NMDA受体对FSGS足细胞细胞骨架改变的作用及机制研究

Focal segmental glomerulosclerosis is clinically characterized (FSGS)by proteinuria and nephrotic syndrome, and is close related with podocyte actin cytoskeleton structural and functional homeostasis failure. Small GTPases, especially RhoA/Rho-kinase which expressed in foot processes and activated by Ca2+, play important roles in actin cytoskeletal dynamic changes. NMDA receptor is expressed in podocytes with significant permeability to Ca2+. Excessive activation of NMDAR can lead significant podocyte cytoskeleton degeneration. To investigate whether NMDAR is activated in FSGS and mediates the disease, FSGS rats and podocyte are involved in this project to measure the changes of the expression and activity of NMDAR subunits, the evocation of downstream Ca2+ dependent cytoskeleton dynamic cell signal pathway, the expressions and rearrangements of functional proteins in foot processes and slit diaphragm by whole cell patch clamp, GST-pull down and confocal microscopy technologies. Establish the NMDAR overexpression and shRNA interference system, evaluate the influences on the integrity of podocyte foot processes and glomerular filtration rate.

局灶节段性肾小球硬化症（FSGS)临床以肾病综合征为主要表现，其发生与足细胞的细胞骨架结构和功能改变密切相关。小分子GTP酶家族是细胞骨架纤维动力学调控的重要转导分子，其受Ca2+调控的RhoA/Rho激酶系统启动影响；NMDAR是谷氨酸的离子型膜受体，为功能性Ca2+通道，足细胞源NMDAR激活可导致细胞骨架的明显改变。本课题将先利用大鼠和足细胞FSGS模型，应用细胞膜片钳、GST-pull down、共聚焦成像等技术，观察FSGS病程中足细胞NMDAR表达和活性变化，及下游Ca2+依赖的骨架功能相关信号途径、结构特异性蛋白表达及分布的影响；然后，建立慢病毒质粒表达体系干扰NMDAR表达与活化，观察对足细胞足突完整性与肾小球滤过的影响。本研究能明确NNMDR对小鼠FSGS模型细胞骨架结构改变的防治效果；解明其在FSGS模型中可能信号转导分子机制；为FSGS蛋白尿防治提供新的可能治疗靶点。

糖尿病肾病（DN）是导致终末期肾病和糖尿病患者死亡的首要原因，其导致的蛋白尿的发生于足细胞结构和功能的改变密切相关。本课题在体小鼠糖尿病模型和离体足细胞高糖模型中观察NMDA受体亚基NR1的表达，显示在高糖下肾源性NR1表达增高，足细胞中表达亦增高。.在高糖下，NMDA受体激活导致了足细胞内钙离子升高，而Ca2+的增高则激活胞内cPKC磷酸化，进而促进内质网NR1成熟释放。在NMDA受体激活后，下游胞内钙信号、细胞骨架相关信号途径分子RhoA、Rac1和Cdc42的GTP化增高，足突结构相关蛋白Synaptopodin表达下降。构建NR1 shRNA慢病毒干扰体系，于离体和在体水平转染，足细胞足突形态、细胞骨架相关蛋白成分及骨架纤维动力学改变均有所改善，同时糖尿病所导致的尿蛋白量亦有所下降。