<scp>RBMX</scp> Transcriptionally Repressed by <scp>EZH2</scp> ‐Associated <scp>H3K27me3</scp> Modification Attenuates Pyroptosis in Renal Ischemia/Reperfusion Injury via Regulating <scp>SIRT3</scp> / <scp>NLRP3</scp> Inflammasome Activation

Ischemic reperfusion injury (IRI) to kidney is a significant clinical factor in acute kidney injury (AKI). This study aimed to investigate the new role of RNA-binding motif protein X-linked (RBMX), a modulator of m6A methylation, in renal IRI and to examine the associated regulatory mechanisms. An in vitro renal IRI model was established using HK-2 cells subjected to hypoxia-reoxygenation (H/R) treatment. To investigate the role of RBMX, RBMX-overexpressing cells were transfected with pcDNA/RBMX. The viability of HK-2 cells was evaluated using the CCK-8 assay. EdU was utilized to evaluate cell proliferation in HK-2 cells. Western blot analysis was conducted to determine the expression levels of proteins involved in NLRP3 inflammasome activation. ELISA was used to measure the secretion of inflammatory cytokines linked to pyroptosis. LDH and PI staining were used to investigate pyroptosis. IP, RIP, and MeRIP assays were performed to detect NLRP3 acetylation and the interaction between NLRP3 and SIRT3. An in vivo IRI mouse model was established to further validate the renoprotective effect of RBMX. Our results showed that RBMX expression was significantly downregulated in IRI mice and in vitro H/R-treated HK-2 cells. In H/R-induced HK-2 cells, RBMX overexpression attenuated NLRP3 inflammasome activation and pyroptosis, shown by reduced expression levels of NLRP3, ASC, cleaved caspase-1, and GSDMD-N, along with decreased levels of IL-18, IL-1β, TNF-α, and IL-6. Additionally, RBMX is associated with the m6A methylation of SIRT3, which is involved in the control of NLRP3 acetylation and activation in H/R-exposed HK-2 cells. SIRT3 knockdown reversed the impacts of RBMX on cell proliferation, NLRP3 inflammasome activation, and pyroptosis. Moreover, EZH2 may be involved in an upstream gene that mediates the H3K27me3 modification of RBMX. Finally, in vivo assays provided evidence suggesting that RBMX overexpression improved renal injury in mice. Taken together, our data support a potential role of the m6A regulator RBMX in suppressing NLRP3 inflammasome activation and pyroptosis possibly through the regulation of m6A methylation of SIRT3 in renal IRI. We hypothesized that targeting the EZH2/RBMX/SIRT3 axis might represent a new therapeutic approach to impede the progression of renal IRI.