Slit Diaphragm and Actin Dynamics

描述（由申请人提供）：本项目的总体目标是有助于理解控制足细胞细胞骨架动力学的细胞和分子机制，并期望了解这些过程将揭示潜在的疾病机制和治疗靶点。我们的假设是，在正常足细胞成熟过程中，在维持分化的足细胞表型中，以及在确定足细胞对肾小球疾病的反应行为中，精确调节的细胞骨架动力学是必不可少的。虽然十年的工作使这一假设成为教条，但对足细胞细胞骨架动力学的详细理解仍然不完整，基于这些机制的治疗靶点也不存在。由于整合的足细胞细胞间连接和足突肌动蛋白细胞骨架重塑几乎总是在肾小球疾病中遇到，我们重点研究了细胞间连接Nephrin-Neph1-Podocin复合物的功能，因为这些受体成分中的任何一种突变或丢失都会导致蛋白尿和足细胞细胞骨架重塑。目前的建议是基于我们最近的观察，足细胞足突的扩散是由类似于培养细胞诱导板足突的机制调节的。通过局灶黏附激酶- p130cas - crk1 /2依赖途径，Nephrin连接诱导板足细胞激活。在小鼠中，足细胞特异性缺失Crk1/2可阻断损伤诱导的足突消退。相对于正常组织，FAK和Cas的磷酸化在人类微小变化疾病患者和膜性肾病患者的足细胞中被诱导。这些结果提供了令人信服的初步证据，表明crk依赖性信号代表了一个治疗靶点，可能有助于阻断人类肾小球疾病的足突消退。鉴于这些结果和额外的初步数据表明，crk依赖性信号通路在足过程扩散中是必要的分子机制，本项目将解决Nephrin-FAK-Cas-Crk信号通路是足过程消退所必需的假设，并且可以在急性和慢性肾小球疾病过程中作为靶点。提出了两个具体目标。在目的1中，我们将从机制上详细研究一个假设，即Nephrin信号传导和功能需要内吞作用和内吞循环，以及随后来自Nephrin信号传导内体的信号传导，这一过程需要Crk1/2。在已完成的工作中，我们已经证明靶向Crk可以减轻急性小鼠肾小球疾病模型中的肾小球疾病表型。在目标2中，我们将扩展临床前工作，通过验证靶向crk依赖性信号通路减弱与慢性肾小球疾病模型相关的疾病表型的假设，该模型更接近于模拟进行性人类慢性肾小球病变。

DESCRIPTION (provided by applicant): The broad goal of this project is to contribute to understanding the cellular and molecular mechanisms that govern podocyte cytoskeletal dynamics with the expectation that understanding these processes will expose potential disease mechanisms and therapeutic targets. We have been guided by the hypothesis that precisely regulated cytoskeletal dynamics are essential during normal podocyte maturation, in maintenance of the differentiated podocyte phenotype, and in determining podocyte behavior in response to glomerular disease. While a decade of work has made this hypothesis dogma, a detailed understanding of mechanisms that govern podocyte cytoskeletal dynamics remains incomplete and therapeutic targets based on these mechanisms do not exist. Because integrated podocyte intercellular junction and foot process actin cytoskeletal remodeling are nearly always encountered in glomerular disease, we have focused on investigating the functions of the intercellular junction Nephrin-Neph1-Podocin complex because mutation or loss of any one of these receptor components causes proteinuria and podocyte cytoskeletal remodeling. The present proposal is based on our recent observations that podocyte foot process spreading is regulated by mechanisms that parallel those used by cultured cells to induce lamellipodial protrusion. Nephrin ligation induces lamellipodial activation by a focal adhesion kinase-p130Cas-Crk1/2-dependent pathway. In mice, podocyte-specific deletion of Crk1/2 blocks injury-induced foot process effacement. Phosphorylation of FAK and Cas are induced in podocytes of patients with human minimal change disease and in membranous nephropathy relative to normal tissue. These results provide compelling initial evidence that Crk-dependent signaling represents a therapeutic target that might be useful in blocking foot process effacement in human glomerular disease. Given these results and additional preliminary data suggesting a molecular mechanism by which Crk-dependent signaling is necessary in foot process spreading, this project will address the hypothesis that Nephrin-FAK-Cas-Crk signaling is necessary for foot process effacement and can be targeted in both acute and chronic glomerular disease processes. Two specific aims are proposed. In aim 1, we will examine in mechanistic detail the hypothesis that Nephrin signaling and function requires endocytosis and endocytic recycling and subsequent signaling from a Nephrin signaling endosome, a process that requires Crk1/2. In completed work, we have demonstrated that targeting Crk attenuates the glomerular disease phenotype in acute murine glomerular disease models. In aim 2, we will extend this pre-clinical work by testing the hypothesis that targeting Crk-dependent signaling attenuates the disease phenotype associated with chronic glomerular disease models that more closely mimic progressive human chronic glomerulopathy.