论著

可溶性Klotho蛋白抑制高糖诱导的STAT3磷酸化通路减轻肾纤维化

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  • 上海交通大学医学院附属新华医院肾脏科,上海 200092

收稿日期: 2017-06-28

  网络出版日期: 2017-08-25

基金资助

上海市科委医学引导类(西医)科技支撑项目(134119a5700)

Soluble Klotho attenuates high glucose-induced renal fibrosis through inhibiting STAT3 pathway

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  • Department of Nephrology, Xinhua Hospital,. Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China

Received date: 2017-06-28

  Online published: 2017-08-25

摘要

目的: 观察可溶性Klotho蛋白对高糖诱导的信号转导及转录激活蛋白3(signal transducer and activator of transcription,STAT3)信号通路磷酸化和肾小管上皮细胞纤维化相关基因表达的影响。方法: 采用体外培养的HK2肾小管上皮细胞,分别设立高糖组(D-葡萄糖30 mmol/L)、正常糖对照组(D-葡萄糖 5 mmol/L)、高渗透压对照组(D-葡萄糖5 mmol/L+甘露醇25 mmol/L)、Klotho干预高糖组和HO3867(STAT3磷酸化抑制剂)干预高糖组,应用蛋白印迹法、实时荧光定量PCR(real time fluorescence quantitative PCR, rt-fqPCR)等检测方法分别观察STAT3磷酸化水平变化和肾小管上皮细胞纤维化相关因子的表达水平变化。结果: ①rt-fqPCR检测结果表明,与正常糖对照组相比,高糖组 HK2细胞内源性Klotho mRNA表达显著下降(P<0.01),而高渗透压对照组无改变(P>0.05);②蛋白印迹检测结果表明,与正常糖组相比,高糖可上调HK2细胞内的STAT3磷酸化水平(P<0.01),而可溶性Klotho蛋白干预可显著抑制高糖引起的STAT3磷酸化激活(P<0.01);STAT3磷酸化抑制剂(HO3867)干预可显著抑制高糖诱导的STAT3磷酸化水平升高(P<0.01);③rt-fqPCR检测结果表明,高糖组HK2细胞表达的纤维化相关因子(α-平滑肌肌动蛋白、Ⅳ型胶原蛋白α1、纤维连接蛋白)较正常糖对照组升高(P<0.01)。Klotho蛋白可抑制高糖诱导的HK2细胞表达纤维化相关因子(α平滑肌肌动蛋白、Ⅳ型胶原蛋白α1、纤维连接蛋白)(P<0.01)。应用STAT3磷酸化抑制剂(HO3867)可部分下调高糖诱导的纤维化相关因子表达(P<0.01)。结论: 可溶性Klotho蛋白可抑制高糖诱导的肾小管上皮细胞纤维化,其机制可能是部分通过抑制STAT3磷酸化通路实现的。

本文引用格式

李慧凛, 吴萍, 刘爽, 蒋更如 . 可溶性Klotho蛋白抑制高糖诱导的STAT3磷酸化通路减轻肾纤维化[J]. 诊断学理论与实践, 2017 , 16(04) : 371 -376 . DOI: 10.16150/j.1671-2870.2017.04.006

Abstract

Objective: To observe the effect of Klotho on high glucose-activated phosphorylation of signal transducer and activator of transcription 3 (STAT3) and expression of fibrosis related gene in renal tubular epithelial cells under high glucose. Methods: Human proximal tubular epithelial cells (HK2) were cultured and incubated with high glucose (HG, D-glucose 30 mmol/L), normal glucose (NG, D-glucose 5 mmol/L), high mannitol (HM, D-glucose 5 mmol/L and mannitol 25 mmol/L), and Klotho or HO3867 in the presence of high glucose. STAT3 activation and expression of target genes were assayed by Western blot or real time fluorescence quantitative PCR(rt-fqPCR). Results: Compared with NG or HM, a significant decrease of endogenous Klotho gene level was detected after HG treatment(P<0.01). Western blot analysis revealed that the level of STAT3 phosphorylation was significantly increased after HG treatment compared with NG or HM(P<0.01). The activation of STAT3 induced by high glucose was markedly reduced by Klotho or HO3867 treatment(P<0.01). The level of mRNA expression of fibrosis related gene(α-SMA, Col4A1, FN) was significantly increased after high glucose treatment in HK-2 cells (P<0.01), while was markedly inhibited by Klotho treatment(P<0.01). HO3867, the inhibitor of STAT3, partially decreased the mRNA levels of above mentioned genes (P<0.01). Conclusions: Soluble Klotho could attenuate high glucose-induced renal fibrosis, which might be achieved partially through inhibition of STAT3 pathway.

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