#2501 Mettl3-mediated m6A methylation of Ndrg1 promotes acute kidney injury sensitivity in diabetic kidney diseases

Abstract Background and Aims The incidence of acute kidney injury (AKI) in patients is associated with diabetic kidney. Nevertheless, the precise underlying mechanism remains unclear. We previously revealed that methyltransferase 3 (METTL3) mediated N6-methyladenosine (m6A) RNA modifications play critical roles in kidney injuries. This study aimed to explore whether METTL3 promotes AKI sensitivity in diabetic kidney diseases (DKD). Method AKI risk in patients with diabetes was assessed through GWAS. The m6A levels and METTL3 were measured in type 1 and type 2 diabetic mice experiencing AKI. METTL3 knockout or silencing was performed to evaluate its impact on ischemia-reperfusion (I/R)-induced AKI in DKD animals and hypoxia-reoxygenation (HR) and high glucose (HG) treated renal tubular epithelial cells (TECs). Furthermore, downstream targets of METTL3 were identified through m6A sequencing to elucidate its role in the pathogenesis of AKI in DKD. Results The GWAS data from 4111 DKD patients and 5908 AKI patients revealed a 16% higher risk of AKI in DN patients. Our findings confirmed that I/R resulted more severe AKI in both STZ induced type 1 and db/db type 2 diabetes models, leading to renal dysfunction, renal tubular damage, and inflammatory response, suggesting an increased susceptibility to AKI in DKD. Induction of AKI by I/R in diabetic mice and HG+H/R-treated TECs significantly elevated m6A methylation levels and METTL3 expression. Specific knockout of Mettl3 in TECs attenuated I/R-induced AKI in STZ-induced diabetic mice. Meanwhile, silencing METTL3 by tail vein injection of adeno-associated virus 9 Mettl3 shRNA alleviated I/R-induced AKI in db/db mice, improving renal function, tubular injury and inflammatory response. MeRIP-seq analysis indicates that N-myc downstream regulated 1 (NDRG1) as the m6A target of METTL3. Knockdown of Mettl3 significantly decreases the distribution of m6A peaks across Ndrg1 mRNA transcript in HG+HR-treated TECs, with m6A-modified stop codon regions of NDRG1 by METTL3 enhancing stability via the IGF2BP2-dependent pathway. NDRG1, a cytoplasmic protein involved in cellular stress response, interacts with NUR77 to regulate AKI by inhibiting p-P65. Knockdown of METTL3 reduced the expression of NDRG1 and inhibited the activity of NUR77, and suppressed the NF-κB pathway activation, thus alleviating kidney injury. Conclusion METTL3 promotes AKI susceptibility in DKD by the m6A modification of NDRG1 and enhancing its stability via IGF2BP2-dependent mechanisms, thereby exacerbating inflammation by the NDRG1/NUR77/NF-κB signaling pathway. Targeting this pathway (METTL3/NDRG1) may hold therapeutic promise for the treatment of AKI in DKD.