Objective

Glomerular injury is a prominent pathological feature of diabetic kidney disease (DKD). Constitutively active NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species that mediates hyperglycemia-induced mesangial cell (MC) fibrotic injury. However, the mechanism that Nox4 utilizes to achieve its biological outcome remains elusive, and the signaling pathways that regulate this isoform oxidase are not well understood. Here, our goal is to study the detailed mechanism by which NAPDH oxidase 4 (Nox4) is post-transcriptionally regulated in MC during diabetic pathology.

Methods

We studied the protein expression of HuR, Nox4 and matrix proteins by western blotting, while we assessed the mRNA stability of Nox4 by RT-PCR and polysomal assay, examined in vitro cultured glomerular mesangial cells treated by high glucose (HG) and diabetic animal induced by STZ. The binding assay between HuR and the Nox4 promoter was done by immuno-precipiating with HuR antibody and detecting the presence of Nox4 mRNA, or by pull-down by using biotinlyated labeled Nox4 promoter RNA and detecting the presence of the HuR protein. The binding was also confirmed in MCs where Nox4 promoter-containing luciferage constructs were transfected. ROS levels were measured with DHE/DCF dyes in cells, or lucigenin chemiluminescence for Nox enzymatic levels, or HPLC assay for superoxide. HuR protein was inhibited by antisense oligo that utilized osmotic pumps for continuous delivery in animal models. The H1bAc1 ratio was measured by an ELISA kit for mice.

Results

We demonstrate that in MCs, high glucose (HG) elicits a rapid upregulation of Nox4 protein via translational mechanisms. Nox4 mRNA 3′ untranslated region (3′-UTR) contains numerous AU-rich elements (AREs) that are potential binding sites for the RNA-binding protein human antigen R (HuR). We show that HG promotes HuR activation/expression and that HuR is required for HG-induced Nox4 protein expression/mRNA translation, ROS generation, and subsequent MC fibrotic injury. Through a series of in vitro RNA-binding assays, we demonstrate that HuR acts via binding to AREs in Nox4 3′-UTR in response to HG. The in vivo relevance of these observations is confirmed by the findings that increased Nox4 is accompanied by the binding of HuR to Nox4 mRNA in kidneys from type 1 diabetic animals, and further suppressing HuR expression showed a reno-protective role in a type 1 diabetic mouse model via reducing MC injury, along with the improvement of hyperglycemia and renal function.

Conclusions

We established for the first time that HuR-mediated translational regulation of Nox4 contributes to the pathogenesis of fibrosis of the glomerular microvascular bed. Thus therapeutic interventions affecting the interplay between Nox4 and HuR could be exploited as valuable tools in designing treatments for DKD.

中文翻译：

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RNA结合蛋白HuR和Nox4之间的相互作用作为糖尿病肾病的新型治疗靶标。

目的

肾小球损伤是糖尿病肾病（DKD）的突出病理特征。组成型活性NADPH氧化酶4（Nox4）是反应性氧物种的主要来源，其介导高血糖诱导的系膜细胞（MC）纤维化损伤。但是，Nox4用来实现其生物学结果的机制仍然难以捉摸，并且调节这种同工型氧化酶的信号传导途径还不是很清楚。在这里，我们的目标是研究在糖尿病病理过程中NAPDH氧化酶4（Nox4）在MC中转录后调控的详细机制。

方法

我们通过蛋白质印迹研究了HuR，Nox4和基质蛋白的蛋白表达，同时通过RT-PCR和多体体分析评估了Nox4的mRNA稳定性，检查了高糖（HG）处理的体外培养肾小球系膜细胞和糖尿病动物诱导的肾小球系膜细胞的表达。由STZ。通过用HuR抗体免疫沉淀并检测Nox4 mRNA的存在，或通过使用生物素标记的Nox4启动子RNA的下拉并检测HuR蛋白的存在，进行HuR与Nox4启动子之间的结合测定。在转染了含有Nox4启动子的荧光素构建体的MC中也证实了结合。用细胞中的DHE / DCF染料或lucingenin化学发光法测定Nox酶水平，或用HPLC法测定过氧化物，测定ROS水平。HuR蛋白受到反义寡核苷酸的抑制，该寡核苷酸利用渗透泵在动物模型中连续递送。通过用于小鼠的ELISA试剂盒测量H1bAc1比率。

结果

我们证明在MCs中，高葡萄糖（HG）通过翻译机制引起Nox4蛋白的快速上调。Nox4 mRNA 3'非翻译区（3'-UTR）包含许多富含AU的元件（ARE），它们是RNA结合蛋白人类抗原R（HuR）的潜在结合位点。我们表明，HG促进HuR激活/表达，并且HuR是HG诱导的Nox4蛋白表达/ mRNA翻译，ROS生成和随后的MC纤维化损伤所必需的。通过一系列的在 体外RNA结合测定法，我们证明了通过的HuR响应于HG结合在的Nox4 3'-UTR的ARE作用。该在 体内 这些发现的相关性被以下发现所证实：Nox4的增加伴随着HuR与1型糖尿病动物肾脏中的Nox4 mRNA结合，并且进一步抑制HuR表达在1型糖尿病小鼠模型中表现出了肾脏保护作用，通过降低MC损伤，以及高血糖症和肾功能的改善。

结论

我们首次确定了HuR介导的Nox4的翻译调控有助于肾小球微血管床纤维化的发病机理。因此，影响Nox4和HuR之间相互作用的治疗性干预措施可被用作设计DKD治疗的有价值的工具。