足细胞分子MGAI-1在儿童激素耐药肾病的致病作用及分子机制研究

Steroid-resistant nephrotic syndrome (SRNS) is a refractory renal disease.Studies have showed that several podocyte molecules,such as nephrin, podocin, CD2AP and α-actinin-4, etc., have been identified in SRNS (NS). Further studies demonstrated that these molecules expressed specifically in podocyte play a critical role on maintaining the normal structural and functional integrity of the glomerular filtration barrier, especially the slit diaphragm (SD) connecting neighboring foot processes of podocyte. In our study before, we found a girl with SRNS carried SD molecule MAGI-1 gene mutation, which was highly suspected causing gene in SRNS.Therefore, in this project, we plan to use podocyte and zebrafish to build MAGI-1 gene mutation and gene knockdown model. In the podocyte and zebrafish model, we will analyze the podocyte foot process morphology, podocyte SD molecules expression, cytoskeleton molecules expression, glomerular basementmembrane(GBM) molecules expression and nephrotic syndrome phenotype in zebrafish.The renal tissue of the patients with MAGI-1 mutation will also be analyzed. At the same time, the molecules downstream of the cAMP-Epac-Rapl pathway will be analyzed, such as GTP-GDP modified Rap1, Cadherin expression, the function of the podocyt adhension and polarity. So as to explore the molecular mechanism of MAGI-1 gene mutation in SRNS. The project will benefit to patients with MAGI-1 mutation,who will be diagnosed and received individual treatment.It will improve our understanding about the molecular pathway of the development of SRNS from the view of podocyte molecules, and further disclose the potential site(s) or molecular target(s) for the intervention of SRNS.

激素耐药肾病（SRNS）为难治性肾病，研究发现肾小球足细胞分子缺陷可引起SRNS，申请人前期通过目标区域捕获及二代测序的研究发现SRNS患儿携带足细胞分子MAGI-1基因罕见突变，而在激素敏感肾病和正常对照中均未发现，提示MAGI-1为SRNS的新致病基因。本研究以MAGI-1基因为切入点，利用MAGI-1突变肾组织、小鼠足细胞系及斑马鱼构建MAGI-1基因突变及敲低挽救模型，来分析MAGI-1突变对肾小球滤过屏障的影响，包括足细胞形态、足细胞分子/基底膜分子的表达、斑马鱼肾病表型；并分析在肾小球滤过屏障完整性方面起重要作用的Rap1信号通路上MAGI-1下游效应分子及细胞生物学行为的改变，即GTP和GDP修饰的Rap1表达、钙粘素表达及足细胞粘附功能，从而揭示MAGI-1突变破坏肾小球滤过屏障的关键分子，该研究有望确定一个新的SRNS致病基因，并从一个新的角度探索激素耐药肾病的分子机制。

课题以足细胞分子MAGI1（激素耐药肾病儿童携带其罕见突变c.T2533C，p.W817R）为切入点，通过构建突变模型来分析其在足细胞的作用。首先敲低MAGI1，观察感兴趣的足细胞分子、Rap1信号通路、足细胞黏附及伸展功能，结果显示MAGI1敲低后Nephrin、JAM4、Rap1、P-Cadherin、actinin4、podocin mRNA及蛋白表达量无变化，Rap1GTP蛋白表达降低，足细胞活力下降、凋亡增加；免疫荧光及扫描电镜显示足细胞形态改变，足突减少，足细胞分子分布不均匀，呈团块状，部分进入细胞核。接着构建MAGI1缺失突变表达载体，即PDZ3区域（MAGI1 814~827aa区域），转染HEK293细胞，实验分正常足细胞、MAGI1低表达组、MAGI1低表达+MAGI1wt组、MAGI1低表达+MAGI1mut组，检测上述足细胞分子mRNA及蛋白水平，结果显示MAGI1低表达及MAGI1低表达+MAGI1mut表达组足细胞分子mRNA及蛋白表达量无改变，Rap1GTP蛋白表达降低，经过MAGI1 wt转染后，Rap1GTP蛋白表达增加，提示MAGI1mut未影响足细胞分子表达量，影响了Rap1GTP蛋白表达。免疫荧光及扫面电镜显示MAGI1低表达组、MAGI1低表达+MAGI1mut组细胞形态改变，足突减少，JAM4及Nephrin分子分布改变，不再定位在细胞膜，α-actinin-4、F-actin蛋白分布亦改变，呈不均匀颗粒、团块状，细胞粘附及伸展能力下降，同样可以被MAGI1wt转染所逆转，而不能MAGI1mut所逆转。.因此，本研究提示MAGI1低表达影响Rap1信号通路、足细胞形态及分子分布及足细胞功能，包括细胞活力、黏附、凋亡及伸展功能；而MAGI1的PDZ3区域（MAGI1 814~827aa）缺失突变同样影响了RAP1信号通路、足细胞功能及分子分布，推测可能是通过影响Rap1信号通路从而影响足细胞功能，研究为探索激素耐药机制提供重要信息。.为了探索跟MAGI1相互作用的分子，采用Illumina技术对上述各组提取mRNA转录组测序，找到NDRG2、NOD2在MAGI1低表达组蛋白表达量下降，转染MAGI1 wt可以逆转，转染MAGI1 mut不能逆转，提示MAGI1敲低及突变均影响NDRG2和NOD2mRNA及蛋白表达量。