Indoxyl Sulfate Affects Glial Function Increasing Oxidative Stress and Neuroinflammation in Chronic Kidney Disease: Interaction between Astrocytes and Microglia - PubMed (original) (raw)

Indoxyl Sulfate Affects Glial Function Increasing Oxidative Stress and Neuroinflammation in Chronic Kidney Disease: Interaction between Astrocytes and Microglia

Simona Adesso et al. Front Pharmacol. 2017.

Abstract

Indoxyl sulfate (IS) is a protein-bound uremic toxin resulting from the metabolism of dietary tryptophan which accumulates in patients with impaired renal function, such as chronic kidney disease (CKD). IS is a well-known nephrovascular toxin but little is known about its effects on central nervous system (CNS) cells. Considering the growing interest in the field of CNS comorbidities in CKD, we studied the effect of IS on CNS cells. IS (15-60 μM) treatment in C6 astrocyte cells increased reactive oxygen species release and decreased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation, and heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase quinone 1 expression. Moreover, IS increased Aryl hydrocarbon Receptor (AhR) and Nuclear Factor-kB (NF-kB) activation in these cells. Similiar observations were made in primary mouse astrocytes and mixed glial cells. Inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) expression, tumor necrosis factor-α and interleukin-6 release and nitrotyrosine formation were increased by IS (15-60 μM) in primary mouse astrocytes and mixed glial cells. IS increased AhR and NF-kB nuclear translocation and reduced Nrf2 translocation and HO-1 expression in primary glial cells. In addition, IS induced cell death in neurons in a dose dependent fashion. Injection of IS (800 mg/kg, i.p.) into mice induced histological changes and increased COX-2 expression and nitrotyrosine formation in thebrain tissue. Taken together, our results show a significant contribution of IS in generating a neurotoxic enviroment and it could also have a potential role in neurodegeneration. IS could be considered also a potential therapeutical target for CKD-associated neurodegenerative complications.

Keywords: chronic kidney disease; indoxyl sulfate; neurodegeneration; neuroinflammation; oxidative stress; uremic toxins.

PubMed Disclaimer

Figures

FIGURE 1

FIGURE 1

Effect of IS (15–60 μM) on ROS formation (A), evaluated by means of the probe H2DCF-DA, in C6 cells in presence of DPI and of NAC. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Values are expressed as mean fluorescence intensity (n = 12). Effect of IS (30 μM) on Nrf2 nuclear translocation in C6 cells in presence of DPI and NAC (B). Nuclear translocation of Nrf2 was detected using immunofluorescence confocal microscopy. Scale bar, 10 μm. Blue and green fluorescences indicate localization of the nucleus (DAPI) and Nrf2, respectively. Analysis was performed by confocal laser scanning microscopy. Effect of IS (15–60 μM) on HO-1 (C), NQO1 (D), and SOD (E) expression in C6 cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Effect of IS (30 μM) on AhR nuclear translocation in presence of DPI in C6 cells (F). Nuclear translocation of AhR was detected using immunofluorescence confocal microscopy. Scale bar, 10 μm. Blue and green fluorescences indicate localization of nucleus (DAPI) and AhR, respectively. Analysis was performed by confocal laser scanning microscopy and values are expressed as mean fluorescence intensity (n = 12). Effect of IS (15–60 μM) on ROS formation (G), evaluated by means of the probe H2DCF-DA, in C6 cells in presence of CH-223191. Values are expressed as mean fluorescence intensity (n = 9). ∘∘∘, ∘∘, and °denote P < 0.001, P < 0.01, and P < 0.05 vs. control. ∗∗∗ denotes P < 0.001, vs. IS alone.

FIGURE 2

FIGURE 2

Effect of IS (30 μM) on p65 nuclear translocation in C6 cells in presence of the antagonists CH-223191, DPI and NAC in C6 cells (A). Nuclear translocation of NF-kB p65 subunit was detected using immunofluorescence confocal microscopy. Scale bar, 10 μm. Blue and green fluorescences indicate localization of the nucleus (DAPI) and p65, respectively. Analysis was performed by confocal laser scanning microscopy. Effect of IS (15–60 μM) on ROS formation (B), evaluated by means of the probe H2DCF-DA, in C6 cells in presence of NF-kB-inhibitor PDTC. Values are expressed as mean fluorescence intensity (n = 9).∘∘∘ denotes P < 0.001 vs. control. ∗∗∗ denotes P < 0.001 and ∗ denotes P < 0.05 vs. IS alone.

