mice. In vitro APC counteracts glucose dependent upregulation of p66shc and inhibits mitochondrial translocation of p66shc in mouse podocytes, but not in endothelial cells in vitro.

This study identifies a novel mechanism through which APC mediates cytoprotection. In glucose stressed podocytes, but not endothelial cells, APC prevents mitochondrial translocation of the ROS-inducing redox-protein p66shc, which is required for protection against DN in vivo.

No relevant conflicts of interest to declare.