PROJECT SUMMARY/ABSTRACT Nephrotic syndrome is a leading cause of end stage kidney disease, which is the eighth leading cause of death in the United States. Thrombin injures podocytes and its inhibition reduces nephrotic-range proteinuria and podocyte injury, two key drivers of nephrotic syndrome progression toward end stage kidney disease. Thus, there is a critical need to discern the molecular mechanisms underlying thrombin-mediated podocyte injury without which, the development of targeted, safe, and effective therapies that slow or halt nephrotic syndrome progression is likely to remain limited. The overall objective of this proposal is to define molecular mechanisms underlying thrombin-dependent, protease-activated receptor-mediated podocyte injury and determine if inhibition of this signaling pathway reduces progression toward end stage kidney disease. The central hypothesis is that thrombin-mediated protease-activated receptor signaling is a modifiable driver of RhoA-dependent podocyte injury during nephrotic syndrome progression. This project will integrate methods from the podocyte and coagulation biology fields including: mouse and rat nephrotic syndrome models, coagulation factor knockdown and conditional protease-activated receptor knockout mice, innovative nanoparticle-mRNA overexpression of coagulation factors, repurposing of Food and Drug Administration approved direct oral anticoagulants to mitigate podocyte injury, a novel flow cytometry approach to quantitate podocyte injury, and molecular biology methods including bimolecular fluorescence complementation and bioluminescence resonance energy transfer in genetically modified podocyte cultures. These complementary methods will be used to investigate the pathophysiologic role of thrombin in podocyte injury and nephrotic syndrome progression toward end stage kidney disease. Our Aims are designed to (1) Reveal the prothrombinase that produces intraglomerular, podocytopathic thrombin to drive podocyte injury and nephrotic syndrome progression, (2) Test the ability of direct oral anticoagulant therapy as a novel method to reduce nephrotic syndrome progression, and (3) Discover the molecular mechanisms by which thrombin-mediated protease-activated receptor signaling stimulates RhoA- dependent podocyte injury. This project is directly responsive to the mission of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) which is to “…support medical research…on kidney…and hematologic diseases, to improve people’s health and quality of life.” In addition, this project directly addresses important research priorities described in the Kidney Research National Dialogue and key aspects of the Healthy People 2030 Chronic Kidney Disease objectives. Completion of the proposed project is expected to establish the mechanisms underlying thrombin-mediated podocyte injury and enable exploitation of existing, Food and Drug Administration approved, direct oral anticoagulants as novel...