m6A-mediated regulation of ECA39 promotes renal fibrosis in chronic kidney disease by enhancing glycolysis and epithelial-mesenchymal transition.

Renal fibrosis is a vital pathological manifestation of chronic kidney disease (CKD). ECA39 is a conserved gene in the regulation of cell behavior; however, its function in renal fibrosis remains unclarified. A murine model of renal fibrosis was established by unilateral ureteral obstruction (UUO) operation. ECA39 expression was significantly upregulated in the kidneys of UUO mice. Prior to UUO operation (14 days), mice were administrated adeno-associated virus serotype 9 (AAV9, 1 × 1011 vector genomes) expressing ECA39 shRNA via tail vein injection. At postoperative day 7, AAV9-mediated inhibition of ECA39 was found to mitigate UUO-induced kidney damage, as manifested by reduced NGAL expression in kidneys, along with reduced serum creatinine and blood urea nitrogen (BUN) levels. Inhibition of ECA39 decreased collagen I, α-SMA and vimentin expression, but increased E-cadherin in kidney tissues. ECA39 inhibition reduced serum lactic acid level, increased ATP production, and suppressed glycolysis-related indicators HK2, PFKM, PKM2, PDK1, and LDHA expression. In parallel, human proximal tubular epithelial cells (HK-2) were treated with TGF-β1 (5 ng/ml, 48 h) to induce a cellular model of injury. ECA39 knockdown inhibit epithelial-mesenchymal transition (EMT) and glycolysis in HK-2 cells. Mechanistically, TGF-β1 treatment increased m6A modification of ECA39 mRNA, and the m6A "reader" IGF2BP2 knockdown reduced ECA39 mRNA stability. IGF2BP2 knockdown reduced lactic acid content and inhibited EMT in HK-2 cells, whereas ECA39 overexpression reversed these effects. Collectively, our studies demonstrated that inhibition of ECA39 suppresses glycolysis and EMT processes, thereby alleviating renal fibrosis in CKD.