METTL3-driven m6A modification of NLRC5 promotes renal fibrosis in chronic kidney disease through Keap1/Nrf2/ARE signaling pathway.

BACKGROUND

METTL3-mediated m6A RNA methylation has been implicated in renal fibrosis, a central pathological feature of chronic kidney disease (CKD). NLRC5, the largest NLR family member, is a direct m6A target of METTL3, but its role in METTL3-driven renal fibrosis remains unclear.

METHODS

An in vitro renal fibrosis model was established using TGF-β1-stimulated human proximal tubular (HK-2) cells. METTL3-mediated m6A modification and stabilization of NLRC5 mRNA were assessed by m6A quantification, RNA stability, MeRIP, and RIP assays. Functional impacts on the Keap1/Nrf2/ARE pathway and fibrotic responses were examined using METTL3 inhibition (STM2457, 10 μM), NLRC5 knockdown or overexpression, Keap1 overexpression, and Nrf2 inhibition (ML385, 5 μM). Fibrotic markers, inflammatory cytokines (IL-1β, TNF-α; ELISA), and oxidative stress (ROS/DCF-DA, SOD, MDA) were measured. NLRC5-overexpression effects on the Keap1/Nrf2/ARE pathway were additionally evaluated. In vivo validation employed a unilateral ureteral obstruction (UUO) mouse model, with kidney injury and fibrosis assessed via H&E, Masson's staining, IHC, ELISA, Western blot, and qRT-PCR.

RESULTS

TGF-β1 upregulated METTL3, NLRC5, and global m6A levels in HK-2 cells. METTL3 directly bound and stabilized NLRC5 mRNA via m6A modification. METTL3 overexpression exacerbated TGF-β1-induced inflammation, oxidative stress, and fibrosis, which were reversed by STM2457. Conversely, METTL3 or NLRC5 inhibition suppressed fibrosis, coinciding with Keap1 downregulation and Nrf2/HO-1/NQO1 upregulation. Keap1 overexpression negated the anti-fibrotic effects of NLRC5 knockdown, while NLRC5 overexpression decreased nuclear Nrf2 and downstream antioxidant targets, confirming NLRC5's inhibitory role on Keap1/Nrf2 signaling. Nrf2 inhibition (ML385) or NLRC5 overexpression rescued METTL3 knockdown phenotypes. In vivo, METTL3 knockdown attenuated UUO-induced renal injury and fibrosis, activating the Keap1/Nrf2/ARE pathway.

CONCLUSIONS

METTL3 promotes renal fibrosis by stabilizing NLRC5 mRNA via m6A modification, leading to suppression of the protective Keap1/Nrf2/ARE pathway. Targeting the METTL3/NLRC5/Keap1/Nrf2/ARE axis may represent a promising therapeutic strategy for CKD-associated fibrosis.