Nephrin蛋白修饰对其內吞结局及足细胞功能的影响及机制

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.

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。

Nephrin是足细胞裂孔隔膜区重要的信号转导分子，可内吞入细胞内，但其內吞的意义、及其对足细胞功能的影响尚不清楚。本项目通过细胞生物学和生物化学的方法，拟研究糖尿病状态下nephrin内吞的变化及可能的机制。研究发现：（1）Nephrin可进入快速转运体（EEA1）、早期内体（Rab5）进入再循环系统参与细胞表面nephrin的自我更新。（2）生理状态下nephrin被泛素化修饰，泛素化nephrin主要分布于核周的内质网。（3）nephrin的蛋白代谢半衰期为6小时，主要通过蛋白酶途径讲解。（4）糖尿病肾病早期主要表现为nephrin分布异常，体内、体外实验证实高糖刺激可增加nephrin泛素化水平，泛素化nephrin进入蛋白酶体途径降解。结论：nephrin通过早期内体进入再循环系统参与足细胞表面nephrin更新，部分nephrin被泛素化后进入蛋白酶体降解；高糖促进nephrin泛素化，增加蛋白酶降解，降低nephrin表达，进一步揭示糖尿病肾病早期足细胞损伤的分子机制。