(A) Domain architecture of DDX18 highlighting putative lactylation sites. (B) MS analysis confirming lactylation at K116. The x axis represents the mass‑to‑charge ratio (m/z), corresponding to the molecular mass divided by the charge state of the peptide. The y axis represents the intensity of the fragment ion peaks. Red peaks correspond to b‑ions and blue peaks to y‑ions. For sequencing, b‑ions are counted from the N‑terminus, while y‑ions are counted from the C‑terminus. The calculation formula is b2-1-m(M) – m(OX) = m(K) + m(La). (C) DDX18 lactylation in HK-2 cells expressing DDX18^WT or multiple K→R mutants of DDX18. (D) Cross-species alignment showing conserved K116 (red) across 7 species. (E) DDX18-K116 lactylation in HK-2 cells expressing DDX18^WT or the DDX18^K116R mutant under H/R stress. (F) DDX18-K116 lactylation in HK-2 cells treated with lactate. (G) Hypoxia treatment induced lactylation of DDX18-K116. (H) DDX18 K116 lactylation was significantly increased in primary renal TECs from BIRI mice. (I and J) Spatial distribution of DDX18-K116 lactylation in vivo. (I) Increased DDX18-K116 lactylation in renal tissues from AKI patients with proteinuria versus without proteinuria. Scale bar: 100 μm. (J) Immunofluorescence staining of DDX18-K116 lactylation in kidney sections from BIRI mice after 24 h of reperfusion. Scale bar: 100 μm. (K–N) In vitro validation using HK-2 cells expressing DDX18^K116R under H/R stress. (K) Wound healing assay showing the migration capacity of DDX18^WT- or DDX18^K116R-expressing HK-2 cells, with wound closure (%) quantified at 0 and 48 h. Fibrotic marker expression (L) was determined by RT-qPCR analysis. The levels of fibrosis-related proteins were determined by Western blotting (M) and immunofluorescence (N). Scale bar: 20 μm. Representative data from 1 of 3 independent experiments are shown. Data in I–K were analyzed using an unpaired 2-tailed Student’s t test and in L using 2-way ANOVA. ***P < 0.001.