Clonal Stabilization Reveals a DAO/3MST-Expressing MDCK Subpopulation With Robust d-Cysteine-Mediated H₂S Production

Hydrogen sulfide (H₂S) functions as a signaling molecule and cytoprotectant. In many tissues, H₂S is produced from l-cysteine, whereas the kidney also produces H₂S from d-cysteine by the sequential activities of d-amino acid oxidase (DAO) and 3-mercaptopyruvate sulfurtransferase (3MST). The d-cysteine pathway produces H₂S more efficiently than the l-cysteine pathway, and d-cysteine significantly reduces ischemia-reperfusion injury in the renal cortex compared with l-cysteine. However, the specific renal cell types responsible for the d-cysteine pathway have not been clearly identified. Here, we show that a subpopulation of MDCK (NBL-2) cells, a renal epithelial cell line, expresses both DAO and 3MST. MDCK cells initially produced H₂S from d-cysteine, but this activity progressively declined with passage. In contrast, clonal cells expressing DAO and 3MST, established from this subpopulation, maintained stable H₂S production. The clonal cells exhibited two distinct patterns of DAO expression: one showed increased DAO levels after confluence, whereas the other displayed high DAO expression even before reaching confluence. These findings suggest that the d-cysteine pathway operates in epithelial cells and plays distinct roles depending on cellular state and dynamics. Moreover, the established clonal cells may provide insight into the role of d-cysteine-mediated H₂S production in the kidney.