title: Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells.
abstract: Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25-250 microg/ml) to determine whether ROI, specifically superoxide (O(2)(*-)), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1-24 h increased O(2)(*-) production 3-6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O(2)(*-) production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O(2)(*-) generation. In conclusion, COM increased mitochondrial O(2)(*-) production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.
authors.affiliation: Centre for Prevention and Treatment of Urinary Stones, Institute of Urology and Nephrology, University College London, UK.
authors.forename: F D
authors.initials: FD
authors.lastname: Khand
authors.forename: M P
authors.initials: MP
authors.lastname: Gordge
authors.forename: W G
authors.initials: WG
authors.lastname: Robertson
authors.forename: A A
authors.initials: AA
authors.lastname: Noronha-Dutra
authors.forename: J S
authors.initials: JS
authors.lastname: Hothersall
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002129:Calcium Oxalate / Q000633:toxicity*; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D007202:Indicators and Reagents; D007686:Kidney Tubules, Distal / Q000187:drug effects* / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D012235:Rhodamines; D013481:Superoxides / Q000378:metabolism*; D013552:Swine
chemical_list: D007202:Indicators and Reagents; D017382:Reactive Oxygen Species; D012235:Rhodamines; D009243:NAD; C058319:dihydrorhodamine 123; D013481:Superoxides; D002129:Calcium Oxalate; D009249:NADP; D000255:Adenosine Triphosphate; D005978:Glutathione