FTO mediates the diabetic kidney disease progression through regulating the m6A modification of NLRP3.

IF 2.2 4区 医学 Q2 UROLOGY & NEPHROLOGY
Qiang Li, Shujuan Mu
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引用次数: 0

Abstract

Background: The objective of our research was to investigate the specific mechanism of FTO in diabetic kidney disease (DKD) progression.

Methods: The DKD model was established with renal tubular epithelial HK-2 cells and mice in vitro and in vivo. The N6-methyladenosine (m6A) content in cells was detected using dot plot assay and the m6A levels of NLRP3 was detected with the MeRIP assay. The mRNA and protein levels were tested with real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and western blot. The IL-1β and IL-18 levels were assessed with enzyme-linked immunosorbent assay (ELISA). The cell viability was measured by cell counting kit (CCK)-8 assay and cell pyroptosis was determined with Annexin V and propidium iodide (PI) double staining followed by flow cytometry analysis. RNA-binding protein immunoprecipitation (RIP) and dual luciferase reporter assays were conducted to detect the interaction between FTO and NLRP3. m6A levels were detected by Me-RIP assay. The renal injury was measured by observing the renal morphology and urine and blood levels of relevant indicators.

Results: The results indicated that high glucose treatment induced HK-2 cell pyroptosis. m6A levels were prominently elevated in high glucose treated HK-2 cells while FTO expression were significantly down-regulated. FTO over-expression promoted cell viability but inhibited pyroptosis of HK-2 cells under high glucose (HG) treatment. Moreover, FTO could inhibit NLRP3 expression. RIP and Me-RIP assays indicated that FTO could bind with NLRP3 and regulate its m6A modification level. Further luciferase assay confirmed that FTO binds with the 233-237 bp region of NLRP3. NLRP3 neutralized the function of FTO in the HG stimulated HK-2 cells. In vivo, the H&E staining showed that FTO over-expression alleviated the kidney injury and suppressed the pyroptosis induced by DKD.

Conclusion: We found that FTO could inhibit the DKD progression in vivo and in vitro by regulated the m6A modification of NLRP3.

FTO 通过调节 NLRP3 的 m6A 修饰介导糖尿病肾病的进展。
研究背景我们的研究目的是探讨 FTO 在糖尿病肾病(DKD)进展中的具体机制:用肾小管上皮 HK-2 细胞和小鼠在体外和体内建立 DKD 模型。方法:利用肾小管上皮 HK-2 细胞和小鼠在体外和体内建立了 DKD 模型,采用点阵图法检测细胞中 N6-甲基腺苷(m6A)的含量,采用 MeRIP 法检测 NLRP3 的 m6A 水平。实时逆转录聚合酶链反应(RT-qPCR)和免疫印迹法检测了 mRNA 和蛋白质水平。用酶联免疫吸附试验(ELISA)评估了 IL-1β 和 IL-18 的水平。细胞活力通过细胞计数试剂盒(CCK)-8测定,细胞凋亡通过Annexin V和碘化丙啶(PI)双染色测定,然后进行流式细胞术分析。进行了 RNA 结合蛋白免疫沉淀(RIP)和双荧光素酶报告实验,以检测 FTO 和 NLRP3 之间的相互作用。通过观察肾脏形态、尿液和血液中相关指标的水平来测量肾脏损伤:结果表明,高糖处理可诱导 HK-2 细胞发生脓毒症。高糖处理的 HK-2 细胞中 m6A 水平显著升高,而 FTO 的表达则明显下调。FTO 的过度表达促进了高糖处理下 HK-2 细胞的存活,但抑制了其热凋亡。此外,FTO 还能抑制 NLRP3 的表达。RIP和Me-RIP实验表明,FTO能与NLRP3结合并调节其m6A修饰水平。进一步的荧光素酶实验证实,FTO与NLRP3的233-237 bp区域结合。在 HG 刺激的 HK-2 细胞中,NLRP3 中和了 FTO 的功能。体内H&E染色显示,FTO的过度表达减轻了DKD对肾脏的损伤,并抑制了DKD诱导的肾脏热解:结论:我们发现 FTO 可通过调节 NLRP3 的 m6A 修饰抑制 DKD 在体内和体外的发展。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
BMC Nephrology
BMC Nephrology UROLOGY & NEPHROLOGY-
CiteScore
4.30
自引率
0.00%
发文量
375
审稿时长
3-8 weeks
期刊介绍: BMC Nephrology is an open access journal publishing original peer-reviewed research articles in all aspects of the prevention, diagnosis and management of kidney and associated disorders, as well as related molecular genetics, pathophysiology, and epidemiology.
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