Phenyl sulfate, indoxyl sulfate and p-cresyl sulfate decrease glutathione level to render cells vulnerable to oxidative stress in renal tubular cells.

In chronic kidney disease patients, oxidative stress is generally associated with disease progression and pathogenesis of its comorbidities. Phenyl sulfate is a protein-bound uremic solute, which accumulates in chronic kidney disease patients, but little is known about its nature. Although many reports revealed that protein-bound uremic solutes induce reactive oxygen species production, the effects of these solutes on anti-oxidant level have not been well studied. Therefore, we examined the effects of protein-bound uremic solutes on glutathione levels. As a result, indoxyl sulfate, phenyl sulfate, and p-cresyl sulfate decreased glutathione levels in porcine renal tubular cells. Next we examined whether phenyl sulfate-treated cells becomes vulnerable to oxidative stress. In phenyl sulfate-treated cells, hydrogen peroxide induced higher rates of cell death than in control cells. Buthionine sulfoximine, which is known to decrease glutathione level, well mimicked the effect of phenyl sulfate. Finally, we evaluated a mixture of indoxyl sulfate, phenyl sulfate, and p-cresyl sulfate at concentrations comparable to the serum concentrations of hemodialysis patients, and we confirmed its decreasing effect on glutathione level. In conclusion, indoxyl sulfate, phenyl sulfate, and p-cresyl sulfate decrease glutathione levels, rendering the cells vulnerable to oxidative stress

Effect of hydrogen peroxide on cellular viability in BSO-treated cells.

<p>Cells were treated with indicated concentrations (0, 0.001, 0.005, 0.02, 0.1 mmol/L) of buthionine sulfoximine for 24 hours and then treated with hydrogen peroxide (0, 10, 20 μmol/L) for 5 hours. After treatment, cellular viability was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193342#pone.0193342.g003" target="_blank">Fig 3</a>. Data are shown as mean ± S.D. (n = 3). Data are representative of two independent experiments. Cont, control; BSO, buthionine sulfoximine; H2O2, hydrogen peroxide. Asterisks indicate significant differences compared to corresponding controls (i.e. each black, grey, or hatched column was compared with its respective black, grey, or hatched control column) (**p < 0.01).</p

Effect of hydrogen peroxide on cellular viability in three uremic solutes-treated cells.

<p>Cells were treated with medium containing indicated concentrations (0, 0.1, 0.2, 0.5, 1 mmol/L) of each protein-bound uremic solute for 24 hours and then treated with hydrogen peroxide (0, 10, 20 μmol/L) for 5 hours. After treatment, cellular viability was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193342#pone.0193342.g003" target="_blank">Fig 3</a>. Data are shown as mean ± S.D. (n = 3). Data are representative of two independent experiments. Cont, control; IS, indoxyl sulfate; PCS, <i>p</i>-cresyl sulfate; PhS, phenyl sulfate; H2O2, hydrogen peroxide. Asterisks indicate significant differences compared to corresponding controls (i.e. each black, grey, or hatched column was compared with its respective black, grey, or hatched control column) (**p < 0.01).</p

Effects of protein-bound uremic solutes on total glutathione level.

<p>Porcine renal tubular cells were treated with each of the four protein-bound uremic solutes for 24 hours. Cells were lysed and glutathione levels were measured using a Glutathione Assay Kit. The total glutathione level of 5 x 10⁵ cells in 50 μL lysate is expressed as molar concentration of reduced glutathione. Data are shown as mean ± S.D (n = 2). Data are representative of two independent experiments. Cont, control; IS, indoxyl sulfate; PCS, <i>p</i>-cresyl sulfate; PhS, phenyl sulfate; IAA, indoleacetic acid. Asterisks indicate significant difference compared to control (**p < 0.01).</p

Effect of mixtures of three protein-bound uremic solutes on total glutathione levels.

