(A) Immunofluorescence showing the subcellular localization of DDX18 (red) relative to the nucleolar marker fibrillarin (FBL; green) in HK-2 cells under normoxia or H/R conditions. Scale bars: 5 μm. (B) Subcellular localization of DDX18 (red) with nucleolar (green) in HK-2 cells expressing the DDX18^WT or DDX18^K116R mutant under H/R. Scale bars: 5 μm. (C) GO analysis (RIP-seq, base mean ≥ 500, IP/input ≥ 2, P < 0.05) of DDX18-bound transcripts in H/R-treated cells. (D) GO analysis (RNA-seq, fold change > 2, P < 0.05) of genes differentially expressed between DDX18^WT- and DDX18^K116R-expressing HK-2 cells under H/R. (E–G) Integrative multiomics identifies CD44 as a key target. (E) Venn diagram of overlapping genes from RNA-seq and RIP-seq. (F) Heatmap analysis of overlapping downregulated genes in DDX18^K116R mutant cells, with cell adhesion–associated genes highlighted (adjusted P < 0.0001, |log₂ fold change|> 0.2). (G) DDX18-deficient HK-2 cells were reconstituted with either DDX18^WT or DDX18^K116R before exposure to H/R conditions. Relative mRNA levels of candidate genes associated with adhesion function were quantified via RT-qPCR. (H) Transcriptomic data (GEO: GSE175759) identified the correlation between CD44 and DDX18 expression levels in microdissected tubulointerstitial tissue from patients with kidney disease. (I and J) The protein level of CD44 was determined by Western blotting (I) and immunofluorescence (J). Scale bar: 20 μm. (K–M) CD44^+/+ and DDX18^−/− HK-2 cells were transfected with DDX18^WT or DDX18^K116R mutant and subsequently subjected to H/R. Fibrotic gene expression (K) was determined by RT-qPCR. The levels of fibrosis-related proteins were determined by Western blotting (L) and immunofluorescence (M). Scale bar: 20 μm. (N) CD44 mRNA stability in DDX18^WT- or DDX18^K116R-expressing HK-2 cells under hypoxic conditions was detected by actinomycin D assay. (O) Schematics showing the DDX18-binding site on the CD44 transcript by RIP-seq. (P) Biotinylated RNA probe and its deletion mutant (probe-Del, 35,229,249–35,229,385 nt deficiency) were incubated with HK-2 or HEK293T cell lysates and pulled down with streptavidin beads, and associated proteins were analyzed by Western blotting. (Q and R) CD44 and DDX18 double-knockout HK-2 cells were cotransfected with DDX18^WT or DDX18^K116R and a CD44-Del plasmid (CD44 region 35,229,249–35,229,385 nt deletion) or CD44 plasmid. (Q) CD44 mRNA stability in HK-2 cells was detected by actinomycin D assay. (R) CD44 protein level was determined by Western blot analysis. Representative data from one of three independent experiments are shown. Data in A, B, G, and Q were analyzed using an unpaired 2-tailed Student’s t test, in K and N using 2-way ANOVA, and in H using Pearson’s correlation analysis. *P < 0.05, **P < 0.01,***P < 0.001.