Indoxyl sulfate induces complex redox alterations in mesangial cells.
Indoxyl sulfate is a protein metabolite that is concentrated in the serum of patients with chronic renal insufficiency. It also is a uremic toxin that has been implicated in the progression of chronic renal disease in rodent models. We have shown previously that mesangial cell redox status is related to activation of mitogen-activated protein kinases and cell proliferation, which are factors related to glomerular damage. We used three methods to examine the ability of indoxyl sulfate to alter mesangial cell redox as a possible mechanism for its toxicity. Indoxyl sulfate increases mesangial cell reduction rate in a concentration-dependent manner as demonstrated by redox microphysiometry. Alterations occurred at concentrations as low as 100 microM, with more marked alterations occurring at higher concentrations associated with human renal failure. We demonstrated that indoxyl sulfate induces the production of intracellular reactive oxygen species (ROS) in mesangial cells (EC50 = 550 microM) by using the ROS-sensitive fluorescent dye CM-DCF. ROS generation was only partially (approximately 50%) inhibited by the NADPH oxidase inhibitor diphenylene iodinium at low (< or = 300 microM) indoxyl sulfate concentrations. Diphenylene iodinium was without effect at higher concentrations of indoxyl sulfate. We also used electron paramagnetic spin resonance spectroscopy with extracellular and intracellular spin traps to show that indoxyl sulfate increases extracellular SOD-sensitive O2-* production and intracellular hydroxyl radical production that may derive from an initial O2-* burst. These results document that indoxyl sulfate, when applied to renal mesangial cells at pathological concentrations, induces rapid and complex changes in mesangial cell redox.
Gelasco
Andrew K
AK
Nephrology Division, Medical Univ. of South Carolina, 96 Jonathan Lucas St., 829 CSB, Charleston, SC 29425-2227, USA. gelascoa@musc.edu
Raymond
John R
JR
D000818:Animals; D002478:Cells, Cultured; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide / Q000378:metabolism; D017665:Hydroxyl Radical / Q000378:metabolism; D007200:Indican / Q000008:administration & dosage / Q000494:pharmacology* / Q000633:toxicity; D008297:Male; D050527:Mesangial Cells / Q000187:drug effects* / Q000378:metabolism*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors; D009861:Onium Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D009861:Onium Compounds; D017382:Reactive Oxygen Species; D013481:Superoxides; D017665:Hydroxyl Radical; C007517:diphenyleneiodonium; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; D007200:Indican; D004121:Dimethyl Sulfoxide