FIGURE 3

FIGURE 3

Effect of IS (15–60 μM) on ROS formation (A), evaluated by means of the probe H2DCF-DA, in astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Effect of IS (15–60 μM) on nitrotyrosine formmation (B) in astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Effect of IS (30 μM) on Nrf2 nuclear translocation in astrocytes and mixed glial cells (C). Nuclear translocation of Nrf2 was detected using immunofluorescence confocal microscopy. Scale bar, 10 μm. Blue and green fluorescences indicate localization of nucleus (DAPI) and Nrf2, respectively. Analysis was performed by confocal laser scanning microscopy. Effect of IS (15–60 μM) on HO-1 expression (D) in astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Values are expressed as mean fluorescence intensity (n = 9). ∘∘∘, ∘∘, and °denote P < 0.001, P < 0.01, and P < 0.05 vs control. ∗∗ and ∗ denote P < 0.01 and P < 0.05 vs. astrocytes.

FIGURE 4

FIGURE 4

Effect of IS (30 μM) on AhR (A) and p65 (B) nuclear translocation in astrocytes and mixed glial cells. Nuclear translocation of AhR and p65 was detected using immunofluorescence confocal microscopy. Scale bar, 10 μm. Blue and green fluorescences indicate localization of nucleus (DAPI) and AhR and p65, respectively. Analysis (n = 9) was performed by confocal laser scanning microscopy.

FIGURE 5

FIGURE 5

Effect of IS (15–60 μM) on iNOS (A), COX-2 (B) expression by astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Values are expressed as mean fluorescence intensity (n = 9). Effect of IS (15–60 μM) TNF-α (C) and IL-6 (D) release by astrocytes and mixed glial cells (n = 9). Cyokine release was assessed by ELISA assay and expressed as pg/ml (n = 9). ∘∘∘, ∘∘, and °denote P < 0.001, P < 0.01, and P < 0.05 vs. control. ∗∗∗,∗∗, and ∗ denote P < 0.001, P < 0.01, and P < 0.05 vs. astrocytes.

FIGURE 6

FIGURE 6

Effect of IS (15–60 μM) on cortical and on hippocampal neuronal cell viability. Values are expressed as percentage of cytotoxicity (n = 9). ∘∘∘and ° denote P < 0.001 and P < 0.05 vs. control, # denotes P < 0.05 vs. hippocampal neurons.

FIGURE 7

FIGURE 7

Histologic and immunohistochemical findings of brain and kidneys in treated mice (IS column). (A) (1) Brain; normal tissue from control mouse. (2) Brain; neuronal pyknosis associated with mild satellitosis. (3) Kidney; normal tissue from control mouse. (4) Kidney; Atrophic glomeruli and severe vacuolar degeneration of tubules with proteinaceous amorphous material and hypereosinophilic concretions within lumen (arrows); Hematoxylin and Eosin (HE) stain. (B) (1) Brain; normal tissue from control mouse. (2) Brain; strong immunoreactivity for COX-2 antibody in degenerating neurons (arrows) from treated mouse. (3) Kidney; normal tissue from control mouse. (4) Kidney; strong immunoreactivity for COX-2 antibody in blood vessels of the glomeruli (arrows) from treated mouse. Immunohistochemistry (HRP-method). (C) (1) Brain; normal tissue from control mouse. (2) Brain; the immunoreactivity with the anti-nitrotyrosine antibody is intensely detected in the neurons of a treated mice (arrows). (3) Kidney; normal tissue from control mouse. (4) Kidney; strong immunoreactivity in blood vessels of an atrophic glomerulus (arrow) from treated mouse. Immunohistochemistry (HRP-method). Data are from two independent experiments and represent mean ± SEM (n = 5–10 per group).

Similar articles

Cited by

References

    1. Adams J. D., Jr., Odunze I. N. (1991). Oxygen free radicals and Parkinson’s disease. Free Radic. Biol. Med. 10 161–169. 10.1016/0891-5849(91)90009-R - DOI - PubMed
    1. Adesso S., Popolo A., Bianco G., Sorrentino R., Pinto A., Autore G., et al. (2013). The uremic toxin indoxyl sulphate enhances macrophage response to LPS. PLoS ONE 8:e76778 10.1371/journal.pone.0076778 - DOI - PMC - PubMed
    1. Allaman I., Belanger M., Magistretti P. J. (2011). Astrocyte-neuron metabolic relationships: for better and for worse. Trends Neurosci. 34 76–87. 10.1016/j.tins.2010.12.001 - DOI - PubMed
    1. Benda P., Lightbody J., Sato G., Levine L., Sweet W. (1968). Differentiated rat glial cell strain in tissue culture. Science 161 370–371. 10.1126/science.161.3839.370 - DOI - PubMed
    1. Bianco G., Russo R., Marzocco S., Velotto S., Autore G., Severino L. (2012). Modulation of macrophage activity by aflatoxins B1 and B2 and their metabolites aflatoxins M1 and M2. Toxicon 59 644–650. 10.1016/j.toxicon.2012.02.010 - DOI - PubMed

LinkOut - more resources