Project Summary The podocyte has become a crucial focus of research and clinical efforts as a target for kidney disease interventions due to its vital role in regulating glomerular permeability and maintaining glomerular structure. Podocyte injury is pathogenetically and prognostically important in diabetic kidney disease (DKD). One of the main factors determining pathological changes in glomerular morphology and permeability is the elevation of basal intracellular calcium ([Ca2+]i) levels in podocytes, which can occur due to activation of various signaling cascades. Protease-activated receptors (PARs) are emerging as proteins of interest for their potential to modulate podocyte [Ca2+]i levels, especially under pathological conditions, such as DKD. Clinical studies have demonstrated that circulating concentrations of PAR-activating proteases are associated with DKD. Furthermore, the recent prospective OPTIMUS-5 study revealed several beneficial effects of the FDA-approved PAR1 antagonist Vorapaxar in type 2 diabetes mellitus. However, despite crucial evidence for the importance of PAR signaling pathways in podocytes in DKD, this area is still understudied. Our preliminary data demonstrate the functional presence of a PAR-GPCR-TRPC6 signaling pathway in rat and human podocytes that is increased under diabetic conditions. Consistent with these findings, we found that serine proteases promote activation of PAR1-TRPC6 cascade in podocytes from freshly isolated rat glomeruli, which triggers a rapid elevation of [Ca2+]i. Furthermore, our pilot studies have revealed that these signaling pathways are highly upregulated in a rat model of type 2 Diabetic Nephropathy (T2DN rats), similar to clinical observations in human patients. The central hypothesis of this proposal is that during the development of DKD in type 2 diabetes, when urinary thrombin and urokinase concentrations increase rapidly, overstimulation of PAR1 promotes excessive [Ca2+]i levels in podocytes through activation of TRPC6 channels, ultimately leading to cell apoptosis, development of albuminuria and glomerular damage. Thus, inhibition of PAR1 activity will mitigate podocyte damage and may be of therapeutic benefit in DKD. Several innovative approaches and unique rat models will be utilized to test the following Specific Aims: Aim 1 will test the hypothesis that PAR1 expression and its activity increase during the progression of DKD in type 2 diabetes and that this pathway contributes to the alterations in Ca2+ homeostasis in podocytes and glomerular damage; and Aim 2 will provide mechanistic insight into the activation of PAR-1 mediated signaling in podocytes and associated glomerular structure and function changes. In addition, the correlation of PAR signaling in podocytes and sex difference in the development of DKD in T2DN rats will be explored.