FTO 通过调节 NLRP3 的 mA 修饰介导糖尿病肾病的进展。
FTO mediates the diabetic kidney disease progression through regulating the mA modification of NLRP3.
机构信息
Department of Nephrology, Guang'anmen Hospital South Campus, China Academy of Chinese Medical Sciences, No.138, Xingfeng Street, Huangcun Village, DaXing District, Beijing, 102600, China.
出版信息
BMC Nephrol. 2024 Oct 11;25(1):345. doi: 10.1186/s12882-024-03741-5.
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 (mA) content in cells was detected using dot plot assay and the mA 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. mA 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. mA 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 mA 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 mA modification of NLRP3.
背景
本研究旨在探讨 FTO 在糖尿病肾病（DKD）进展中的具体作用机制。
方法
通过体外和体内的肾小管上皮 HK-2 细胞和小鼠建立 DKD 模型。使用点图分析检测细胞中的 N6-甲基腺苷（mA）含量，并使用 MeRIP 分析检测 NLRP3 的 mA 水平。使用实时逆转录-聚合酶链反应（RT-qPCR）和蛋白质印迹法检测 mRNA 和蛋白水平。使用酶联免疫吸附测定法（ELISA）评估 IL-1β 和 IL-18 水平。通过细胞计数试剂盒（CCK-8）测定法测量细胞活力，并通过 Annexin V 和碘化丙啶（PI）双重染色后进行流式细胞术分析来确定细胞焦亡。进行 RNA 结合蛋白免疫沉淀（RIP）和双荧光素酶报告基因测定以检测 FTO 与 NLRP3 之间的相互作用。使用 Me-RIP 分析检测 mA 水平。通过观察肾脏形态和尿液及血液中相关指标的水平来评估肾脏损伤。
结果
结果表明，高糖处理诱导 HK-2 细胞发生焦亡。高糖处理的 HK-2 细胞中 mA 水平显著升高，而 FTO 表达显著下调。在高糖（HG）处理下，FTO 过表达促进细胞活力，但抑制 HK-2 细胞的焦亡。此外，FTO 可抑制 NLRP3 的表达。RIP 和 Me-RIP 分析表明，FTO 可与 NLRP3 结合并调节其 mA 修饰水平。进一步的荧光素酶测定证实，FTO 与 NLRP3 的 233-237 bp 区域结合。NLRP3 中和了 FTO 在 HG 刺激的 HK-2 细胞中的作用。在体内，H&E 染色显示，FTO 过表达可减轻肾脏损伤并抑制 DKD 诱导的焦亡。
结论
我们发现，FTO 可通过调节 NLRP3 的 mA 修饰来抑制体内和体外的 DKD 进展。
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