In this study, we present a novel epigenetic regulatory mechanism implicated in podocytes dysfunction during DKD. Currently, the role of m6A in podocyte injury under diabetic conditions remains relatively unexplored. Initially, high glucose cultured mature mice podocytes and diabetic STZ mice showed a significant increase in METTL3 expression, contributing to podocyte injury and inflammatory factor release under diabetic conditions, which aggravated renal pathology and increased urinary albumin excretion. METTL3 contributed to renal inflammation and podocytes injury by enhancing MDM2 m6A RNA methylation and increasing its RNA stability through IGF2BP2-dependent mechanisms. Suppression of MDM2 relieved podocyte injury driven by Notch signaling. These findings collectively indicated that targeting the METTL3/MDM2 axis promised as a therapeutic strategy for DKD.
M6A modification is a critical post-transcriptional modification found in eukaryotic mRNA, exhibiting significant involvement in the pathogenesis of various diseases (30, 31). Yet, the researches related to m6A methylation in DKD is limited. METTL3 plays a crucial role as an essential component of the m6A methyltransferase complex. It catalyzes methylation reactions and has been been reported in kidney disease (20, 32, 33). For example, METTL3 enhances TAB3 mRNA m6A methylation, reduces mRNA decay, and accelerates acute kidney injury (AKI) progression in an IGF2BP2-dependent manner (26). The interaction between METTL3 and DGCR8 thus contributed to the maturation of miR-873-5p, leading to a protective effect against colistin-induced oxidative stress and apoptosi (34). Additionally, increased METTL3 levels in podocytes trigger TIMP2 mRNA m6A modification, and the m6A reader IGF2BP2 directly binds to the m6A site on TIMP2 mRNA, thereby contributing to podocyte dysfunction in DKD (28). Firstly, we conducted a screening of methylation-related genes that contribute to the elevated m6A modification in DKD. We noted a significant elevation in METTL3 expression in the renal tissues of STZ-induced diabetic mice, and METTL3 contributed to increased m6A levels in both vivo and vitro. We investigated the effect of METTL3 on DKD. Conditional METTL3 knockout of podocytes notably reduced apoptosis and inflammatory cytokine release under diabetic conditions and alleviated podocyte dedifferentiation, renal pathological damage, and urinary albumin excretion. In contrast, METTL3 overexpression in podocytes exacerbated apoptosis and increased the release of inflammatory cytokines. Notably, METTL3 knockout did not affect blood glucose levels. Recent studies on acute injury have explored the therapeutic potential of Cpd-564, which is a novel METTL3 inhibitor. Cpd-564 showed remarkable efficacy in restoring kidney function, inhibiting kidney injury, and alleviating inflammatory responses (26). A METTL3 inhibitor exhibited therapeutic efficacy against myeloid leukemia according to previous studies. This discovery highlights the potential efficacy of METTL3-targeted therapies for various diseases. Nevertheless, the exploration of METTL3 inhibitors is in the early phases of investigation currently. Our study shows the crucial role of podocytes in DKD onset and progression. Targeting METTL3-mediated m6A modifications is important for the diagnosis and treatment of DKD.
Moreover, we investigated the underlying mechanism of METTL3-mediated podocyte injury. The fate of target transcripts relies on the recognition of m6A readers, which includes IGF2BP1, IGF2BP2, and IGF2BP3.This unique family of m6A readers specifically targets thousands of mRNA transcripts by recognizing a consistent GGC (m6A) sequence. These proteins recognize m6A modification sites on mRNA transcripts, influence their stability, protein expression, and exert functional regulatory roles under stress conditions (35, 36). In contrast to the YTH domain-containing family protein 2, which promotes mRNA degradation, IGF2BP2 functions by preserving mRNA stability (36). For instance, IGF2BP2, which recognizes METTL3-mediated m6A modification of MIS12, enhances MIS12 stability, increases its expression, and reverses senescence in human mesenchymal stem cells (37). In our study, we observed increased IGF2BP2 expression in the renal tissues of DKD mice. IGF2BP2 is directly associated with a specific m6A site in MDM2, which plays a critical role in maintaining MDM2 mRNA stability under METTL3 control.
Further investigations aimed to decipher which downstream gene are modified by METTL3 in a manner leading to podocyte injury. Using RIP-seq analysis revealed the presence of mRNA modification sites of METTL3 around the 3' UTR of MDM2 mRNA. Subsequent RIP-PCR and meRIP-qPCR analyses validated the effect of METTL3 on MDM2 expression through m6A modification. MDM2, which is an E3 ubiquitin ligase, identifies and ubiquitinates various protein substrates and plays a crucial role in cell cycle regulation, tumorigenesis, and cellular differentiation via p53-dependent and p53-independent pathways (38, 39). Podocytes and renal tubular epithelial cells display high expression levels of MDM2, which is known to have a critical impact on kidney disease (40).40 Increased MDM2 expression has been observed in both unilateral ureteral obstruction (UUO) rat models and mice with ischemia-reperfusion-induced acute kidney injury, where the suppression of MDM2 overexpression effectively mitigates renal fibrosis, inflammation, and albuminuria (41, 42). Additionally, differentiated podocytes possess other inherent obstacles to mitosis, weaken their proliferative response and impeding recovery from injury. While under stress condition, they tend to develop irregularly shaped nuclei, abnormal mitotic spindles, and in cytokinesis, ultimately leading to aneuploidy (43). Mature podocytes exit the cell cycle, as evidenced by the lower expression of the proliferation markers (i.e., Ki67, PCNA, and Cyclin B1) (13, 14). Consistent with these findings, our data showed that podocytes re-entered the cell cycle and progressed to the S and G2/M phases under diabetic conditions. These abnormal cell cycle events were caused by the activation of METTL3-mediated MDM2-Notch1 signaling under diabetic conditions, which helped mature podocytes overcome the G2/M checkpoint. METTL3 knockout inhibited the abnormal activation of the MDM2-Notch1 pathway, which prevented podocyte death during the initial phases of glomerular injury and alleviated glomerulosclerosis.
In conclusion, our study suggest the novel finding that METTL3 exhibits elevated expression in type 1 DKD, leading to increased m6A modification of MDM2 in podocytes through an IGF2BP2-dependent mechanism, Which subsequently triggering inflammation and apoptosis in DKD podocytes through the upregulation of the Notch1 signaling pathway. These findings reveals a previously unrecognized pathogenic mechanism of DKD and provide new impetus for the exploration of new therapeutic strategies