Antioxidant Pre-Treatment Reduces the Toxic Effects of Oxalate on Renal Epithelial Cells in a Cell Culture Model of Urolithiasis

Abstract

Urolithiasis is characterized by the formation and retention of solid crystals within the urinary tract. Kidney stones are mostly composed of calcium oxalate, which predominantly generates free radicals that are toxic to renal tubular cells. The aim of the study is to explore possible effects of antioxidant pre-treatment on inhibition of oxidative stress. Three cell lines were used as in vitro model of urolithiasis: MDCK I, MDCK II and LLC-PK1. Oxidative stress was induced by exposure of cells to sodium oxalate in concentration of 8 mM. In order to prevent oxidative stress, cells were pre-treated with three different concentrations of l-arginine and vitamin E. Oxidative stress was evaluated by determining the expression of superoxide dismutase (SOD), osteopontin (OPN), and by the concentration of glutathione (GSH). In all three cell lines, pre-treatment of antioxidants increased cell survival. Positive correlation of SOD and OPN expression as well as GSH concentration was observed in all groups of cells. Our results indicate that an antioxidant pre-treatment with l-arginine and vitamin E is able to hamper oxalate-induced oxidative stress in kidney epithelial cells and as such could play a role in prevention of urolithiasis.

Keywords: ">l-arginine; LLC-PK1 cells; Madin-Darby canine kidney cells; urolithiasis; vitamin E.

Figure 1

Effects of l-arginine on cell survival in MDCK I, MDCK II and LLC-PK1 cell lines. Three controls of untreated cells, cells treated with l-arginine only and cells treated with sodium oxalate only were used. Increasing concentrations of l-arginine (0.05, 0.01 and 0.5 ng/mL) were used for pretreatment of cells prior to exposure to sodium oxalate to evaluate the effects on cell survival. The values are presented as means ± standard deviation. Plus (+) and minus (−) sign underneath the x-axis indicate addition of l-arginine and sodium oxalate, respectively. Bars assigned with asterisk (*) are statistically significantly different (p < 0.05) compared to control treated with sodium oxalate only, as determined with Student’s t-test. The data shown are representative of at least three independent experiments.

Figure 2

Effects of vitamin E on cell survival in MDCK I, MDCK II and LLC-PK1 cell lines. Three controls of untreated cells, cells treated with vitamin E only and cells treated with sodium oxalate only were used. Increasing concentrations of vitamin E (5, 15 and 25 µM) were used for pretreatment of cells prior to exposure to sodium oxalate to evaluate the effects on cell survival. The values are presented as means ± standard deviation. Plus (+) and minus (−) sign underneath the x-axis indicate addition of vitamin E and sodium oxalate, respectively. Bars assigned with asterisk (*) are statistically significantly different (p < 0.05) compared to control treated with sodium oxalate only, as determined by Student’s t-test. The data shown are representative of at least three independent experiments.

Figure 2

Effects of vitamin E on cell survival in MDCK I, MDCK II and LLC-PK1 cell lines. Three controls of untreated cells, cells treated with vitamin E only and cells treated with sodium oxalate only were used. Increasing concentrations of vitamin E (5, 15 and 25 µM) were used for pretreatment of cells prior to exposure to sodium oxalate to evaluate the effects on cell survival. The values are presented as means ± standard deviation. Plus (+) and minus (−) sign underneath the x-axis indicate addition of vitamin E and sodium oxalate, respectively. Bars assigned with asterisk (*) are statistically significantly different (p < 0.05) compared to control treated with sodium oxalate only, as determined by Student’s t-test. The data shown are representative of at least three independent experiments.

Figure 3

Effects of l-arginine and Vitamin E pretreatment on the expression on superoxide dismutase (SOD) gene in MDCK I, MDCK II and LLC-PK1 cell lines. (A,B) panels: SOD expression was inhibited in controls treated with sodium oxalate only, and was promoted in controls treated with antioxidants (l-arginine and vitamin E) only. SOD was stronger expressed in cells pretreated with antioxidant and then exposed to sodium oxalate compared to control cells treated with sodium oxalate only, but somewhat weaker expressed compared to cells treated with antioxidant only. The gene expression analysis was done by RT-PCR and obtained results were semi-quantified by ImageLab software. The values are represented as means ± SD. The data shown are representative of at least three independent experiments. Panel (C): Representative figures of Southern blot analysis of SOD expression compared to LDHA expression.

Figure 4

Effects of l-arginine and vitamin E pretreatment on the expression on osteopontin (OPN) gene in MDCK I, MDCK II and LLC-PK1 cell lines. (A,B) panels: OPN expression was promoted in controls treated with sodium oxalate only, and was inhibited in controls treated with antioxidants (l-arginine and vitamin E) only. OPN gene expression was suppressed in cells pretreated with antioxidant and later exposed to sodium oxalate when compared to OPN gene expression in control cells treated with sodium oxalate only, but somewhat higher expressed than in cells treated with antioxidant only. The gene expression analysis was done by reverse transcriptase PCR and semi quantified by ImageLab software. The values are represented as means ± SD. The data shown are representative of at least three independent experiments. (C) panel: Representative figures of Southern blot analysis of OPN gene expression compared to LDHA gene expression.

Figure 5

Effects of l-arginine and vitamin E pretreatment on the levels of GSH in MDCK I, MDCK II and LLC-PK1 cell lines. GSH levels were lower in oxalate-only treated cells compared to untreated controls, cells pretreated with l-arginine and vitamin E showed recovery of GSH levels. GSH measurement was done using spectrophotometry. The values are represented as means ± SD. The data shown are representative of at least three independent experiments.