Identification and functional validation of mitochondria-related genes associated with tubular injury in kidney transplantation and ischemia–reperfusion injury

Ischemia–reperfusion injury (IRI) limits graft function and long-term outcomes after kidney transplantation. Proximal tubular (PT) cells are highly mitochondria-rich and metabolically active, making them vulnerable to ischemic and oxidative stress. However, the molecular mechanisms linking mitochondrial dysfunction to graft function remain incompletely understood. We integrated single-nucleus RNA sequencing, bioinformatics analyses, and experimental validation to identify mitochondria-associated genes in PT cells related to graft injury and vulnerability in the transplant setting. Mitochondria-related differentially expressed genes (Mito-DEGs) were used to construct machine learning models for delayed graft function (DGF) risk stratification. The candidate gene HAO2 was further evaluated by immunohistochemistry (IHC) in kidney transplant biopsies, a murine IRI model, and H₂O₂-treated HK-2 cells. Human biopsy samples were obtained at or prior to reperfusion and may not fully capture the extent of post-transplant IRI. Both overexpression and siRNA-mediated knockdown were performed to assess its function. Cell viability, apoptosis, and mitochondrial function were assessed using standard assays. PT cells from acute kidney injury samples exhibited mitochondrial dysfunction and metabolic impairment. Donor kidney clustering suggested heterogeneity in DGF risk associated with mitochondrial bioenergetic capacity. An eight-gene Mito-DEG signature demonstrated moderate performance in stratifying DGF risk. IHC analysis demonstrated that HAO2 expression was reduced in DGF recipient biopsies. Consistently, HAO2 was downregulated in murine IRI kidneys and injured HK-2 cells. Functionally, HAO2 overexpression alleviated oxidative stress, apoptosis, and mitochondrial dysfunction, whereas HAO2 knockdown exerted opposite effects and further aggravated H₂O₂-induced injury. Regulatory analysis identified 14 miRNAs and four TFs potentially controlling HAO2, while downstream pathways linked HAO2 to amino acid and fatty acid metabolism, extracellular matrix organization, and immune responses. These findings suggest that HAO2 may serve as a functional indicator of mitochondrial metabolic capacity associated with graft vulnerability rather than being specific to IRI alone. Given the observational nature of the clinical data, further studies are required to determine whether HAO2 provides added value beyond established histopathological assessment and to clarify its potential role in guiding mitochondrial-targeted conditioning strategies, such as oxygenated machine perfusion, aimed at preserving graft function during transplantation. Not applicable.