Molecular mechanism of oxalate-induced free radical production and glutathione redox imbalance in renal epithelial cells: effect of antioxidants.
BACKGROUND
Peroxidation of renal cells is a critical event in the nucleation and formation of calcium oxalate crystals under hyperoxaluric conditions. We previously demonstrated that oxalate-induced peroxidative injury is one of the major mechanisms in promoting crystal attachment to renal epithelial cells.


METHODS
In this study we have demonstrated that the mechanism of oxalate-induced peroxidative injury is through the induction of TGF-beta1 and glutathione (GSH) redox imbalance in LLC-PK1 cells.


RESULTS
LLC-PK1, renal epithelial cells exposed to oxalate had significantly higher reactive oxygen species (ROS) production; higher TGF-beta1 levels, as measured by ELISA (1.89 +/- 0.035 fold increase) or Western blot (1.65 +/- 0.01 fold increase); increased malondialdehyde formation; increased LDH release, and loss of cell viability. In addition, oxalate exposure significantly decreased GSH content, glutathione reductase, glucose-6-phosphate dehydrogenase activities, and increased oxidized GSH content. Treatment with vitamin E, neutralizing anti-TGF-beta antibody, or diphenylene iodium, an inhibitor of NAD(P)H oxidase, significantly inhibited oxalate-induced ROS production and prevented peroxidative injury and cytolysis. Vitamin E, catalase, or desferoxamine treatment also significantly restored the oxalate-induced cellular GSH redox status toward the control level, and vitamin E treatment significantly attenuated the oxalate-mediated increase in TGF-beta1 protein in cultured LLC-PK1 cells.


CONCLUSIONS
This is the first study to demonstrate that the mechanism of oxalate-induced free radical production in renal tubular epithelial cells is through the activation of NAD(P)H oxidase via cytokine TGF-beta1 induction. These results also provide direct evidence that antioxidant therapy might prevent calcium oxalate nucleation and kidney stone formation by preventing oxalate-mediated peroxidative injury and GSH redox imbalance.
Rashed
Tanvir
T
Department of Urology, Vattikuti Urology Institute and Henry Ford Health Sciences Center, Detroit, Mich. 48202, USA.
Menon
Mani
M
Thamilselvan
Sivagnanam
S
D000975:Antioxidants / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000187:drug effects*; D005609:Free Radicals / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology* / Q000187:drug effects*; D010070:Oxalates / Q000494:pharmacology*; D010084:Oxidation-Reduction / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D053773:Transforming Growth Factor beta1; D019459:Urothelium / Q000166:cytology / Q000187:drug effects
D000975:Antioxidants; D005609:Free Radicals; D010070:Oxalates; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D005978:Glutathione