Sulfite-mediated oxidative stress in kidney cells

Sulfite-mediated oxidative stress in kidney cells.BackgroundChronic renal failure has been associated with oxidative stress. Serum sulfite, sulfate, cysteine, homocysteine, cysteine sulfinic acid, and γ-glutamylcysteine are elevated in patients on hemodialysis, suggesting an accelerated catabolism of sulfur-containing amino acids or a reduced elimination of sulfite/sulfate, or both. Administration of metabisulfite has also been shown to damage kidney cells.MethodsMeasurement of reactive oxygen species (ROS) was performed with the fluorescence of dichlorofluorescein (DCF), and that of intracellular ATP was by the luciferin-luciferase reaction. Oxidation of sulfite and succinate by isolated mitochondria from rat kidney was monitored polarographically. The fluorescent probe, 5, 5′, 6, 6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) was employed to assess any loss in membrane potential in energized respiring mitochondria. Activities of glutamate and malate dehydrogenases (GDH, MDH, respectively) were assayed by the spectrophotometric measurement of NADH. Sulfite was determined by HPLC-fluorimetric measurement of monochlorobimane-sulfite and cell viability was by the MTT procedure.ResultsAn immediate increase in ROS followed exposure of Madin-Darby canine kidney (MDCK), type II, and opossum kidney (OK) cells to 5–500 μmol/L sulfite. Depletion of intracellular ATP was also observed. A low rate of oxidation of 100 μmol/L sulfite was observed polarographically in isolated kidney mitochondria, but ADP-stimulated State 3 respiration was not apparent. ATP biosynthesis from the oxidation of glutamate in rat kidney mitochondria was significantly inhibited by coincubation with 100 μmol/L sulfite; this was not the case with malate, succinate, and TMPD/ascorbate. However, activities of both GDH and MDH in kidney mitochondrial extract were inhibited. The mitochondrial membrane potential and cell viability were not compromised.ConclusionMicromolar sulfite elicited an immediate increase in ROS in MDCK, type II, and OK cells. This was accompanied by a depletion of intracellular ATP, which could be explained by its inhibitory effect on mitochondrial GDH. Although MDH was similarly inhibited, the impact was buffered by the high level of this enzyme in kidney mitochondria