Molecular mechanism of ALKBH5‐mediated m6A demethylation regulating lipopolysaccharide‐induced epithelial–mesenchymal transition in sepsis‐induced acute kidney injury

Abstract This study explored the mechanism by which the m6A demethylase ALKBH5 mediates epithelial–mesenchymal transition (EMT) in sepsis‐associated acute kidney injury (SA‐AKI) and AKI‐chronic kidney disease (CKD) transition. HK‐2 cells were stimulated with lipopolysaccharide (LPS) to establish an in vitro model of SA‐AKI. ALKBH5 expression was reduced through the transfection of si‐ALKBH5. Cell viability, apoptosis, and migration were detected by CCK‐8 assay, TUNEL staining, and Transwell. The levels of TNF‐α, IL‐1β, and IL‐6 were measured by enzyme‐linked immunosorbent assay. Quantitative real‐time polymerase chain reaction or Western blotting was performed to determine the expressions of ALKBH5, miR‐205‐5p, DDX5, E‐cadherin, and α‐SMA. The m6A level was quantitatively analyzed. The expression of pri‐miR‐205 bound to DGCR8 and m6A‐modified pri‐miR‐205 after intervention with ALKBH5 expression was detected by RNA immunoprecipitation. A dual‐luciferase assay confirmed the binding between miR‐205‐5p and DDX5. ALKBH5 was highly expressed in LPS‐induced HK‐2 cells. Inhibition of ALKBH5 increased cell viability, repressed apoptosis, and reduced EMT. Inhibition of ALKBH5 increased the m6A modification level, thereby promoting DGCR8 binding to pri‐miR‐205 to increase miR‐205‐5p expression and eventually targeting DDX5 expression. Low expression of miR‐205‐5p or overexpression of DDX5 partially abolished the inhibitory effect of ALKBH5 silencing on EMT. In conclusion, ALKBH5 represses miR‐205‐5p expression by removing m6A modification to upregulate DDX5 expression, thereby promoting EMT and AKI‐CKD transition after SA‐AKI.