DESCRIPTION (provided by applicant): Project Summary Diabetic kidney disease (DKD) is the common cause of kidney failure. Elucidating mechanisms that mediate the early stage of DKD may help to identify novel preventive and therapeutic measures for patients with DKD. In the previous funding period, we demonstrated a critical role of NF-κB and STAT3 acetylation in DKD through the regulation of inflammatory gene expression (see progress report). To further understand the mechanism of early DKD injury, we performed proteomic studies in the glomeruli of streptozotocin (STZ)-induced diabetic rats and identified protein S (PS) as one of the proteins which were highly regulated in early DKD. PS, coded by Pros1, is a cofactor for the formation of activated protein C (APC). APC formation has been shown to protect glomerular cells from apoptosis in DKD. In addition, PS shares structural similarities with GAS6, which is known to be involved in the pathogenesis of kidney disease. Both PS and GAS6 bind to TAM receptors (Tyro3, Axl, and Mer), which belong to a family of receptor tyrosine kinases that mediates regulation of inflammation as well as cell survival and migration. However, PS and GAS6 have different binding affinity to individual TAM receptors and appear to have different functions. The role of PS and TAM receptors in kidney disease is not known. Our preliminary data suggest that PS expression increased in the glomeruli of diabetic rats and in human kidneys with early DKD, but decreased in those with advanced DKD. Plasma concentrations of PS were not different between diabetic and non-diabetic rats or humans, suggesting a local regulation of PS expression. We found that knockdown of PS in podocytes enhanced high glucose-induced apoptosis while overexpression of PS inhibited TNF-α-induced pro-inflammatory gene expression, suggesting a protective role of PS against diabetes-induced podocyte injury. We found that Tyro3, one of the TMA receptors, was upregulated by high glucose and knockdown of Tyro3 expression abolished the protective effects of PS in podocytes. Tyro3 expression is also upregulated in human glomeruli at early DKD but downregulated at late DKD. We developed podocyte-specific Pros1 knockout mice (KO) and found that diabetic KO mice developed more proteinuria, mesangial expansion, and foot process effacement than diabetic wild-type mice. Based on these findings, we hypothesize that PS may protect glomerular cell injury in early DKD through the formation of APC and/or effects mediated by Tyro3. We will test our hypothesis by two aims: In the aim 1, we will determine the factors which upregulate PS and Tyro3 at early DKD and those suppress PS and Tyro3 at late DKD and the effects of PS and Tyro3 in podocytes cultured in diabetic condition. In the aim 2 we will determine the role of PS and Tyro3 in DKD progression in vivo using both conditional overexpression and knockout mouse models for PS and Tyro3. We believe that the propose...