地塞米松对肾小球足细胞瞬时受体电位阳离子通道蛋白6表达和分布的影响

Influence of dexamethasone on the expression and distribution of transient receptor potentialcation channel 6 in glomerular podocytes

目的以嘌呤霉素（PAN）损伤足细胞，以地塞米松（DEX）干预，观察不同时间点瞬时受体电位阳离子通道蛋白6（TRPC6）表达和分布的变化，探讨在足细胞损伤过程中，DEX对TRPC6表达与分布的影响及与足细胞损伤的关系。方法体外培养足细胞，设立对照组、PAN刺激组和DEX干预组。对照组用含0．02％二甲亚砜（DMSO）的RPM11640培养液培养，PAN刺激组加入PAN（50μg／m1），DEX干预组同时加入PAN（50μg/m1）和溶解于0．02％DMSO的DEX（1μmol/L），处理8h、24h、48h后观察细胞形态并拍摄，采用图像处理软件分析各组细胞形态及胞体面积的差别。采用流式细胞仪检测足细胞凋亡率，采用实时荧光定量PCR、Western印迹和间接免疫荧光染色检测各时间点TRPC6的mRNA、蛋白表达及分布变化。结果经PAN刺激后细胞体显著缩小（P〈0．05），足突回缩、消失，细胞间失去相互连接，刺激后48h足细胞凋亡率显著升高（P〈0．05）。PAN刺激组在刺激24h和48h后TRPC6mRNA及蛋白表达量显著上升（P〈0．05），TRPC6在足细胞上的分布亦出现异常。DEX干预后细胞胞体显著大于PAN刺激组（P〈0．05），可维持正常的足突形态；干预48h后足细胞凋亡率显著低于PAN刺激组，TRPC6mRNA及蛋白的表达、分布均趋于正常水平。结论DEX通过稳定重要的足细胞分子TRPC6的表达及分布来抑制足细胞损伤，从而发挥保护足细胞和抗蛋白尿的作用。

Objective To observe the changes of foot processes, expression and distribution of transient receptor potential cation channel 6 （TRPC6） in podocytes by puromycin aminonucleoside （PAN） and dexamethasone （DEX） intervention, then to investigate the function of TRPC6 in podocytes and its relation to proteinuria in kidney diseases. Methods Podocytes cultured in vitro were divided into three group： control group, PAN stimulation group and DEX intervention group. Mouse podocyte cell line （MPC5） were cultured in 0.02% dimethyl sulfoxide （DMSO） in control group, subjected to PAN （50 μg/ml） treatment alone or with DEX （1 μmol/L） in other two groups for 8 h, 24 h, 48 h. The podocyte morphology was observed and took pictures by phase contrast microscope, then the differences of morphology and areas were analyzed. The distribution, mRNA expression and protein expression of TRPC6 were detected by indirect immunocyto-fluorescence, real-time quantitative PCR and Western blotting, respectively. Results The well-developed podocyte arborization and interconnection wasformed in control group, but PAN led to significant shrinkage of podocytes （P 〈 0.05）, together with podocyte foot process retraction, effacement and loss of cell contact. DEX significantly prevented the shrinkage and apoptosis of podocytes. The apoptosis rate was significantly increased after PAN stimulated 48 h （P 〈 0.05）. Real-time quantitative PCR and Western blotting found TRPC6 mRNA and protein expression were prone to increase in PAN group compared with control group （P 〈 0.05）. The distribution of TRPC6 becamed abnormal in PAN group. DEX decreased TRPC6 mRNA and protein expression at 48 h compared with PAN group （P 〈 0.05）. The abnormal distribution of TRPC6 was also alleviated by the protection of DEX. Conclusion DEX exerts a direct action to podocyte which retains the integrity of slit diaphragm against podocyte injury, and alleviates proteinuria via stabilizing mRNA, protein expression and distribution of TRPC6.