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目的探讨Klotho抑制成纤维细胞生长因子2(fibroblast growth factor2,FGF2)诱导的肾小管上皮细胞(HK-2)上皮-间充质转分化(epithelia-mesenchymal transition,EMT)的作用及机制。方法重组人Klotho蛋白预孵育前后,倒置显微镜观察FGF2对HK-2细胞形态的影响,免疫荧光检测上皮细胞标志物cytokeratin和间充质细胞标志物vimentin的表达变化,Western blot检测α-SMA、E-cadherin和ERK1/2的表达变化。结果 100 ng/mL的FGF2作用于HK-2细胞48 h后,HK-2细胞变成长梭形,细胞间隙明显增大,上皮细胞标志物E-cadherin的表达明显下降,间充质细胞标志物α-SMA表达明显升高(P<0.05),提示FGF2可以诱导HK-2细胞发生EMT。而Klotho蛋白预孵育可以显著抑制FGF2诱导的EMT,并且FGF2/FGFR(FGFs受体)下游信号分子pERK1/2的表达明显减弱(P<0.05)。结论 Klotho可以通过减弱FGF2信号通路活化抑制FGF2诱导的HK-2细胞EMT,Klotho可能参与了肾间质纤维化的发生和发展。
Objective To investigate the effect and mechanism of Klotho on inhibition of epithelial mesenchymal transition (EMT) induced by fibroblast growth factor 2 (FGF2) in renal tubular epithelial cells (HK-2). Methods The effect of FGF2 on the morphology of HK-2 cells was observed by inverted microscope before and after preincubation with Klotho protein. The expression of cytokeratin and vimentin, a marker of mesenchymal cells, was detected by immunofluorescence. The expressions of α-SMA, E -cadherin and ERK1 / 2 expression changes. Results After HK-2 cells were treated with 100 ng / mL of FGF2 for 48 h, HK-2 cells became long fusiform, the number of intercellular spaces was significantly increased, the expression of E-cadherin, a marker of epithelial cells, was markedly decreased. Mesenchymal cell markers Α-SMA expression was significantly increased (P <0.05), suggesting that FGF2 can induce HK-2 cells EMT. However, Klotho preincubation could significantly inhibit FGF2-induced EMT, and the expression of pERK1 / 2 downstream of FGF2 / FGFR (FGFs receptor) was significantly decreased (P <0.05). Conclusion Klotho can inhibit the FGF2-induced EMT in HK-2 cells by attenuating the activation of FGF2 signaling pathway. Klotho may be involved in the development and progression of renal interstitial fibrosis.