Thrombin-Mediated Podocyte Injury Mechanisms

项目摘要/摘要 肾病综合征是终末期肾病的主要原因，是第八大主要死亡原因。 在美国。凝血酶损伤足细胞，其抑制作用可减少肾病综合征范围的蛋白尿和 足细胞损伤是肾病综合征进展为终末期肾病的两个关键驱动因素。因此， 迫切需要弄清凝血酶介导的足细胞损伤的分子机制。 如果没有它，靶向、安全和有效的延缓或阻止肾病综合征的治疗方法的发展 进展可能仍然有限。这项提议的总体目标是定义分子机制 潜在的凝血酶依赖的、蛋白酶激活的受体介导的足细胞损伤并确定是否抑制 这一信号通路的缺失可减少终末期肾病的进展。中心假设是 凝血酶介导的蛋白水解酶激活受体信号是RhoA依赖的足细胞的可修饰驱动因素 肾病综合征进展过程中的损伤。该项目将整合来自足细胞和 凝血生物学领域包括：小鼠和大鼠肾病综合征模型、凝血因子基因敲除 和条件性蛋白酶激活受体基因敲除小鼠，创新的纳米颗粒-mRNA过表达 凝血因子，食品和药物管理局批准的直接口服抗凝剂以缓解 足细胞损伤--一种新的定量足细胞损伤的流式细胞术方法和分子生物学方法 包括双分子荧光互补和生物发光共振能量转移 转基因足细胞培养。这些互补的方法将被用来调查 凝血酶在足细胞损伤和肾病综合征向终末期发展中的病理生理作用 肾脏疾病。我们的目标是(1)揭示产生肾小球内的凝血酶原酶， 足细胞病理性凝血酶驱动足细胞损伤和肾病综合征进展，(2)检测 直接口服抗凝治疗作为减少肾病综合征进展的新方法，以及(3)发现 凝血酶介导的蛋白酶激活受体信号刺激RhoA-A的分子机制 依赖性足细胞损伤。该项目直接响应国家糖尿病研究所的使命 和消化和肾脏疾病(NIDDK)，即“…”支持医学研究…关于肾脏…和 血液病，以改善人们的健康和生活质量。此外，这个项目直接解决了 肾脏研究国家对话中描述的重要研究重点和健康的关键方面 2030人慢性肾脏疾病目标。拟议项目完成后，预计将建立 凝血酶介导的足细胞损伤的潜在机制和对现有、食物和 美国药品监督管理局批准的直接口服抗凝剂作为延缓或阻止NS进展的新疗法。

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 therapeutics to slow or halt NS progression.