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

Akari Miyamoto1, Haruna Ueno1, Hideo Kimura2

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The kidney produces hydrogen sulfide (H2S) from d-cysteine via d-amino acid oxidase (DAO) and 3-mercaptopyruvate sulfurtransferase (3MST) in epithelial cells. This pathway is crucial for renal function and cytoprotection.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Renal Physiology

Background:

  • Hydrogen sulfide (H2S) is a vital signaling molecule and cytoprotectant.
  • The kidney uniquely produces H2S from d-cysteine using d-amino acid oxidase (DAO) and 3-mercaptopyruvate sulfurtransferase (3MST).
  • The d-cysteine pathway offers more efficient H2S production than the l-cysteine route, with d-cysteine reducing renal ischemia-reperfusion injury.

Purpose of the Study:

  • To identify the specific renal cell types involved in the d-cysteine H2S production pathway.
  • To characterize the expression and activity of DAO and 3MST in renal epithelial cells.
  • To establish stable cell models for studying d-cysteine-mediated H2S production in the kidney.

Main Methods:

  • Utilized a renal epithelial cell line (MDCK NBL-2) to investigate H2S production from d-cysteine.
  • Established clonal cell lines expressing both DAO and 3MST from a subpopulation of MDCK cells.
  • Analyzed DAO expression patterns in relation to cell confluence and passage number.

Main Results:

  • A subpopulation of MDCK cells expressed both DAO and 3MST, producing H2S from d-cysteine.
  • H2S production from d-cysteine declined with cell passage in unselected MDCK cells.
  • Established clonal cells maintained stable H2S production, exhibiting distinct DAO expression patterns.
  • DAO expression increased post-confluence in some clones, while others showed high expression pre-confluence.

Conclusions:

  • The d-cysteine pathway for H2S production is active in renal epithelial cells.
  • Cellular state and dynamics influence the role of d-cysteine-mediated H2S production.
  • The developed clonal cell lines serve as valuable tools for understanding renal H2S metabolism and its protective roles.