高糖通过结缔组织生长因子诱导足细胞减少podocalyxin的表达

Connective tissue growth factor mediates high glucose-induced down-regulation of podocalyxin expression in mouse podocytes

目的探讨高糖对小鼠足细胞podocalyxin表达的影响及结缔组织生长因子(CTGF)参与其损伤的可能机制。方法构建针对CTGF基因的特异性siRNA质粒并转染至小鼠足细胞。将体外培养的足细胞分为6组(1)正常对照组,正常足细胞其培养基含D-葡萄糖1g/L;(2)等渗对照组,正常足细胞其培养基含D-葡萄糖1g/L,甘露醇3.5g/L;(3)高糖组,正常足细胞其培养基含D-葡萄糖4.5g/L;(4)高糖+空白对照组:足细胞转染空白质粒后于高糖培养基中培养;(5)高糖+阴性对照组:足细胞转染含无关序列的重组质粒后于高糖培养基中培养;(6)高糖+干扰组:足细胞转染针对CTGF的siRNA表达质粒后于高糖培养基中培养。Western blot方法检测小鼠足细胞Podocalyxin、CTGF蛋白表达及细胞外信号调节激酶(ERK1/2)磷酸化水平。结果高糖培养足细胞24、48h时均可下调其Podocalyxin蛋白表达量(P<0.05),并可诱导CTGF蛋白表达增加(P<0.05)。同时高糖可以活化足细胞ERK1/2信号途径,在高糖刺激30min时开始活化,持续活化至24h。特异性siRNA干扰CTGF合成后,ERK1/2活化减少,并且足细胞Podocalyxin表达升高。结论 CTGF是高糖诱导小鼠足细胞损伤的重要介质,可通过ERK1/2途径诱导podocalyxin表达下降,针对CTGF的siRNA能明显改善podocalyxin表达量,为DN的治疗提供新的思路。

Objective To detect the effect of connective tissue growth factor（CTGF） on podocalyxin expression in mouse potocytes exposed to high glucose in vitro and explore the possible pathway involved. Methods The expression vector carrying a small interfering RNA（siRNA） targeting CTGF was transfected into mouse podocytes cultured in the presence of 1 g/L glucose（normal control） ,4.5 g/L glucose（high glucose group） ,1 g/L glucose ＋ 3.5 g/L mannitol（iso-osmolar control group） . The changes in the protein expression levels of podocalyxin,CTGF and ERK1/2 in the cells in response to the treatments were investigated using Western blotting. Results High glucose exposure for 24 and 48 h resulted in significantly decreased expression of podocalyxin and increased CTGF in the podocytes（P0.05） . Phosphorylation of ERK1/2 occurred as early as 30 min after the exposure,and the activation was maintained till 24 h. Transfection of the cells with siRNA targeting CTGF significantly inhibited these changes. Conclusion CTGF is an important mediator of high glucose-induced potocyte damage and decreases the protein level of podocalyxin by the ERK1/2 pathway. CTGF-specific siRNA can alleviate high glucose-induced podocyte injury,suggesting its potential value in treatment of diabetic nephropathy.