醛糖还原酶对大鼠肾脏系膜细胞表达纤连蛋白和Ⅳ型胶原蛋白的影响

目的探讨醛糖还原酶(AR)对大鼠肾脏系膜细胞(MsC)表达细胞外基质成分纤连蛋白(FN)和Ⅳ型胶原(ColⅣ)蛋白的影响,以及AR在肾小球硬化病变过程中的可能作用及其机制。方法采用酶切、连接方法构建真核表达质粒pCDNA3-AR,采用脂质体介导以及G418筛选的方法,将pCDNA3-AR稳定转染至MsC中,应用逆转录聚合酶链反应(RT-PCR)、Western印迹法和免疫荧光检测转染MsC表达AR情况;以Western印迹法检测正常MsC、AR转基因MsC、应用AR抑制剂Sorbinil和Zopolrestat的正常MsC、转化生长因子-β1(TGF-β1)作用后的正常MsC的FN和ColⅣ蛋白表达情况。结果与正常MsC相比,TGFβ1作用后的正常MsCFN和ColⅣ蛋白表达水平分别增高1.6和1.7倍(P<0.01);AR转基因MsC的FN、ColⅣ表达分别增加1.8倍和1.5倍;应用Sorbinil时,FN和ColⅣ分别降低1.8倍和1.4倍(P<0.05);应用Zopolrestat时,FN和ColⅣ分别降低1.7倍和1.4倍(P<0.05)。结论AR高表达或活性受抑制时可以明显影响MsCFN、ColⅣ的蛋白表达,提示AR可能与肾小球硬化的病理过程有关。

Effects of aldose reductase on the expression of fibronectin and collagen IV in cultured rat renal mesangial cells

OBJECTIVE: To study the effect of aldose reductase (AR) on expression of fibronectin and collagen IV in cultured rat renal mesangial cells (MsC). METHODS: AR expression plasmid vector (pCDNA3-AR) was constructed by restriction endonuclease digestion and ligation procedures. Stable expression of AR in MsC was established by Lipofectin transfection. Western blot and immunofluorescence analyses were performed to verify the transfection efficiency. Expression of fibronectin and collagen IV proteins were analyzed using Western blot. RESULTS: Expression of fibronectin and collagen IV in naive MsC treated with TGF-beta1 was upregulated in comparison to that of the untreated naive MsC (P < 0.01). MsC transfected with pCDNA3-AR showed a remarkable increase of expression of fibronectin and collagen IV (P < 0.01). Aldose reductase inhibitors (Sorbinil and Zopolrestat) significantly inhibited the expression of fibronectin and collagen IV in naive MsC (P < 0.05). CONCLUSIONS: Overexpression or inhibition of AR activity significantly alters the expression of fibronectin and collagen IV proteins in cultured rat MsC, suggesting that AR plays a significant role in the pathogenesis of glomerulosclersis.