Skip to main content
Journal ArticleOPEN ACCESS

Differential effects of indoxyl sulfate and inorganic phosphate in a murine cerebral endothelial cell line (bEnd.3)

Toxins (2014) 6(6) 1742-1760
DOI: 10.3390/toxins6061742
47Citations
Citations of this article
33Readers
Mendeley users who have this article in their library.

Abstract

Endothelial dysfunction plays a key role in stroke in chronic kidney disease patients. To explore the underlying mechanisms, we evaluated the effects of two uremic toxins on cerebral endothelium function. bEnd.3 cells were exposed to indoxyl sulfate (IS) and inorganic phosphate (Pi). Nitric oxide (NO), reactive oxygen species (ROS) and O2•- were measured using specific fluorophores. Peroxynitrite and eNOS uncoupling were evaluated using ebselen, a peroxide scavenger, and tetrahydrobiopterin (BH4), respectively. Cell viability decreased after IS or Pi treatment (p < 0.01). Both toxins reduced NO production (IS, p < 0.05; Pi, p < 0.001) and induced ROS production (p < 0.001). IS and 2 mM Pi reduced O2•- production (p < 0.001). Antioxidant pretreatment reduced ROS levels in both IS- and Pi-treated cells, but a more marked reduction of O2•- production was observed in Pi-treated cells (p < 0.001). Ebselen reduced the ROS production induced by the two toxins (p < 0.001); suggesting a role of peroxynitrite in this process. BH4 addition significantly reduced O2•- and increased NO production in Pi-treated cells (p < 0.001), suggesting eNOS uncoupling, but had no effect in IS-treated cells. This study shows, for the first time, that IS and Pi induce cerebral endothelial dysfunction by decreasing NO levels due to enhanced oxidative stress. However, Pi appears to be more deleterious, as it also induces eNOS uncoupling. © 2014 by the authors; licensee MDPI, Basel, Switzerland.

