{"title":"FTO mediates the diabetic kidney disease progression through regulating the m<sup>6</sup>A modification of NLRP3.","authors":"Qiang Li, Shujuan Mu","doi":"10.1186/s12882-024-03741-5","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>The objective of our research was to investigate the specific mechanism of FTO in diabetic kidney disease (DKD) progression.</p><p><strong>Methods: </strong>The DKD model was established with renal tubular epithelial HK-2 cells and mice in vitro and in vivo. The N6-methyladenosine (m<sup>6</sup>A) content in cells was detected using dot plot assay and the m<sup>6</sup>A 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. m<sup>6</sup>A 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.</p><p><strong>Results: </strong>The results indicated that high glucose treatment induced HK-2 cell pyroptosis. m<sup>6</sup>A 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 m<sup>6</sup>A 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.</p><p><strong>Conclusion: </strong>We found that FTO could inhibit the DKD progression in vivo and in vitro by regulated the m<sup>6</sup>A modification of NLRP3.</p>","PeriodicalId":9089,"journal":{"name":"BMC Nephrology","volume":"25 1","pages":"345"},"PeriodicalIF":2.2000,"publicationDate":"2024-10-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11468296/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"BMC Nephrology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1186/s12882-024-03741-5","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"UROLOGY & NEPHROLOGY","Score":null,"Total":0}
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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.
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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