红细胞生成素对白蛋白诱导HK-2细胞转分化中TGFβ1/ILK通路的调控作用

Effect of erythropoietin on TGFβ1/ILK signaling pathway in albumin induced transition of human kidney proximal tubular epithelial cell-2

目的研究TGFβ1/ILK(转化生长因子-β1/整合素连接激酶)通路在人白蛋白诱导的人近端肾小管上皮细胞(HK-2细胞)转分化过程中的作用,探讨红细胞生成素(EPO)是否通过抑制TGFβ1/ILK通路而产生保护效应。方法体外培养HK-2细胞,RT-PCR法检测TGFβ1、ILK mRNA的表达;细胞免疫荧光法检测E-钙黏蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)的蛋白表达;ELISA法检测纤连蛋白(FN)的蛋白表达。结果白蛋白诱导组TGFβ1、ILK mRNA较空白对照组显著升高,EPO干预组TGFβ1、ILKmRNA显著下调;白蛋白诱导组E-cadherin的蛋白表达较空白对照组下降,α-SMA、FN的蛋白表达上升,而在EPO干预组及TGFβ1拮抗剂组E-cadherin的蛋白表达较白蛋白诱导组显著上调,α-SMA、FN的蛋白表达显著下调,且与EPO的浓度有关;Person直线相关分析显示白蛋白诱导组及EPO干预组TGFβ1 mRNA与ILK mRNA呈正相关。结论 TGFβ1/ILK通路参与了白蛋白诱导的HK-2细胞转分化过程,EPO通过抑制TGFβ1/ILK通路而抑制转分化过程,产生肾保护效应。

【Objective】To study the effects of TGFβ1/ILK signaling pathway on albumin induced epithelial-mesenchymal transition（EMT） of normal human kidney proximal tubular epithelial cells（HK-2）,and to investigate whether erythropoietin（EPO） produces protect effect by inhibiting the TGFβ1/ILK signaling pathway.【Methods】 HK-2 cells which cultured in vitro were divided into several groups randomly： blank control group,EPO induced group,albumin induced group,EPO inhibited group（5,10,20 U/mL）,inhibitor of TGF-β1 receptor group.24 hours later,reverse transcription polymerase chain reaction（RT-PCR） was used to evaluate the mRNA levels of TGF-β1 and ILK.The levels of E-cadherin and α-smooth muscle actin（α-SMA） proteins were assessed by immunofluorescent.ELISA was used to measure the levels of FN proteins in supernatant.【Results】Compared with blank control group the expression of TGF-β1,ILK mRNA was significantly increased in the albumin induced group（P 0.05）.EPO inhibited group TGF-β1,ILK mRNA expressions significantly decreased（P 0.05）.Immunofluorescent showed that the expression of E-cadherin proteins decreased and the expression of α-SMA proteins increased in the albumin induced group（P 0.05）.Meanwhile,the expression of E-cadherin proteins significantly increased and the expression of α-SMA proteins significantly decreased in the EPO inhibited group and inhibitor of TGF-β1 receptor group as compared to the albumin induced group（P 0.05）,and the effects were related to the concentration of EPO.ELISA method showed the expression of FN proteins significantly decreased in the EPO inhibited group and inhibitor of TGF-β1 receptor group when comparing with the albumin induced group（P 0.05）.There was positive correlation between the expression of TGF-β1 mRNA and ILK mRNA in the albumin induced group and EPO inhibited group by pearson correlation analysis.【Conclusion】TGF-β1/ILK signaling pathway is involved in albumin induced EMT.EPO can inhibit the process of EMT,and then protect the kidney,which may be achieved through its inhibition of TGF-β1/ILK signaling pathway.