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

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.