Egr-1下调Klotho促进糖尿病肾病进展的作用及机制

Diabetic nephropathy (DN) is one of the most common causes of chronic kidney Disease (CKD) leading to premature death and end-stage renal disease (ESRD). The progress of DN are characterized by the accumulation of extracellular matrix (ECM) in the glomeruli (glomerular fibrosis, glomerulosclerosis) and the tubular interstitium (tubulointerstitial fibrosis). Recently, early growth response protein 1 (Egr-1) has been proven to play a key role in the organ fibrosis by combination to the gene promoters of not only most components of ECMs but also transforming growth factor beta (TGF-β). Our pilot study show that the expressions of Egr-1 in the kidneys of OLETF rats, a kind of spontaneous type 2 diabetic animal model, were upregulated, while the Klotho, a newly recognized hormone-like protein from kidney with kidney profective effects, was downregulated. In vitro, we found that overexpression of Egr-1 by transfecting with M61-Egr-1 plasmid aggravated the kidney damage accompanied by the decreased expression of Klotho, which can be reversed by siEgr-1. Further, we found that there were Egr-1 binding sites in the gene promoter of Klotho. Thus, We hypothesis that Egr-1 promotes the progress of DN through negative transcriptional regulation of klotho via binding to the gene promoter of Klotho. In the present study, we employ the rat glomeruler mesangial cells incubated with high glucose, advanced glycation end-products (AGEs) or TGF-β1 respectively to mimic the diabetic condition. Then, the cells were transfected by Ad-Egr-1 or shEgr-1 to overexpress or knock-down Egr-1 gene. The fibronectin (FN), collagen (Col) I, III and IV are determined by RT-qPCR and Western Blotting respectively. The expressions of Egr-1, klotho, TGF-β, p-/t-Smad3, p-/t-ERK1/2 and p-/t-p38MAPK were analyzed. Further, the cells were transfected by Ad-Klotho and shKlotho to verify the protective role of Klotho and the relationship between Klotho and Egr-1. The cells with Egr-1 overexpression will be treated by exogenous klotho protein of different concentrations. Then ChIP, luciferase report assay, site-specific mutation of Klotho gene promoter will be employed to analyze the direct relationship between the klotho gene promoter and Egr-1 protein. At last, Ad-Klotho, shKlotho, Ad-Egr-1, shEgr-1 will be injected to db/db mice with DN respectively to verify the role and the relationship of klotho and Egr-1 in the progress of DN. Finally, we will elucidate the mechanisms of Egr-1 in progress of DN through negative transcriptional regulation of Klotho by directly binding to the gene promoter of Klotho, which is a potential target for the prevention of DN progress.

早期生长反应蛋白-1（Egr-1）是器官纤维化的关键调控因子，我们在前期研究中发现，糖尿病肾病（DN）大鼠肾脏Egr-1显著升高而Klotho降低，过表达Egr-1明显下调系膜细胞Klotho，siEgr-1能上调Klotho，而Klotho启动子区存在多个Egr-1结合位点，推测糖尿病时Egr-1上调使Klotho抑制而促进DN进展，但目前未见报道。本课题拟利用Egr-1稳定过表达和基因敲减的大鼠系膜细胞，分别以Klotho过表达或基因敲减质粒转染上述细胞，观察细胞增殖及功能变化，探讨Egr-1对Klotho的调控作用；以ChIP、荧光素酶报告基因及启动子定位突变验证Egr-1对Klotho的负调控；最后分别以Klotho或Egr-1过表达或基因敲减腺病毒转染DN小鼠模型，在动物模型验证上述作用。本课题将阐明Egr-1下调Klotho促进DN进展的作用及机制，可能是防治DN潜在的新靶点。

糖尿病肾脏病（DKD）已成为目前导致终末期肾病的主要原因，如何有效延缓 DKD 进展是目前研究的热点和难点，然而目前尚无有效的治疗措施。因此，深入探讨 DKD 发病机制，寻找调控 DKD 进展的关键靶点，对于防治 DKD 进展具有十分重要的意义。最近研究发现，Egr-1和Klotho是器官纤维化的重要调控因子，但它们在DKD中的作用及其之间的调控机制并不十分清楚。本研究主要围绕Egr-1和Klotho开展相关实验。我们主要采用高糖诱导的系膜细胞和TGF-β1诱导的肾小管上皮细胞为细胞模型、高脂饮食和链脲佐菌素诱导的糖尿病小鼠为动物模型，并用RT-PCR、western blot、免疫组化、染色质免疫共沉淀等方法进行相关实验。主要结果如下：1、DM小鼠肾脏Klotho和Egr-1成负相关关系2、Klotho可通过 TGF-β1/Smad3信号通路下调人系膜细胞系中Egr-1的表达，进而抑制高糖诱导的肾小球系膜基质沉积；3、在糖尿病小鼠模型中敲减Egr-1可延缓糖尿病肾脏病进展，而过表达Egr-1则可促进糖尿病肾病进展，这可能是通过NOX4介导的氧化应激产生的作用。4、Egr-1可能通过上调lncRNA Arid2-IR促进肾小球系膜基质沉积。5、在糖尿病小鼠模型中过表达Klotho延缓糖尿病肾脏病进展，可能通过LncNEAT1调控ERK1/2 通路抑制肾小管上皮细胞EMT发生。本研究深入地阐明在DKD状态下，Klotho和Egr-1之间的调控机制以及Klotho和Egr-1在DKD肾脏纤维化中的作用及可能的作用机制，为防治DKD提供新的潜在靶点和治疗途径。