Diabetic Kidney Disease Progression Alleviated in Mice by ALKBH5-Mediated UC-MSCs-Derived Exosomes That Inhibit TRAF6 m6A Modification and Promote M2 Macrophage Polarisation.

BACKGROUND: Diabetic kidney disease (DKD) is a major diabetes complication with limited treatment options. Exosomes (Exo) from umbilical cord mesenchymal stem cells (UC-MSCs) have shown therapeutic promise. The role of alkylation repair homologue protein 5 (ALKBH5)-modified UC-MSCs Exo in regulating macrophage polarisation and alleviating DKD is investigated. METHODS: DKD-associated inflammation was modelled by Lipopolysaccharide (LPS)/interferon-gamma (IFN-γ)-stimulated RAW264.7 macrophages. RT-qPCR and western blotting were employed to analyse mRNA and protein expression. Exosomes from ALKBH5-modified UC-MSCs were isolated and characterised. Macrophage polarisation (M1/M2) was assessed by flow cytometry, RT-qPCR, and enzyme-linked immunosorbent assay (ELISA). Tumor necrosis factor receptor-associated factor 6 (TRAF6) N6-methyladenosine (m6A) modification and expression were analysed via methylated RNA immunoprecipitation (MeRIP) and RNA immunoprecipitation (RIP) assays. The DKD model was established using spontaneous diabetic db/db mice. The renal function of mice was evaluated by ELISA and commercial assay kits. Hematoxylin-eosin (HE), periodic acid-Schiff (PAS), and Masson's trichrome staining were performed to assess the renal histopathology of mice. RESULTS: ALKBH5 overexpression promoted M2 and inhibited M1 macrophage polarisation. ALKBH5 downregulated TRAF6 via m6A demethylation. ALKBH5-modified UC-MSCs Exo enhanced M2 polarisation and suppressed M1 phenotype in vitro. In DKD mice, ALKBH5-modified UC-MSCs Exo mitigated renal injury. Moreover, these exosomes enhanced anti-inflammatory responses and promoted M2 macrophage polarisation in DKD mice. CONCLUSION: ALKBH5-modified UC-MSCs Exo reduced TRAF6 expression by demethylating its m6A sites, promoting M2 macrophage polarisation and alleviating DKD progression. These findings suggested that ALKBH5-modified UC-MSCs Exo might represent a promising therapeutic approach for DKD.