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 expressionsof 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.