血管内皮细胞生长因子激活Rac1引起肾小球内皮细胞通透性增高的机制

Mechanism of hyperpermeability induced by vascular endothelial growth factor in glomerular endothelial cells through Racl activation

目的探讨细胞内Racl信号激活是否在血管内皮细胞生长因子（VEGF）增加肾小球内皮细胞的通透性和导致紧密连接酪氨酸磷酸化中起作用。方法采用原代培养的大鼠肾小球内皮细胞作为实验对象。通过体外研究，检测跨内皮细胞电阻抗观察不同浓度VEGF（5和50μg／L）对内皮细胞通透性的影响。内皮细胞转染野生型Racl和显性负性Racl质粒后，采用免疫沉淀和免疫印迹等方法观察上述效应是否源自Racl信号的激活，并观察紧密连接occludin蛋白酪氨酸磷酸化状态的改变。结果高浓度的VEGF（50μg／L）刺激可引起大鼠肾小球内皮细胞单层通透性显著增高（P〈0．05），并引起肾小球内皮细胞中GTP结合的Racl和膜结合的Hacl显著增加（P〈0．01），同时紧密连接蛋白occludin的酪氨酸磷酸化也增加（P〈0．05）。用Racl显性负性突变体转染肾小球内皮细胞能显著减弱VEGF对occludin蛋白酪氨酸磷酸化和内皮细胞通透性的影响（P〈0．05）。结论高浓度的VEGF可导致肾小球内皮细胞通透性增高，其与紧密连接蛋白occludin的酪氨酸磷酸化相关，这一作用需要Racl信号途径的激活。糖尿病中增高的VEGF可能通过Racl激活-occludin磷酸化导致肾小球内皮通透性增高，这可能是糖尿病肾病的发病机制之一。

Objective To investigate if Racl GTPase activation plays an important role in hyperpermeability and tyrosine phosphorylation of tight junction induced by vascular endothelial growth factor （VEGF） in glomerular endothelial cells （GEnCs）. Methods Primary cultured rat endothelial cells were used as experimental model. The effect of VEGF at different concentrations （5 or 50 μg/L） on endothelial permeability was investigated by transendothelial electrical resistance （TEER）. The permeability of GEnCs transfected with wild type Racl （wtRacl） or dominant negative Racl （N17Rac1） was also detected. Immune precipitation and immune blotting were used to detect the tyrosine phosphor-occludin in GEnCs. Results VEGF at high concentration （50 μg/L） induced hyperpermeability in GEnCs （P〈0.05）. At the same time, GTP-binding and membrane- bound Racl GTPase significantly increased（P〈0.01）in GEnCs. Tyrosine phosphor-occludin was also increased （P〈0.05） under VEGF stimulation. However, transfection of GEnCs with N17Racl dramatically attenuated the effect of VEGF on tyrosine phospho-occludin and endothelial cell permeability. Conclusions Increased VEGF can induce hyperpermeability in glomerular endothelial cells, which is related to occludin tyrosine phosphorylation through Racl activation. It provides a framework for understanding the role of VEGF-induced Racl-phospho-occludin pathway in the integrity of barrier function in the diabetic milieu.