Single-Nucleus Transcriptome Analysis Reveals Chac1-Driven Ferroptosis as a Key Mediator in Adriamycin-Induced Kidney Injury

Background: Although genome-wide association studies (GWAS) have identified Chac1 as a risk gene for chronic kidney disease (CKD), its cell type-specific roles in disease pathogenesis remain elusive, despite its known critical function in stress responses through glutathione metabolism regulation. Methods: We established a murine model of Adriamycin-induced kidney injury in four groups of male mice: (1) wild-type (WT), (2) Chac1 knockout (CH) , (3) WT mice treated with Adriamycin (WT_Adr), and (4) Chac1 knockout mice treated with Adriamycin (CH_Adr). Single-nucleus RNA sequencing (snRNA-seq) was performed on kidney tissues, followed by quality control, integration with published murine kidney reference datasets using scVI and scANVI to remove batch effects and annotate cell types. Differential gene expression (DEG) analysis was conducted at single-cell and pseudobulk levels, complemented by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and cellular composition analysis. Results: Our single-cell and pseudo-bulk analyses identified high-confidence DEGs significantly enriched in pathways governing fundamental kidney processes, including development, ion transport, cell migration, metabolic regulation, and tissue homeostasis. Additionally, our analysis revealed a strong upregulation of multiple ferroptosis-related genes specifically within the injPT cells during disease induction. Consistently, pathway enrichment analysis highlighted the significant activation of the ferroptosis pathway, involving crucial elements of glutathione metabolism, iron ion transport, reactive oxygen species (ROS), oxidative stress, and lipid transport. Conclusion: Our comprehensive single-nucleus RNA sequencing analysis provides novel insights into the cell-type-specific roles of Chac1 in Adriamycin-induced kidney injury. These findings not only reveal the complex role of Chac1 in ADR-induced nephropathy, but also hold broader implications for understanding chronic kidney disease pathogenesis and propose Chac1 as a promising target for future therapeutic strategies aimed at mitigating kidney injury and preventing CKD progression. Funding: NIDDK Support