Wang Yi-Han, Teng Yan, Yao Fang-Lan, Wei Shu-Qi, Li Yan
Department of Kidney Transplantation, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China.
Emergency Intensive Care Unit, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China.
Kaohsiung J Med Sci. 2026 Mar 2:e70188. doi: 10.1002/kjm2.70188.
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.
肾脏缺血再灌注损伤（IRI）是急性肾损伤（AKI）的一个重要临床因素。本研究旨在探讨m6A甲基化调节剂RNA结合基序蛋白X连锁（RBMX）在肾脏IRI中的新作用，并研究相关的调控机制。使用经缺氧复氧（H/R）处理的HK-2细胞建立体外肾脏IRI模型。为了研究RBMX的作用，用pcDNA/RBMX转染RBMX过表达细胞。使用CCK-8法评估HK-2细胞的活力。利用EdU评估HK-2细胞的增殖。进行蛋白质印迹分析以确定参与NLRP3炎性小体激活的蛋白质的表达水平。ELISA用于测量与细胞焦亡相关的炎性细胞因子的分泌。LDH和PI染色用于研究细胞焦亡。进行IP、RIP和MeRIP实验以检测NLRP3的乙酰化以及NLRP3与SIRT3之间的相互作用。建立体内IRI小鼠模型以进一步验证RBMX的肾脏保护作用。我们的结果表明，RBMX在IRI小鼠和体外H/R处理的HK-2细胞中的表达显著下调。在H/R诱导的HK-2细胞中，RBMX过表达减弱了NLRP3炎性小体的激活和细胞焦亡，表现为NLRP3、ASC、裂解的caspase-1和GSDMD-N的表达水平降低，以及IL-18、IL-1β、TNF-α和IL-6水平的下降。此外，RBMX与SIRT3的m6A甲基化有关，SIRT3参与了H/R处理的HK-2细胞中NLRP3乙酰化和激活的控制。SIRT3敲低逆转了RBMX对细胞增殖、NLRP3炎性小体激活和细胞焦亡的影响。此外，EZH2可能参与介导RBMX的H3K27me3修饰的上游基因。最后，体内实验提供的证据表明RBMX过表达改善了小鼠的肾损伤。综上所述，我们的数据支持m6A调节剂RBMX在肾脏IRI中可能通过调节SIRT3的m6A甲基化来抑制NLRP3炎性小体激活和细胞焦亡的潜在作用。我们推测靶向EZH2/RBMX/SIRT3轴可能代表一种新的治疗方法来阻止肾脏IRI的进展。
