Activated Protein C Targets PI3K-p85/XBP1 Pathway to Inhibit Hyperglycemia Induced Endoplasmic Reticulum Stress in Diabetic Nephropathy

Abstract 3354

Diabetic nephropathy (DN) is a multifactorial disease associated with substantial changes in the haemostatic system. A hallmark of diabetes induced haemostatic dysfunction is impaired thrombomodulin (TM) dependent-protein C (PC) activation. Impaired PC activation triggers glomerular podocyte and endothelial cell dysfunction, thus promoting DN. The intracellular mechanism through which loss of TM and PC activation contributes to DN is not known. Here we show that the haemostatic mediator activated PC (aPC) regulates cellular homeostasis by inhibiting hyperglycemia induced endoplasmic reticulum (ER)-stress in DN.

Hyperglycaemia was induced in wild-type (wt) mice or mice with altered activity of the TM-PC system (loss of function secondary to impaired PC activation (TM^Pro/LacZ) or gain of function with high aPC plasma levels (APC^high)). Subsets of diabetic mice were treated with the chemical ER-chaperone TUDCA. After 26 weeks of persistent hyperglycaemia markers of DN were determined and tissue samples were isolated for ex vivo analysis. Supplementary in vitro assays were performed in podocytes and endothelial cells.

Persistent hyperglycaemia in wt mice caused severe ER-stress and DN. Ex vivo analysis of transcription factors regulating the ER-stress response showed an increase of the ER-stress markers CHOP and ATF6, while nuclear translocation of the highly conserved transcription factor X-box binding protein-1 (XBP1) was reduced in DN. These changes were aggravated in diabetic TM^Pro/LacZ mice. Conversely, in a mouse model with constitutively higher aPC levels (APC^high mice) nuclear levels of XBP1 were normalized and expression of ATF6 and CHOP was reduced despite persistent hyperglycaemia. In addition, aPC reverses the pathological ER-stress alterations in diabetic TM^Pro/LacZ mice. Pharmacological inhibition of ER-stress by TUDCA normalized nuclear levels of XBP1, inhibited CHOP/ATF6 expression, and protected against DN in diabetic wt and TM^Pro/LacZ mice. In vitro hyperglycaemia inhibited nuclear translocation of XBP1 in endothelial cells and podocytes, the two cellular components of glomerular filtration barrier. Activated PC directly promotes the nuclear translocation of XBP1 in these cells, which is required to inhibit hyperglycaemia induced ER-stress. Deletion of XBP1 in podocyte or endothelial cells abolished the cytoprotective effect of aPC. Furthermore, aPC regulates the interaction of PI3K regulatory subunit p85α with XBP1 which is known to mediate its nuclear translocation and diminished hyperglycaemia induced ER-stress. These studies demonstrate that hyperglycemia induced ER-stress is causally linked to DN and establish a novel link between haemostatic system and ER function in regulating cellular homeostasis in chronic kidney disease.