Nephrin是足细胞连接相邻足突和信号转导的主要分子，在磷酸化和非磷酸化状态均被內吞，但其內吞的意义、內吞的结局及其对足细胞功能的影响尚不清楚。本项目拟研究nephrin是否通过磷酸化-泛素化-內吞-溶酶体途径降解，降低其表达、影响下游信号及足细胞功能。通过nephrin稳定转染MDCK细胞与nephrin胞外段融合蛋白共培养，模拟nephrin连接的生理状态，用点突变和抑制剂方法，评价nephrin磷酸化和泛素化修饰对nephrin表达、內吞途径、下游信号和细胞骨架的影响；在高糖刺激的小鼠足细胞中，用不同方法干扰內吞和蛋白修饰，观察不同蛋白修饰和內吞途径对nephrin表达、下游信号和细胞骨架的影响；在链脲佐菌素糖尿病模型中进一步验证nephrin修饰、內吞与蛋白尿的关系。明确不同蛋白修饰对nephrin內吞途径、表达和足细胞功能的影响，进一步探讨足细胞损伤与蛋白尿的发病机制。

It is well known that nephrin plays an indispensable role in maintaining the integrity of slit diaphragm and signal transduction in podocyte. It is reported that unphosphorylated and phosphorylated nephrin is internalized in a clathrin-dependent (CDE) and a clathrin- idependent (CIE) manner respectively. However, the pathophysiological role of nephrin endocytosis in podocyte injury is still unclear. Phosphorylation and ubiquitination are important signals to guide the endocytic sorting of proteins and modified proteins are preferable to suffer degradation in lysosomes. It is proposed that the phosphorylaed nephrin is subjected to ubiquitination and degradation in lysosomes, which suppresses expression and downstream signaling of nephrin and disturbs the normal function of podocyte. The nephrin-transfected MDCK cells(MDCK-nephrin) is exposed to the fusion protein of extracellular domain of nephrin with Fc portion of human Ig, which mimics the physiological interaction of nephrin in vivo. The inhibitors and mutation vectors are introduced to evaluate the effects of phosphorylation and ubiquitination of nephrin on endocytosis, downstream signals and cytoskeleton of MDCK-nephrin. In mouse podocytes with high glucose exposure, the expression and downstream signaling of nephrin and cytoskeleton remodeling are assessed under conditions of various modification and endocytosis regulators. A STZ-induced diabetes model is used to determine the relationship between the modification of nephrin with the onset of proteinuria. The purpose of the project is to explore the roles of post-translational modification of nephrin on endocytic pathways, downstream signaling of nephrin and podocyte homeostasis in the pathogenesis of proteinuria.