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目的 探讨终末期糖基化终产物(AGEs)介导肾小管上皮细胞转分化和胶原 (Col)I合成的分子机制。方法 体外培养正常大鼠近端肾小管上皮细胞系(NRK52E),应用自 制的AGE-牛血清白蛋白(BSA)刺激NRK52E细胞。免疫细胞化学方法检测不同时间磷酸化(p) Smad2／3核转位情况。ELISA方法检测细胞培养上清TGF-β1的水平。RT-PCR方法检测α-平滑 肌肌动蛋白(SMA)、E-钙黏着糖蛋白(cadherin)和Col I mRNA表达。Western印迹检测α-SMA、 E-cadherin和Col I蛋白的表达。同时观察TGF-β1中和抗体对AGE-BSA上述效应的阻断作用。 结果 基础状态下,NRK52E细胞存在低水平p-Smad2／3核表达(16％)。与BSA对照组比较, AGE-BSA以时间依赖方式上调NRK52E细胞p-Smad2／3核转位,其高峰出现在30 min(68％比 30．5％,P<0．01)和24 h(76％比31．3％,P<0．01)。AGE-BSA显著上调α-SMA和Col I mRNA和 蛋白表达;下调E-cadherin mRNA和蛋白的表达;并能促进NRK52E细胞合成和分泌TGF-β1。 TGF-β1中和抗体能明显抑制AGE-BSA介导的24 h p-Smad2／3核转位(25．2％,P<0．01),但不 能阻抑30 min活化高峰;能明显抑制AGE-BSA介导的α-SMA和Col I mRNA和蛋白表达。以及 显著地上调E-cadherin mRNA和蛋白的表达。结论 AGEs通过TGF-β依赖和非依赖途径诱导 肾小管上皮细胞Smads信号通路活化,促进其向肌成纤维母细胞转分化和Col I的合成。
Objective To investigate the molecular mechanism of end-stage glycosylation end products (AGEs) -mediated renal tubular epithelial cell transdifferentiation and collagen (Col) I synthesis. Methods Normal rat proximal tubular epithelial cell line (NRK52E) was cultured in vitro and NRK52E cells were stimulated with homemade AGE-BSA. Immunocytochemistry was used to detect phosphorylated (p) Smad2 / 3 nuclear translocation at different time points. ELISA method to detect the level of TGF-β1 in the cell culture supernatant. The mRNA expression of α-smooth muscle actin (SMA), E-cadherin and Col I were detected by RT-PCR. Western blot was used to detect the expression of α-SMA, E-cadherin and Col I protein. At the same time, the blocking effect of the TGF-β1 neutralizing antibody on the above effect of AGE-BSA was observed. Results Under the basal conditions, NRK52E cells had low level of p-Smad2 / 3 nuclear expression (16%). AGE-BSA upregulated the p-Smad2 / 3 nuclear translocation in NRK52E cells in a time-dependent manner at 30 min (68% vs. 30.5%, P <0.01) and 24 h 76% vs. 31.3%, P <0.01). AGE-BSA significantly up-regulated the mRNA and protein expression of α-SMA and Col I, down-regulated the expression of E-cadherin mRNA and protein, and promoted the synthesis and secretion of TGF-β1 in NRK52E cells. The neutralizing antibody of TGF-β1 could significantly inhibit AGE-BSA-mediated 24 h p-Smad2 / 3 nuclear translocation (25.2%, P <0.01), but could not inhibit the activation peak at 30 min; AGE-BSA-mediated α-SMA and Col I mRNA and protein expression. As well as significantly up-regulate E-cadherin mRNA and protein expression. Conclusion AGEs can induce the activation of Smads signal pathway in renal tubular epithelial cells via TGF-β-dependent and non-dependent pathways, and promote their transdifferentiation into muscle myofibroblasts and the synthesis of Col I.