c-Abl在肾小球足细胞nephrin信号转导中的作用及机制

Podocytes are major cellular components of glomerular filtration barrier. Nephrin is an important structural and signal molecule. Expression and/or phosphorylation of nephrin is closely related to pococyte injury, but its specific mechanisms of the signal transduction remain unclear. c-Abl is a nonreceptor tyrosine kinase，which plays a key role in cell survival and cytoskeleton regulation. This project intends to study the role of c-Abl in podocyte injury and cytoskeleton rearrangement regulated by nephrin signaling. Firstly, through the establishment of angiotensinⅡ-infused animal model, podocyte culture and the analysis of renal biopsy specimens of podocyte disease patients, we will evaluate c-Abl expression, phosphorylation and activity change, and its interaction with nephrin phosphorylation and podocyte injury; Secondly, through the changes of c-Abl expression and activity with its recombinant plasmid, siRNA, or its specific inhibitor,we will evaluate the effect of c-Abl on podocyte injury and cytoskeleton rearrangement regulated by nephrin signaling; Finally, through the c-Abl mutant plasmids construction and cotransfection with nephrin in HEK293 cell, we will idnetify the interaction between nephrin and c-Abl, and further elucidate the molecular mechanism. This investigation will provide a theoretical evidence for seeking a new therapeutic target of podocyte injury.

足细胞是肾小球滤过屏障的主要细胞成分，Nephrin是足细胞裂隙膜重要的结构和信号转导分子，其表达和/或磷酸化改变与足细胞损伤密切相关，但其信号转导机制仍不清楚。c-Abl是一种非受体酪氨酸激酶，在调节细胞存活及细胞骨架重排信号中起关键作用。本项目拟研究c-Abl在nephrin信号介导足细胞损伤及骨架重排中的作用及机制。通过建立血管紧张素Ⅱ输注动物模型、培养足细胞及足细胞病病人肾活检标本，评价c-Abl表达、分布及活性改变及其与nephrin磷酸化和足细胞损伤的相互作用；通过c-Abl表达质粒、siRNA转染或特异性抑制剂改变c-Abl表达及活性，评价其对nephrin信号调节足细胞凋亡及细胞骨架重排的影响；通过构建缺失c-Abl不同功能域的突变质粒，与nephrin共转染HEK293细胞，进一步明确c-Abl与nephrin之间的作用关系，为寻求有效防治足细胞损伤的新靶点提供理论依据。

Nephrin是足细胞裂隙膜重要的结构和信号分子，其表达和/或磷酸化水平改变与足细胞损伤密切相关，但其信号转导机制仍不清楚。本项目旨在探讨c-Abl在足细胞nephrin信号介导足细胞损伤中的作用及分子机制。本研究发现正常大鼠肾小球及体外培养小鼠足细胞中nephrin与c-Abl存在一定程度的共定位及相互作用，AngⅡ刺激可显著减少两者的共定位及结合，并伴随足细胞nephrin-Akt信号改变及细胞损伤。利用c-Abl质粒转染上调足细胞c-Abl表达可进一步增强AngⅡ诱导的Akt去磷酸化，加重足细胞损伤；而用c-Abl siRNA转染或STI571预处理抑制c-Abl表达/活性可显著逆转AngⅡ诱导的Akt去磷酸化，改善AngⅡ所致的足细胞损伤。构建CD16/7-nephrin-myc嵌合质粒，转染足细胞后以CD16诱导nephrin磷酸化，可显著增加CD16/7-nephrin-myc嵌合质粒与c-Abl的共定位及相互作用，表明与c-Abl结合的是nephrin的磷酸化形式。构建不同结构域（SH2/SH3或FABD）缺失的c-Abl突变载体，与CD16/7-nephrin-myc嵌合质粒共转染COS7细胞，发现c-Abl通过其SH2/SH3结构域直接与p-nephrin相结合，参与COS7细胞骨架重排的调控。正常肾组织中nephrin和c-Abl呈线性共定位于肾小球毛细血管袢，在IgAN、MCD、FSGS及MN等病理状态下，c-Abl与nephrin共定位显著减弱。结论：c-Abl是nephrin的下游信号分子，通过其SH2/SH3结构域与p-nephrin相结合，在AngⅡ刺激或足细胞病等病理条件下，c-Abl由nephrin解离并活化，通过Akt通路调控足细胞表型改变。