Figures

  • Figure 1. Effect of indoxyl sulfate (IS) (a) and inorganic phosphate (Pi) (b) on cerebral endothelial cell viability. bEnd.3 cells were incubated with IS or Pi at 37 °C for 24 h, and then treated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) for 3 h. Cell viability was determined by measuring absorbance at 570 nm. Cell viability of untreated control cells was taken as 100%. Data are expressed as mean ± SEM of three independent experiments. ** p < 0.01 vs. control. ISn, normal IS concentration; ISu, uremic IS concentration; ISm, maximal IS concentration; Pi2, 2 mM Pi; Pi3, 3 mM Pi.
  • Figure 2. Effect of IS (a) and Pi (b) on NO production in cerebral endothelial cells. bEnd.3 cells were incubated with 0.1 µM DAF-FM in D-PBS at 37 °C for 1 h and then treated with IS or Pi. NO was determined immediately by measuring fluorescence (λEx 492 nm, λEm 510 nm). NO production in untreated control cells was taken as 100%. Data are expressed as mean ± SEM of four independent experiments. (a) * p < 0.05—ISm vs. Control, ISu + L-Name vs. ISu, *** p < 0.001—ISn vs. Control, ISn + L-Name vs. ISn; (b) *** p < 0.001—Pi2 + L-Name vs. Pi2, Pi3 vs. Control, Pi3 + L-Name vs. Pi3. ISn, normal IS concentration; ISu, uremic IS concentration; ISm, maximal IS concentration; Pi2, 2 mMPi; Pi3, 3 mM Pi; L-Name (N-nitro-L-arginine methyl ester), an inhibitor of eNOS activity.
  • Figure 3. Effect of IS (a) and Pi (b) on ROS production in cerebral endothelial cells. bEnd.3 cells were incubated with 1 µM DCFH-DA in D-PBS at 37 °C for 30 min and then treated with IS or Pi. ROS was determined immediately by measuring fluorescence (λEx 492 nm, λEm 535 nm). ROS production in untreated control cells was taken as 100%. Data are expressed as mean ± SEM of four independent experiments. *** p < 0.001 vs. control. ISn, normal IS concentration; ISu, uremic IS concentration; ISm, maximal IS concentration; Pi2, 2 mM Pi; Pi3, 3 mM Pi.
  • Figure 4. Effect of antioxidants on IS- (a) and Pi- (b) induced ROS production in cerebral endothelial cells. bEnd.3 cells were incubated with 1 µM DCFH-DA in D-PBS at 37 °C for 30 min and then treated with ISm or 3 mM Pi, with or without the antioxidants α-tocopherol (Vit E, 10 µg/mL) and N-acetyl-L-cysteine (NAC, 10 mM), respectively. ROS levels were determined immediately by measuring fluorescence (λEx 492 nm, λEm 535 nm). ROS levels in untreated control cells were taken as 100%. Data are expressed as mean ± SEM of four independent experiments. (a) * p < 0.05 vs. ISm, *** p < 0.001 vs. control; (b) *** p < 0.001—Pi3 vs. control, Pi3 + NAC vs. Pi3 and Pi3 + Vit E vs. Pi3. ISn, normal IS concentration; ISu, uremic IS concentration; ISm, maximal IS concentration; Pi2, 2 mM Pi; Pi3, Pi 3 mM Pi.
  • Figure 5. Effect of IS (a) and Pi (b) on O2•– production in bEnd.3 cells. bEnd.3 cells were incubated with 10 µM DHE in D-PBS at 37 °C for 1 h and then treated with IS and Pi. O2•– was determined immediately by measuring fluorescence (λEx 492 nm, λEm 615 nm). O2•– production in untreated control cells was taken as 100%. Data are expressed as mean ± SEM of four independent experiments. *** p < 0.001 vs. Control. ISn, normal IS concentration; ISu, uremic IS concentration; ISm, maximal IS concentration; Pi2, 2 mM Pi; Pi3, 3 mM Pi.
  • Figure 6. Effect of antioxidants on IS- (a) and Pi- (b) induced O2•– production in bEnd.3 cells. bEnd.3 cells were incubated with 10 µM DHE in D-PBS at 37 °C for 1 h and then treated with IS and Pi with or without the antioxidants α-tocopherol (Vit E, 10 µg/mL) and N-acetyl-L-cysteine (NAC, 10 mM). O2•– was determined immediately by measuring fluorescence (λEx 492 nm, λEm 615 nm). O2•– production in untreated control cells was taken as 100%. Data are expressed as mean ± SEM of four independent experiments. (a) * p < 0.05 vs. IS; (b) *** p < 0.001 vs. Pi3. ISn, normal IS concentration; ISu, uremic IS concentration; ISm, maximal IS concentration; Pi2, 2 mM Pi; Pi3, 3 mM Pi.
  • Figure 7. Effect of ebselen on IS (a) and Pi (b) induced ROS production in bEnd.3 cells. bEnd.3 cells were incubated with 1 µM DCFH-DA alone or with ebselen (10 µM) at 37 °C for 30 min and then treated with IS and Pi. ROS was determined immediately by measuring fluorescence (λEx 492 nm, λEm 535 nm). ROS production in untreated control cells was taken as 100%. Data are expressed as mean ± SEM of four independent experiments. (a) *** P<0.001—ISn vs. Control, ISu vs. Control, ISm vs. Control, Control + Eb vs. Control, ISn + Eb vs. ISn, ISu + Eb vs. ISu, ISm + Eb vs. ISm, Isn vs. Control, Isu vs. Control, Ism vs. Control; (b) *** p < 0.001—Control + Eb vs. Control, Pi2 + Eb vs Pi2, Pi3 + Eb vs. Pi3. ISn, normal IS concentration; ISu, uremic IS concentration; ISm, maximal IS concentration; Pi2, 2 mM Pi; Pi3, 3 mM Pi; Eb, Ebselen, a peroxide scavenger.
  • Figure 8. eNOS uncoupling in IS- and Pi- treated bEnd.3 cells. bEnd.3 cells were incubated overnight with 10 µM BH4. The cells were subsequently incubated with 10 µM DHE or 0.1 µM DAF-FM in D-PBS at 37 °C for 1 h and then treated with ISm and Pi3. O2•– (Figure 8a) and NO (Figure 8b) were determined immediately by measuring fluorescence: λEx 492 nm, λEm 615 nm and λEx 492 nm, λEm 510 nm, respectively. O2•– and NO production in untreated control cells were taken as 100%. Data are expressed as mean ± SEM of four independent experiments. (a) *** p < 0.001—Control + BH4 vs. Control, Pi3 + BH4 vs. Pi3. (b) *** p < 0.001—Pi + BH4 vs. Pi3. ISm, maximal IS concentration; Pi3, 3 mM Pi; BH4, ((6R)-5,6,7,8-tetrahydro-L-biopterin), a redox cofactor of Enos.

Author supplied keywords

References Powered by Scopus

Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays

Journal of Immunological Methods
51120Citations
N/AReaders
Get full text

Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.

Proceedings of the National Academy of Sciences of the United States of America
4566Citations
N/AReaders
Get full text

Clinical epidemiology of cardiovascular disease in chronic renal disease

American Journal of Kidney Diseases
3130Citations
N/AReaders
Get full text
View more at Scopus

Cited by Powered by Scopus

Uremic toxic blood-brain barrier disruption mediated by AhR activation leads to cognitive impairment during experimental renal dysfunction

Journal of the American Society of Nephrology
112Citations
N/AReaders
Get full text

Crosstalk between the nervous system and the kidney

Kidney International
98Citations
N/AReaders
Get full text

Indoxyl sulfate, a uremic endotheliotoxin

Toxins
96Citations
N/AReaders
Get full text
View more at Scopus

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Cite

CITATION STYLE

APA

Stinghen, A. E. M., Chillon, J. M., Massy, Z. A., & Boullier, A. (2014). Differential effects of indoxyl sulfate and inorganic phosphate in a murine cerebral endothelial cell line (bEnd.3). Toxins, 6(6), 1742–1760. https://doi.org/10.3390/toxins6061742

Readers over time

‘15‘16‘17‘18‘19‘20‘21‘22‘2402468

Readers' Seniority

Tooltip

PhD / Post grad / Masters / Doc 15

75%

Researcher 3

15%

Lecturer / Post doc 2

10%

Readers' Discipline

Tooltip

Agricultural and Biological Sciences 8

42%

Medicine and Dentistry 5

26%

Pharmacology, Toxicology and Pharmaceut... 3

16%

Biochemistry, Genetics and Molecular Bi... 3

16%

Save time finding and organizing research with Mendeley

Sign up for free
0