Integrating Bulk and Single-Cell RNA Sequencing Identifies and Validates Lactylation-Related Signatures in Acute Kidney Injury

Abstract

Background: Acute kidney injury (AKI) is a severe clinical syndrome characterized by metabolic stress and profound inflammation. However, the landscape of lactylation-associated molecular alterations and their potential relevance in AKI remain incompletely understood. Methods: Bulk transcriptomes (GSE30718) were analyzed using the limma package, weighted gene co-expression network analysis (WGCNA), and consensus clustering to characterize AKI-associated molecular patterns linked to lactylation-associated signatures. Hub genes, prioritized through least absolute shrinkage and selection operator (LASSO) regression and the random forest algorithm, were integrated into a diagnostic nomogram and evaluated in an external cohort (GSE139061). Immune infiltration analysis was performed, and single-cell RNA sequencing data (GSE183276) were used to resolve cell-type-specific expression patterns of the hub genes. A cisplatin-induced murine AKI model validated global protein lactylation and hub gene expression by immunohistochemistry and Western blot. Results: Three hub genes (CKLF, ACLY, and SLC13A3) reliably discriminated AKI from controls (training AUC = 0.897). Their diagnostic performance varied in the external validation cohort. These genes correlated significantly with diverse immune infiltrates. Single-cell analysis localized CKLF predominantly to immune cells, ACLY broadly across renal populations, and SLC13A3 to proximal tubules. In vivo validation demonstrated increased global protein lysine lactylation levels in injured kidneys and confirmed expression alterations of CKLF, ACLY, and SLC13A3 consistent with transcriptomic observations. Conclusions: Integrating transcriptomic analysis with in vivo experimental validation, this study identified CKLF, ACLY, and SLC13A3 as candidate lactylation-associated signatures linked to immune and metabolic alterations in AKI.