Cell junction proteins in podocyte injury repair

描述（由申请方提供）：成年肾脏足细胞的主要细胞-细胞连接是狭缝隔膜，是肾脏滤过屏障的重要组成部分。足突消失期间缝隙隔膜的丧失会导致蛋白尿，并导致许多慢性肾脏疾病（例如糖尿病和艾滋病毒相关肾病）的进展。足细胞连接的发育形成和成体稳态是尚未完全理解的过程，但对于开发治疗策略以修复或替换在疾病中受损或丢失的足细胞是重要的。我们最近已经确定了封闭蛋白样细胞连接蛋白TM 4SF 10和Fyn结合蛋白ADAP作为足细胞表达的蛋白质，影响板状伪足的形成，Fyn活性和Nephrin磷酸化。TM 4SF 10在足细胞发育期间瞬时表达，并在损伤修复期间重新表达。因此，我们推测，在疾病中，TM 4SF 10可以通过保留简化的细胞连接和通过修改Fyn的活动来保护足突消失期间的足细胞。我们还假设，ADAP，一个大的适配器蛋白与已知的作用，在整合素信号，功能作为一个脚手架中心的足细胞足过程整合肌动蛋白细胞骨架动力学在两个狭缝隔膜和整合素附件的足过程。我们将通过使用标准突变分析和体外结合试验确定ADAP与TM 4SF 10和狭缝隔膜复合物的其他关键细胞内组分的结合来测试这一假设，并基于板状伪足延伸、整合素结合和肾蛋白磷酸化来评估功能效应。拟定的研究还将包括进一步表征ADAP敲除小鼠的肾脏表型，以及进一步开发和表征TM 4SF 10敲除小鼠模型。将建立来自这些小鼠的足细胞系，以研究足细胞中TM 4SF 10和ADAP在调节整联蛋白细胞附着和Nephrin磷酸化方面的必要功能。此外，我们已经建立了TM 4SF 10的功能，直接抑制Fyn的活动，可能是通过螯合Fyn远离质膜。这将用真实的时间成像和共定位方法进行研究，在存在和不存在TM 4SF 10的情况下评估Fyn的细胞内运输。这些研究描述了第一个整合的分子机制，连接细胞-细胞粘附在狭缝隔膜与细胞-基质粘附与整联蛋白结合在足突，事件需要同步足突形成和收缩。这些研究将对开发针对以足突消失为特征的肾小球疾病的新疗法具有重要意义，其中预防或修复受损的足细胞将是有益的。 公共卫生相关性：治疗损害肾脏过滤装置（肾小球）的疾病需要开发鼓励新肾细胞再生的策略。这需要对肾脏的特化细胞如何更新和形成其独特结构的基本理解。在肾小球修复过程中重要的是足细胞之间如何建立和维持细胞-细胞连接，因为这些细胞连接是血液过滤装置的关键组成部分。这些研究将提供关于足细胞在发育过程中如何形成以及它如何修复自身以应对疾病造成的损伤的重要信息。

DESCRIPTION (provided by applicant): The major cell-cell junction of the adult kidney podocyte is the slit diaphragm, an important component of the kidney filtration barrier. The loss of the slit diaphragm during foot process effacement results in proteinuria and contributes to the progression of many chronic kidney diseases such as diabetes and HIV-associated nephropathy. The developmental formation and adult homeostasis of podocyte cell junctions are processes that are not fully understood, but are important for the development of therapeutic strategies to repair or replace podocytes that are damage or lost in disease. We have recently identified the claudin-like cell junction protein TM4SF10 and the Fyn binding protein ADAP as podocyte-expressed proteins that affect lamellipodia formation, Fyn activity, and Nephrin phosphorylation. TM4SF10 is transiently expressed during podocyte development and is re-expressed during injury repair. Thus, we hypothesize that in disease, TM4SF10 may function to protect the podocyte during foot process effacement by preserving a simplified cell junction and by modifying the activities of Fyn. We also hypothesize that ADAP, a large adapter protein with known roles in integrin signaling, functions as a scaffolding center in the podocyte foot process integrating actin cytoskeleton dynamics at both the slit diaphragm and in integrin attachments of the foot process. We will test this hypothesis by determining the association of ADAP binding to TM4SF10 and other key intracellular components of the slit diaphragm complex using standard mutational analysis and in vitro binding assays, and assess functional effects based on lamellipodia extension, integrin binding, and Nephrin phosphorylation. Proposed studies also will include further characterize of the renal phenotype in ADAP knockout mice and further development and characterization of a knockout mouse model for TM4SF10. Podocyte cells lines from these mice will be established to investigate the requisite functions of TM4SF10 and ADAP in podocytes with regard to regulating integrin cell attachments and Nephrin phosphorylation. In addition, we have established TM4SF10 functions to directly suppress Fyn activity, possibly by sequestering Fyn away from the plasma membrane. This will be investigated with real time imaging and co-localization methods assessing intracellular trafficking of Fyn in the presence and absence of TM4SF10. These studies describe the first integrated molecular mechanism connecting cell-cell adhesion at the slit diaphragm with cell-matrix adhesion with integrin binding in the foot process, events that require synchronization during foot process formation and retraction. These studies will have important implications toward the development of novel therapies directed at glomerular diseases characterized by foot processes effacement where preventing or repairing damaged podocytes would be beneficial. PUBLIC HEALTH RELEVANCE: Lay Summary to treat diseases that damage the kidney's filtering apparatus (glomerulus) requires the development of strategies that encourage regeneration of new kidney cells. This requires a fundamental understanding of how the specialized cells of the kidney renew and form their unique structure. Important in the glomerulus repair process is how cell-cell junctions are established and maintained between podocytes, as these cell junctions are a key component of the blood filtering apparatus. These studies will provide important information on how the podocyte forms during development, and how it repairs itself in response to damage caused by disease.