2,8-Dihydroxyadenine disrupts epithelial integrity and alters kidney cell phenotype in vitro

Adenine phosphoribosyltransferase (APRT) deficiency is an autosomal recessive disorder that causes accumulation of 2,8-dihydroxyadenine (DHA) in the urinary tract, leading to kidney stones and chronic kidney disease. Progression to end-stage kidney disease can occur without timely treatment. The xanthine oxidoreductase inhibitors, allopurinol and febuxostat, block DHA generation and halt or delay stone formation and disease progression. Some patients cannot tolerate these drugs, necessitating new therapeutic approaches. This study aimed to investigate how DHA influences structural and molecular changes in HK-2, HEK293, and MDCK kidney cells. DHA exposure reduced cell viability and impaired migration in all cell lines. Increased expression of the adhesion protein CD44 was observed in HEK293 and HK-2 cells, adjacent to DHA crystals. Transepithelial electrical resistance measurements of MDCK cells indicated reduced epithelial integrity following DHA exposure. RNA sequencing of DHA-treated HK-2 cells revealed gene expression changes, with upregulation of TNF-α and mTORC1 signaling and downregulation of epithelial–mesenchymal transition and oxidative phosphorylation. Functional enrichment analysis highlighted pathways related to cell cycle regulation, inflammation, and metabolic processes. These findings show that dose-dependent toxic effects of DHA toxicity interrupt cell migration and epithelial integrity. CD44 may contribute to crystal adhesion, making it a potential therapeutic target in DHA crystal nephropathy.