Coagulation Protease Signaling in Glomerular Disease

 描述(申请人提供)：迫切需要确定分子信号通路，这些信号通路是凝血酶介导的足细胞损伤的关键调节因子，如果没有这些通路，针对肾病综合征的靶向、安全和有效的治疗方法的开发可能仍然有限。这项建议的总体目标是发展一名止血内科医生兼科学家的职业生涯，他将学习应用凝血生物学、分子生物学、分子药理学和系统生物学技术来确定凝血酶介导的肾小球损伤的分子机制。中心假说是，在蛋白尿期间，足细胞暴露于凝血酶，凝血酶通过激活蛋白酶激活受体(PAR)来加重足细胞损伤。利用已建立的足细胞培养系，将通过抗PAR抗体阻断、shRNA敲除和邻近连接实验来确定对凝血酶介导的细胞凋亡诱导至关重要的PAR种类。补充抗凝血酶，一种非特异性凝血酶抑制物，已知可以减少实验性肾病模型的蛋白尿。因此，抗凝血酶改变蛋白尿的作用机制将通过在每个分子水平上对凝血酶信号轴进行药理操作并检查每种情况对蛋白尿测量的肾小球损伤的影响来确定。利用大鼠和人类培养的足细胞，将评估基因表达谱和转录网络，以确定足细胞对凝血酶暴露的特异性转录反应。随后，这些图谱和网络将与分离的大鼠肾小球和从人类NS肾活检中分离的肾小球的现有基因表达谱相关联，以确定与人类肾病综合征相关的凝血酶依赖足细胞转录反应，并同时为选择这些过程的适当临床前模型提供信息。将采用优先策略来指导对高价值候选基因及其生物学途径的进一步研究。这一职业发展奖直接响应了国家糖尿病、消化和肾脏疾病研究所(NIDDK)的使命，即“支持肾脏、泌尿外科和血液疾病的医学研究和研究培训”。完成这一职业发展奖的培训计划和研究，预计将产生令人信服的初步数据，使获奖者能够成功竞争R01资金，以便系统地分析肾小球疾病中的凝血信号。

 DESCRIPTION (provided by applicant): There is a critical need to define the molecular signaling pathways that are the crucial regulators of thrombin- mediated podocyte injury without which, the development of targeted, safe and effective therapies for nephrotic syndrome is likely to remain limited. The overall objective of this proposal is to develop the career of a hemostasis physician-scientist who will learn to apply coagulation biology, molecular biology, molecular pharmacology, and systems biology techniques to define the molecular mechanisms of thrombin-mediated glomerular injury. The central hypothesis is that, during proteinuria, podocytes are exposed to thrombin which exacerbates podocyte injury via activation of protease activated receptors (PAR). Using established podocyte cell culture lines, the PAR species that are essential for thrombin-mediated apoptosis induction will be determined through anti-PAR antibody blockade, shRNA knock-down, and proximity ligation experiments. Supplementation with antithrombin, a non-specific thrombin inhibitor, is known to attenuate proteinuria in experimental nephrosis models. Thus, the mechanism of action by which antithrombin modifies proteinuria will be determined by pharmacologically manipulating the thrombin signaling axis at each molecular level and examining the impact of each condition on glomerular injury as measured by proteinuria. Using rat and human cultured podocytes, gene expression profiles and transcriptional networks will be evaluated to determine podocyte-specific transcriptional responses to thrombin exposure. Subsequently, these profiles and networks will be correlated with those of isolated rat glomeruli and existing gene expression profiles of glomeruli isolated from human NS renal biopsies to determine thrombin-dependent podocyte transcriptional responses that are relevant to human nephrotic syndrome and simultaneously inform the selection of appropriate preclinical models of these processes. A prioritized strategy will be employed to guide further investigation of high-value candidate genes and their biological pathways. This career development award is directly responsive to the mission of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) which is to "support medical research and research training" on "kidney, urologic, and hematologic diseases." Completion of the training program and research in this career development award is expected generate compelling preliminary data for the awardee to successfully compete for R01 funding to systematically analyze coagulation signaling in glomerular diseases.