Cell junction proteins in podocyte injury repair

描述(申请人提供)：成人肾足细胞的主要细胞-细胞连接是裂隙横隔膜，它是肾滤过屏障的重要组成部分。在足突消失过程中，裂隙横隔膜的丢失会导致蛋白尿，并导致许多慢性肾脏疾病的进展，如糖尿病和HIV相关性肾病。足细胞连接的发育形成和成体内稳态是尚未完全了解的过程，但对于制定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。我们最近发现了Claudin样细胞连接蛋白TM4SF10和Fyn结合蛋白ADAP是足细胞表达的影响板脂形成、Fyn活性和Nephin磷酸化的蛋白质。TM4SF10在足细胞发育过程中瞬时表达，在损伤修复过程中重新表达。因此，我们推测，在疾病中，TM4SF10可能通过保留简化的细胞连接和改变Fyn的活性来保护足突消失过程中的足细胞。我们还假设，ADAP是一种在整合素信号转导中具有已知作用的大型适配蛋白，在足细胞足突中发挥支架中心的作用，整合足突缝隙和足突整合素附着处的肌动蛋白细胞骨架动力学。我们将通过使用标准突变分析和体外结合分析来确定ADAP与TM4SF10和缝隙隔膜复合体的其他关键细胞内成分的结合来验证这一假设，并基于片层脂膜延伸、整合素结合和NePhin磷酸化来评估功能效应。拟议的研究还将包括进一步鉴定ADAP基因敲除小鼠的肾脏表型，以及进一步开发和鉴定TM4SF10基因敲除小鼠模型。将建立这些小鼠的足细胞系，以研究足细胞中TM4SF10和ADAP在调节整合素细胞附着和Nephin磷酸化方面的必要功能。此外，我们已经建立了TM4SF10的功能来直接抑制Fyn的活性，可能是通过将Fyn从质膜上隔离出来。这将通过实时成像和共定位方法来研究，以评估在存在和不存在TM4SF10的情况下Fyn的细胞内运输。这些研究描述了第一个整合的分子机制，将缝隙隔膜上的细胞-细胞黏附与细胞-基质黏附与足部过程中的整合素结合联系起来，这些事件需要在足突形成和回缩过程中同步发生。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，在这种疾病中，预防或修复受损的足细胞将是有益的。

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.