Identification of Ferroptosis-Related Hub Genes and Immune Infiltration Landscape in Chronic Kidney Disease via Bioinformatics and Experimental Verification.

BACKGROUND

Chronic kidney disease (CKD) is a serious global health problem with increasing incidence. Ferroptosis plays a crucial role in kidney diseases, but limited studies have elucidated the mechanism and role of ferroptosis in CKD.

METHODS

CKD data sets and ferroptosis-related genes were acquired from the Gene Expression Omnibus (GEO) database and FerrDB V2. By integrating bioinformatics including weighted gene coexpression network analysis (WGCNA), enrichment analyses, protein-protein interaction (PPI) network and GeneMANIA analysis, ferroptosis-related hub genes were identified in CKD. Validation of hub genes was conducted using an external data set, and diagnostic potential capability was evaluated through receiver operating curve (ROC) analysis. Subsequently, the relationship between hub genes and clinical traits was performed using Nephroseq v5 database. Gene set enrichment analysis (GSEA) of hub genes was performed. The CIBERSORT algorithm was employed to examine the infiltration of 22 distinct immune cell types in CKD. Western blotting, RT-qPCR and HPA database were utilized to further validate the role of NFE2L2, NNMT and GDF15 in CKD.

RESULTS

By integrating bioinformatics including WGCNA, PPI, and GeneMANIA, 7 hub genes were identified including NNMT, GDF15, ACSL1, DLD, NFE2L2, PARP1, and NR4A1. These hub genes have been validated in the validation set and clinical correlation analysis established a clear link between hub gene expression and renal function deterioration. ROC analysis demonstrated excellent diagnostic efficacy. GSEA indicated that these hub genes affect CKD progress or prognosis through the posttranscriptional modifications and transport of pathogenic factors, immune dysfunction, and cell cycle dysregulation. The CKD samples exhibited elevated levels of CD8+ T cells and M0 macrophages, while memory B cells, resting memory CD4+ T cells, Tregs, and resting mast cells showed decreased levels. Moreover, in vitro experiments revealed that NFE2L2, NNMT and GDF15 were upregulated in TGF-β1-treated HK-2 cells.

CONCLUSION

Our study confirms NNMT, GDF15, ACSL1, DLD, NFE2L2, PARP1, and NR4A1 as significantly upregulated biomarkers associated with ferroptosis in CKD progression, suggesting their potential as novel targets for CKD diagnosis and treatment.