Klotho-Egr-1-miR-31介导糖尿病肾脏病肾间质纤维化的分子机制

Tubulointerstitial fibrosis (TIF) is crucial in the development of renal fibrosis in diabetic kidney disease (DKD), but the mechanism is not clear yet. In the preliminary experiments, we have demonstrated that Klotho protein level was obviously declined and miR-31 expression was up-regulated in kidney of diabetic nephropathy mouse model. Klotho decreased luciferase activity of miR-31 and weakened the binding of Egr-1 and miR-31 promoter in high glucose induced HK-2 cells. HK-2 cells transfected with miR-31 mimic revealed remarkably increased expression of FN, COL1 and αSMA mRNA and protein levels. HK-2 cells transfected with inhibitor revealed the opposite result. The data from Dual-luciferase activity assay demonstrated that miR-31 can directly target the 3’UTR of FIH-1 mRNA. Based on these findings, we speculate that Klotho could prevent tubulointerstitial fibrosis in diabetic kidney disease via downregulation of Egr-1-miR-31. We will use the RTEC-specific klotho-knockout mice Kap-cre and clarify the mechanism of klotho in regulating RTEC miR-31 by both in vitro and in vivo experiments, explore and verify the molecular mechanism of Klotho down-regulates Egr-1-miR-31 and miR-31 aggravate fibrosis via downregulating FIH-1 expression. This study will provide a theoretical basis for delaying DKD progression and searching for novel marker.

肾小管间质纤维化在糖尿病肾脏病(DKD)进展中起着非常重要的作用，然而其机制尚未完全阐明。预实验发现：糖尿病肾病小鼠模型肾皮质Klotho水平明显下降，而miR-31表达升高；高糖环境中对人肾小管上皮细胞过表达Klotho可以负性调控Egr-1荧光素酶活性并减弱高糖环境中Egr-1和miR-31启动子的结合；且发现miR-31能够靶作用于FIH-1发挥致肾纤维化的作用。基于这些发现，我们推测：Klotho可能通过Egr-1-miR-31介导糖尿病肾脏病肾间质纤维化。围绕该假说，我们拟使用近曲小管特异性Klotho敲除小鼠和原代肾小管上皮细胞进一步探索和验证Klotho通过Egr-1-miR-31影响糖尿病肾脏病肾间质纤维化，以及miR-31靶向调节FIH-1基因的表达介导肾间质纤维化的分子机制，以期为延缓DKD进展寻找潜在靶点提供理论基础。

肾小管间质纤维化在糖尿病肾脏病(DKD)进展中有非常重要的作用，然而其机制尚未完全阐明。本项目研究发现：（1）高糖诱导的HK-2细胞中miR-31表达逐渐升高，FIH-1表达逐渐下降,呈负性相关关系；miR-31mimic转染HK-2细胞后可见FIH-1表达下降，纤维化因子表达增加。miR-31 inhibitor转染后可见FIH-1表达升高，纤维化因子表达下降。（2）共转染miR-31 mimic和野生型载体可以减弱野生型载体的荧光素酶活性；而共转染miR-31 inhibitor后得到相反结果。（3）肾小管上皮细胞敲除Egr-1后，再给予低糖和高糖刺激，结果显示敲除Klotho通过上调Egr-1的活性从而影响细胞内miR-31的表达水平；随着高糖刺激时间延长，Klotho的蛋白表达逐渐减低；而Egr-1和miR-31表达逐渐增加；在高糖中增加Klotho浓度可以减低miR-31的表达；高糖可以增加Egr-1和miR-31promotor的结合；而klotho可以减弱Egr-1和miR-31promotor的结合。本项目揭示了Klotho通过Egr-1调控miR-31表达的分子机制，为Klotho参与肾纤维化的分子机制提供了较为充分的实验依据，也为未来的临床转化研究提供了新的研究靶点。