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Indoxyl sulfate induces complex redox alterations in mesangial cells

Department of Medicine, Nephrology Division, Medical University of South Carolina, and Research Service, Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina

Submitted 30 July 2004 ; accepted in final form 12 January 2006

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 µM, 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 µM) by using the ROS-sensitive fluorescent dye CM-DCF. ROS generation was only partially (50%) inhibited by the NADPH oxidase inhibitor diphenylene iodinium at low (300 µM) 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.

electron paramagnetic spin resonance; fluorescence; microphysiometry; superoxide; NADPH oxidase; reactive oxygen species; uremic toxins