Slit Diaphragm and Actin Dynamics

描述（由申请人提供）：本项目的广泛目标是帮助理解控制足细胞细胞骨架动力学的细胞和分子机制，期望理解这些过程将揭示潜在的疾病机制和治疗靶点。我们一直受到这样一个假设的指导，即在正常的足细胞成熟过程中，在维持分化的足细胞表型，并确定足细胞的行为，以应对肾小球疾病，精确调节细胞骨架动力学是必不可少的。 虽然十年的工作已经使这一假说教条，足细胞细胞骨架动力学的机制的详细了解仍然不完整，基于这些机制的治疗目标并不存在。由于整合足细胞间连接和足突肌动蛋白细胞骨架重塑几乎总是遇到肾小球疾病，我们一直专注于研究细胞间连接Nephrin-Neph 1-Podocin复合物的功能，因为突变或损失的任何一个这些受体成分引起蛋白尿和足细胞骨架重塑。 目前的建议是基于我们最近的观察，足细胞足突扩展的机制，平行的培养细胞诱导板状突起。Nephrin连接通过黏着斑激酶-p130 Cas-Crk 1/2依赖性途径诱导板状伪足活化 在小鼠中，足细胞特异性Crk 1/2缺失阻断损伤诱导的足突消失。 相对于正常组织，FAK和Cas的磷酸化在患有人类微小病变疾病的患者和膜性肾病中的足细胞中被诱导。 这些结果提供了令人信服的初步证据表明，Crk依赖性信号转导代表了一个治疗靶点，可能有助于阻断人类肾小球疾病中的足突消失。鉴于这些结果和额外的初步数据表明，Crk依赖性信号传导在足突扩散中是必要的分子机制，该项目将解决Nephrin-FAK-Cas-Crk信号传导对于足突消失是必要的并且可以在急性和慢性肾小球疾病过程中靶向的假设。提出了两个具体目标。 在目标1中，我们将详细研究这样的假设：肾蛋白信号传导和功能需要内吞和内吞再循环以及来自肾蛋白信号内体的后续信号传导，这一过程需要Crk 1/2。在已完成的工作中，我们已经证明，在急性小鼠肾小球疾病模型中，靶向Crk可减弱肾小球疾病表型。 在目标2中，我们将通过测试靶向Crk依赖性信号转导减弱与慢性肾小球疾病模型相关的疾病表型的假设来扩展该临床前工作，所述慢性肾小球疾病模型更接近地模拟进行性人类慢性肾小球病。

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.