METTL3-Mediated N6-Methyladenosine mRNA Modification and cGAS-STING Pathway Activity in Kidney Fibrosis

Key Points METTL3 was upregulated in CKD, leading to increased m 6 A modification levels. M 6 A modifications were enriched in genes related to the cyclic guanosine monophosphate–AMP synthase-stimulator of IFN genes pathway in CKD. Normalizing heightened METTL3 and m 6 A modification levels showed promise as a strategy to combat kidney disease. Background Chemical modifications on RNA profoundly affect RNA function and regulation. m 6 A, the most abundant RNA modification in eukaryotes, plays a pivotal role in diverse cellular processes and disease mechanisms. However, its importance is understudied in human CKD samples regarding its influence on pathological mechanisms. Methods Liquid chromatography–tandem mass spectrometry and methylated RNA immunoprecipitation sequencing were used to examine alterations in m 6 A levels and patterns in CKD samples. Overexpression of the m 6 A writer METTL3 in cultured kidney tubular cells was performed to confirm the effect of m 6 A in tubular cells and explore the biological functions of m 6 A modification on target genes. In addition, tubule-specific deletion of Mettl3 (Ksp-Cre Mettl3 f/f ) mice and antisense oligonucleotides inhibiting Mettl3 expression were used to reduce m 6 A modification in an animal kidney disease model. Results By examining 127 human CKD samples, we observed a significant increase in m 6 A modification and METTL3 expression in diseased kidneys. Epitranscriptomic analysis unveiled an enrichment of m 6 A modifications in transcripts associated with the activation of inflammatory signaling pathways, particularly the cyclic guanosine monophosphate–AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway. m 6 A hypermethylation increased mRNA stability in cGAS and STING1 as well as elevated the expression of key proteins within the cGAS-STING pathway. Both the tubule-specific deletion of Mettl3 and the use of antisense oligonucleotides to inhibit Mettl3 expression protected mice from inflammation, reduced cytokine expression, decreased immune cell recruitment, and attenuated kidney fibrosis. Conclusions Our research revealed heightened METTL3-mediated m 6 A modification in fibrotic kidneys, particularly enriching the cGAS-STING pathway. This hypermethylation increased mRNA stability for cGAS and STING1, leading to sterile inflammation and fibrosis.