Cell junction proteins in podocyte injury repair

足细胞损伤修复中的细胞连接蛋白

• 批准号: 8342329
• 负责人: Leslie A Bruggeman
• 金额: $ 33.55万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-20 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8342329
• 关键词: AIDS-Associated Nephropathy; Actins; Adult; Affect; Binding; Binding Proteins; Biochemical; Biological Assay; Cell Adhesion; Cell Line; Cell membrane; Cell-Cell Adhesion; Cell-Matrix Junction; Cells; Chronic Kidney Failure; Co-Immunoprecipitations; Complex; Cytoskeleton; Development; Diabetes Mellitus; Disease; Event; Filtration; Focal Adhesions; Foot Process; Homeostasis; Image; Immunologics; In Vitro; Injury; Integrin Binding; Integrins; Intercellular Junctions; Kidney; Kinetics; Knockout Mice; Light; Mediating; Methods; Molecular; Mouse Cell Line; Mus; Natural regeneration; Pathology; Phenotype; Phosphorylation; Phosphotyrosine; Process; Property; Proteins; Proteinuria; Receptor Signaling; Regulation; Renal function; Renal glomerular disease; Role; Severities; Signal Transduction; Structure; Synapses; T-Cell Receptor; Testing; Time; Tyrosine; adapter protein; base; blood filter; in vivo; inhibitor/antagonist; injury and repair; kidney cell; migration; mouse model; nephrin; novel; podocyte; prevent; repaired; response; scaffold; slit diaphragm; src-Family Kinases; therapeutic development; trafficking

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.

描述（由申请人提供）：成人肾脏足细胞的主要细胞电池连接是缝隙隔膜，这是肾脏过滤屏障的重要组成部分。脚步过程中缝隙膜片的丧失导致蛋白尿，并导致许多慢性肾脏疾病（例如糖尿病和与HIV相关的肾病）的进展。足细胞连接的发育形成和成人体内稳态是尚未完全理解的过程，但对于制定修复或替代疾病损害或疾病损失的治疗策略很重要。我们最近已经将类似克劳丁的细胞连接蛋白TM4SF10和FYN结合蛋白ADAP确定为影响层状脂蛋白形成，FYN活性和肾素磷酸化的足细胞表达的蛋白。 TM4SF10在足细胞发育过程中瞬时表达，并在损伤修复过程中重新表达。因此，我们假设在疾病中，TM4SF10可以通过保留简化的细胞连接并修改FYN的活性来在脚步过程中起作用。我们还假设ADAP是一种大型衔接蛋白，在整联蛋白信号传导中具有已知作用，在缝隙隔膜和脚部过程的整合蛋白附件上整合了肌动蛋白细胞骨架动力学的足迹中的脚手架中心。我们将使用标准突变分析和体外结合测定法确定ADAP与TM4SF10与TM4SF10的结合以及其他关键的细胞内成分的关联来检验该假设，并根据层状脂蛋白扩展，整合素结合和肾磷脂磷酸化评估功能效应。拟议的研究还将包括ADAP敲除小鼠中肾脏表型的进一步特征，以及对TM4SF10的敲除小鼠模型的进一步发展和表征。将建立来自这些小鼠的足细胞系，以研究调节整联蛋白细胞附着和肾素磷酸化的TM4SF10和足细胞中ADAP的必要功能。此外，我们已经建立了TM4SF10功能，以直接抑制FYN活性，这可能是通过隔离质膜来隔离Fyn的。这将通过实时成像和共定位方法进行研究，以评估在存在和不存在TM4SF10的情况下Fyn的细胞内运输。这些研究描述了在裂缝隔膜处与细胞 - 矩阵粘附连接细胞细胞粘附的第一个整合分子机制，并在脚步过程中与整合素结合，在脚步过程中需要同步在脚步过程中需要同步的事件。这些研究将对针对肾小球疾病的新型疗法的发展具有重要意义，该疗法的特征是脚步过程，预防或修复受损的足细胞是有益的。 公共卫生相关性：总结以治疗损害肾脏过滤器（肾小球）的疾病，需要制定鼓励新肾细胞再生的策略。这就需要对肾脏的专业细胞续签并形成其独特结构的基本了解。在肾小球修复过程中重要的是如何在足细胞之间建立和维持细胞 - 细胞连接，因为这些细胞连接是血液过滤设备的关键组成部分。这些研究将提供有关在发育过程中如何形成足细胞的重要信息，以及如何根据疾病造成的损害进行修复。