<p>Cells were treated with medium containing indicated concentrations (0, 0.2, 1 mmol/L) of each protein-bound uremic solute or 10 mmol/L PhS for 24 hours. Cells were lysed and glutathione levels were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193342#pone.0193342.g001" target="_blank">Fig 1</a>. Data are shown as mean ± S.D. (n = 3). Data are representative of two independent experiments. Cont, control; IS, indoxyl sulfate; PCS, <i>p</i>-cresyl sulfate; PhS, phenyl sulfate. Asterisks indicate significant differences compared to control (*p < 0.05, **p < 0.01).</p

Effect of exogenous reduced glutathione on hydrogen peroxide-induced cell death in PhS-treated cells.

<p>Cells were treated with 10 mmol/L PhS for 24 hours and then treated with hydrogen peroxide (0 or 20 μmol/L) for 5 hours in the absence or presence of reduced glutathione (0, 0.1, 0.2, 0.5 mmol/L). After treatment, cell death was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193342#pone.0193342.g005" target="_blank">Fig 5</a>. Data are shown as mean ± S.D. (n = 2). Data are representative of two independent experiments. Cont, control; PhS, phenyl sulfate; Glu, glutathione; H2O2, hydrogen peroxide. Asterisks indicate significant differences between the indicated pairs (**p < 0.01).</p

Dose-dependent effect of buthionine sulfoximine on total glutathione level.

<p>Cells were treated with indicated concentrations (0, 0.001, 0.005, 0.02, 0.1 mmol/L) of buthionine sulfoximine or 10 mmol/L PhS for 24 hours. Cells were lysed and glutathione levels were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193342#pone.0193342.g001" target="_blank">Fig 1</a>. Data are shown as mean ± S.D. (n = 3). Data are representative of two independent experiments. Cont, control; BSO, buthionine sulfoximine; PhS, phenyl sulfate. Asterisks indicate significant differences compared to control (**p < 0.01).</p

Effect of exogenous reduced glutathione on cellular vulnerability to hydrogen peroxide in PhS-treated cells.

<p>Cells were treated with 0 or 10 mmol/L of PhS for 24 hours and then treated with hydrogen peroxide (0, 10, 20 μmol/L) for 5 hours in the absence or presence of reduced glutathione (0, 0.05, 0.1, 0.2, 0.5 mmol/L). After treatment, cellular viability was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193342#pone.0193342.g003" target="_blank">Fig 3</a>. Data are expressed as percent of control. Data are shown as mean ± S.D. (n = 3). Data are representative of two independent experiments. Cont, control; PhS, phenyl sulfate; Glu, glutathione; H2O2, hydrogen peroxide. Asterisks indicate significant differences compared to respective 0 μmol/L of hydrogen peroxide (i.e. grey or hatched column was compared with respective black columns) (*p < 0.05, **p < 0.01).</p

Effects of hydrogen peroxide on cell death in PhS-treated cells.

<p>Cells were treated with 0 or 10 mmol/L PhS for 24 hours and then treated with hydrogen peroxide (0, 10 or 20 μmol/L) for 5 hours. After treatment, cells were stained with annexin V-FITC and propidium iodide (PI). Percent of annexin V / PI-positive cells was analyzed and calculated. Data are shown as mean ± S.D. (n = 2). Data are representative of two independent experiments. Cont, control; PhS, phenyl sulfate; H2O2, hydrogen peroxide. Asterisk indicates significant difference between the indicated pair (*p < 0.05).</p

Effect of hydrogen peroxide on cellular viability in PhS-treated cells.

<p>Cells were treated with indicated concentrations (0, 0.2, 0.5, 2, 5, 10 mmol/L) of PhS for 24 hours and then treated with hydrogen peroxide (0, 10, 20 μmol/L) for 5 hours. After treatment, cellular viability was evaluated using Cell Counting Kit-8. Data are expressed as percent of control. Data are shown as mean ± S.D. (n = 3). Data are representative of two independent experiments. Cont, control; PhS, phenyl sulfate; H2O2, hydrogen peroxide. Asterisks indicate significant differences compared to corresponding controls (i.e. each black, grey, or hatched column was compared with its respective black, grey, or hatched control column) (*p < 0.05, **p < 0.01).</p