Abstract

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, a class of systemic autoimmune diseases, results in damage of various critical organs including kidneys, lungs, eyes, and nervous system. MicroRNA-340-5p was confirmed to be downregulated in autoimmune pathogenesis. However, the role of miR-340-5p remains unknown in ANCA-induced glomerulonephritis (GN). The current study aimed to explore the role of miR-340-5p in ANCA-induced GN. The animal models of ANCA-induced GN was established by experimental autoimmune vasculitis (EAV) operation. The primary glomerular endothelial cells (PGEnCs) were treated with anti-myeloperoxidase (anti-MPO) to mimic cell injury in vitro. The renal function was analysed by measuring serum creatinine, blood urea nitrogen, urine blood, urine protein and urine leukocytes. The levels of RNA and proteins were examined by RT-qPCR and western blot analysis, respectively. The binding capacity between miR-340-5p and KDM4C was detected by luciferase reporter assay. Cell apoptosis was analysed by flow cytometry in vitro. Cell viability was determined by CCK-8 assay. The cleaved caspase-3 activity was analysed by immunofluorescent assay. Cell inflammation was measured by western blot. Cell procoagulant activity was assessed by FXa generation assay. The histological changes of renal tissues were assessed by Haematoxylin and eosin (H&E) staining assay. The correlation between miR-340-5p and KDM4C level (or content of TNF-α and IL-6) was analysed by Pearson correlation analysis. The injection of anti-MPO IgG induced a significant elevation of Serum creatinine and blood urea nitrogen in serum, as well as urine blood, urine protein and urine leukocytes. Importantly, KDM4C was downregulated in model group. In mechanism, we identified that miR-340-5p bound with KDM4C 3'untranslated region (UTR), negatively regulated KDM4C in endothelial cells and negatively correlated with KDM4C in serum of GN rats. In function, we found that miR-340-5p promoted B cell activation and proliferation by downregulating KDM4C. The in vitro assays showed that the decrease of cell viability induced by anti-MPO was reversed by miR-340-5p overexpression, and further reduced by KDM4C overexpression. Inversely, the suppressive effects of miR-340-5p mimics on cell apoptosis, cleaved caspase-3 activity, inflammatory response and pro-coagulation were countervailed by KDM4C overexpression in anti-MPO-treated cells. The in vivo assays validated that miR-340-5p overexpression mitigated the impairment of renal function, and histological changes induced by anti-MPO IgG injection in model group. Finally, we found the negative correlation between miR-340-5p and TNF-α (or IL-6) content in serum of GN rats. In conclusion, we found that miR-340-5p inhibited endothelial apoptosis and inflammatory response by targeting KDM4C in ANCA-mediated GN through activation of B cells, implying a potential novel insight for treatment of ANCA-mediated GN.

摘要

抗中性粒细胞胞浆抗体（ANCA）相关性血管炎是一类全身性自身免疫性疾病，会导致包括肾、肺、眼睛和神经系统在内的各种重要器官受损。MicroRNA-340-5p 被证实在自身免疫发病机制中被下调。然而，miR-340-5p 在 ANCA 诱导的肾小球肾炎 (GN) 中的作用仍然未知。本研究旨在探讨 miR-340-5p 在 ANCA 诱导的 GN 中的作用。通过实验性自身免疫性血管炎（EAV）手术建立ANCA诱导的GN动物模型。用抗髓过氧化物酶 (anti-MPO) 处理原代肾小球内皮细胞 (PGEnCs) 以模拟体外细胞损伤. 通过测量血清肌酐、血尿素氮、尿血、尿蛋白和尿白细胞来分析肾功能。分别通过 RT-qPCR 和蛋白质印迹分析检查 RNA 和蛋白质的水平。miR-340-5p与KDM4C的结合能力通过萤光素酶报告基因检测。体外流式细胞仪分析细胞凋亡. 通过CCK-8测定确定细胞活力。通过免疫荧光测定分析切割的caspase-3活性。通过蛋白质印迹测量细胞炎症。通过 FXa 生成测定评估细胞促凝血活性。通过苏木精和伊红（H＆E）染色测定评估肾组织的组织学变化。通过Pearson相关分析分析miR-340-5p与KDM4C水平（或TNF-α和IL-6含量）的相关性。注射抗 MPO IgG 导致血清中血清肌酐和血尿素氮以及尿血、尿蛋白和尿白细胞显着升高。重要的是，KDM4C 在模型组中被下调。在机制上，我们发现 miR-340-5p 与 KDM4C 3'非翻译区 (UTR) 结合，内皮细胞中的 KDM4C 负调控，与 GN 大鼠血清中的 KDM4C 呈负相关。在功能上，我们发现 miR-340-5p 通过下调 KDM4C 促进 B 细胞活化和增殖。这体外试验表明，抗 MPO 诱导的细胞活力降低被 miR-340-5p 过表达逆转，KDM4C 过表达进一步降低。相反，抗 MPO 处理的细胞中 KDM4C 过表达抵消了 miR-340-5p 模拟物对细胞凋亡、切割的 caspase-3 活性、炎症反应和促凝血的抑制作用。体内_试验证实 miR-340-5p 过表达减轻了模型组中抗 MPO IgG 注射引起的肾功能损害和组织学变化。最后，我们发现 GN 大鼠血清中 miR-340-5p 与 TNF-α（或 IL-6）含量呈负相关。总之，我们发现 miR-340-5p 通过激活 B 细胞靶向 ANCA 介导的 GN 中的 KDM4C 来抑制内皮细胞凋亡和炎症反应，这意味着治疗 ANCA 介导的 GN 的潜在新见解。