内质网应激在足细胞相关分子突变引起局灶节段肾小球硬化中所起作用及机制研究

Focal segmental glomeruloslerosis (FSGS) is a podocytopathy with its clinical features including proteinuria and renal failure. No specific treatment is available for FSGS patients. Recently, we identified two INF2 mutations(S85W, S129_Q130ins VRQLS). Structure analysis suggested these mutations leaded to protein misfolding. In vitro studies showed mice podocyte transfected with INF2-S85W leaded to aggregation of INF2 in the cytoplasm, podocyte apoptosis and increased expressing of GPR78 which is a biomarker of endoplasmic reticulum stress (ERS). We hypothesis ERS maybe a common pathway for podocyte injury and apoptosis, and inhibitors of ERS/mTOR may have protective effect for podocyte injury. To this end, we will measure localization of mutant genes, changes of cell skeleton, ERS signal pathways and apoptosis in mice podocytes over-expressed of mutant INF2 before and after adding inhibitors of ERS/mTOR. Next, we will establish transgenic (INF2-S85W) mice to validate results from in vitro studies. At last, we will test the expression of biomarkers of ERS in kidney tissue from FSGS patients and analyze their clinical relevants. This study is important for us to understand the mechanism of podocyte injury and discovery new treatment targets from this new perspective of ERS.

局灶节段肾小球硬化（FSGS）是足细胞病，表现为蛋白尿及肾衰竭，无特异治疗。我们最近在FSGS家系中成功确定2个INF2突变（S85W，S129_Q130ins VRQLS）。结构模拟显示突变可致INF2折叠异常，足细胞转染S85W后INF2在胞浆内团块样聚集，细胞凋亡增加，内质网应激（ERS）标志分子GPR78表达增加。我们提出ERS在突变致足细胞损伤及凋亡中起重要作用，ERS/mTOR抑制剂对突变引起足细胞损伤具保护作用。为此，我们在小鼠足细胞中过表达突变INF2，应用ERS/mTOR抑制剂干预，检测突变INF2在细胞中定位，细胞骨架、ERS、信号通路、凋亡相关分子改变。构建高表达INF2-S85W转基因小鼠，体内验证上述结果。最后检测ERS相关基因在FSGS患者肾组织表达，分析其与临床相关性。本研究将从ERS这个新视点为深入了解足细胞损伤机制奠定基础，为探索足细胞疾病治疗提供新思路。

局灶节段肾小球硬化（FSGS）是足细胞病，表现为蛋白尿及肾衰竭，无特异治疗。本课题组前期研究发现内质网应激（ERS）可能在突变致足细胞损伤及凋亡中起重要作用。为此，我们利用CRISPR/Cas9技术成功构建COL4A3突变的细胞及小鼠模型，发现IV胶原截短突变（p.Arg1631Stop) 小鼠肾组织的GRP78、PERK、IRE1α均明显增高，凋亡CHOP、Bax指标升高，提示突变的IV型胶原会引起ERS，进而导致细胞凋亡的发生。蛋白酶体抑制剂（MG132）能够显著提高截短突变（c.4317delA)组细胞内COL4A3水平，并可改善足细胞ERS、凋亡，提示通过调节蛋白酶体降解途径可潜在治疗足细胞损伤。此外，我们发现人脐带间充质干细胞（UCMSC）注入截短突变小鼠后尿蛋白和血清肌酐水平明显降低。HE染色图像显示肾小球硬化延迟。免疫荧光显示IV型胶原α3链的分泌显著增加，足细胞和基底膜损伤有所恢复，提示UCMSC移植可能是FSGS的潜在治疗方法。本研究从ERS这个新视点为深入了解足细胞损伤机制奠定基础，提出调节蛋白酶体降解途径和UCMSC两个足细胞损伤潜在新治疗方法。