激活素A在高糖诱导的人肾小管上皮细胞中的表达变化

目的 通过体外高糖刺激的人近端肾小管上皮细胞株(HK-2),探讨激活素A(ACT A)的表达变化及卵泡抑素(FS)的干预作用.方法 HK-2细胞生长于DMEM培养基,待细胞生长至亚融合状态时,更换为无血清DMEM培养基使细胞生长同步化24h,然后将细胞分为以下5组:正常对照组(NG,5.5 mmol/L葡萄糖)、甘露醇对照组(MG,5.5 mmol/L葡萄糖+25 mmol/L甘露醇)、高糖组(HG,25 mmol/L葡萄糖)、HG+FS100组(25 mmol/L葡萄糖+100 μg/L FS)、HG+FS500组(25 mmol/L葡萄糖+500 μg/L FS).12、24、48 h后收集各组细胞及细胞上清液.Western印迹法检测各组细胞ACT A和p-Smad2/3的表达；ELISA检测各组细胞上清液转化生长因子β(TGF-β)和纤连蛋白(FN)的含量.结果 NG组细胞ACT A有微量表达,而HG组表达显著增加(P＜0.05),呈时间依赖性.与HG组相比,FS干预组ACT A表达显著减少(P＜0.05),呈剂量依赖性.与NG组相比,p-Smad2/3在HG组培养24 h后表达显著增加(P＜0.05)；FS可抑制p-Smad2/3的高表达(P＜0.05).ELISA检测结果显示,与NG组相比,HG组培养12 h后TGF-β表达即显著增加(P＜0.05),呈时间依赖性,FS干预对高糖诱导的TGF-3高表达没有影响.与NG组相比,HG组培养24h后FN表达显著增高(P＜0.01),FS对此有明显抑制作用,呈剂量依赖性(P＜0.01).结论 高糖能刺激HK-2细胞ACT A表达增加,从而促进肾小管上皮细胞FN的合成；FS可通过阻断ACT A的活化,减轻肾小管上皮细胞FN的合成.

Expression of activin A in high glucose-induced human kidney tubular epithelial cells

Objective To investigate the expression of activin A and the intervention effect of follistatin on high glucose-cultured human proximal tubular epithelial cells (HK-2) in vitro. Methods HK-2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM). By 80% confluent, the cells were incubated in a serum-free medium for 24 h and then exposed to the following experimental conditions for different time periods (12, 24 and 48 h): 5 mmol/L glucose (normal glucose group, NG), 5 mmol/L glucose plus 25 mmol/L mannitol (osmotic control, mannitol group, MG), 25 mmol/L glucose (high glucose group, HG), 25 mmol/L glucose plus 100 μg/L rh-follistatin (100 μg/L rh-follistatin intervention group, HG+FS100) or 25 mmol/L glucose plus 500 μg/L rh-follistatin (500 μg/L rh-follistatin intervention group, HG+FS500). Then, the cells and cellular supernatant were collected for further detection. Activin A and p-Smad2/3 expression were determined by Western blotting. TGF-β and fibronectin released into media were quantitatively analyzed by enzyme-linked immunosorbent assay(ELISA). Results Activin A had very slight activation in the NG group, but its production was enhanced in the HG group after 12 h culture (P<0.05) and increased gradually until study completion. Follistatin blocked its production in a dose-dependent manner. p-Smad2/3 was significantly increased after 24 h culture in the HG group compared with the NG group and follistatin treatment decreased its overexpression in the HG group (P<0.05）. TGF-β was markedly higher in the HG group than that in the NG group after 12 h culture(P<0.05） and increased in a time-dependent manner, but both doses of follistatin failed to modify the overexpression of TGF-β in HG. Compared with the NG group, fibronectin in the HG group was obviously upregulated after 24 h culture(P<0.01). Follistatin reduced its overexpression in a dose-dependent manner. Conclusions High glucose can induce activin A activation and then promote FN production in HK-2 cells. Decreased fibronectin production inhibited by follistatin in HK-2 cells in high glucose condition is associated with blocking of activin A activation.