Novel cuproptosis-related genes as predictors of diabetic kidney disease: bioinformatics and experimental validation

Diabetic kidney disease (DKD) is the most severe microvascular complication of diabetes and a major cause of end-stage renal disease; however, its pathogenesis remains incompletely understood. Cuproptosis, a recently identified type of copper-dependent cell death, provides a novel perspective for exploring the mechanisms underlying DKD. This study aimed to investigate the role of cuproptosis-related genes (CRGs) in DKD and identify potential diagnostic markers. We integrated renal tubule gene expression data from the Gene Expression Omnibus database with 17 core CRGs to identify differentially expressed CRGs (DE-CRGs). Immunohistochemistry (IHC) and immunofluorescence (IF) staining were performed on 30 DKD and 20 normal renal tissues to validate these findings. Furthermore, in vitro experiments were conducted using HK-2 cells under high glucose conditions to examine dynamic changes at the mRNA and protein levels by RT-qPCR and Western blot analysis, respectively. Diagnostic efficacy was evaluated using logistic regression and receiver operating characteristic (ROC) analysis. Bioinformatics analysis identified six DE-CRGs. Among these, FDX1, DLAT, LIAS, DBT, and DLST were significantly downregulated, whereas NFE2L2 was upregulated in DKD. Protein-level validation by IHC and IF revealed markedly reduced expression of FDX1, NRF2 (encoded by NFE2L2), DLAT, LIAS, DBT, and DLST in DKD tissues compared with the controls. Moreover, in vitro experiments demonstrated that under high glucose conditions, the protein levels of FDX1, DLAT, LIAS, DBT, and DLST decreased in a time-dependent manner, whereas NRF2 exhibited a biphasic response with initial upregulation followed by a decline. Logistic regression analysis revealed decreased expression of FDX1, NRF2, and DLAT as factors significantly associated with DKD. ROC analysis revealed the diagnostic potential of these genes in this cohort, with area under the curve values of 0.693 (IHC) and 0.847 (IF) for FDX1, 0.758 (IHC) and 0.727 (IF) for NRF2, 0.768 (IHC) and 0.856 (IF) for DLAT, and 0.888 (IHC) and 0.957 (IF) for the combined panel. This study suggests an association between cuproptosis and pathogenesis of DKD. Three core CRGs (FDX1, NFE2L2, and DLAT) show potential as candidate biomarkers for DKD and warrant further investigation.