Indoxyl Sulfate Induces IL-6 Expression in Vascular Endothelial and Smooth Muscle Cells through OAT3-Mediated Uptake and Activation of AhR/NF-&#954;B Pathway

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Abstract

Background/Aims: Interleukin-6 (IL-6) is one of the inflammation biomarkers with highest predictive value for outcome in chronic kidney disease (CKD) patients. The present study aimed to determine the effects of indoxyl sulfate (IS) on IL-6 expression in vascular cells. Methods: IS was administered to normo- and hypertensive rats. Human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) were incubated with or without IS. Results: Immunohistochemistry revealed that IS-administered rats showed increased expression of IL-6 in the aortic tissues. IS increased IL-6 expression in HUVECs and HASMCs in a time- and dose-dependent manner. Knockdown of organic anion transporter 3 (OAT3) using small interfering RNA (siRNA) inhibited IS-induced expression of IL-6 in HUVECs and HASMCs. IS induced activation of aryl hydrocarbon receptor (AhR) and nuclear factor-κB (NF-κB) subunit p65 in HUVECs and HASMCs. Both AhR siRNA and p65 siRNA inhibited IS-induced expression of IL-6. AhR siRNA inhibited IS-induced phosphorylation and nuclear translocation of p65 without change in total p65 level. However, p65 siRNA did not inhibit IS-induced nuclear translocation of AhR. Thus, AhR is responsible for IS-induced p65 signaling transduction. Conclusion: IS induces IL-6 expression in vascular endothelial and smooth muscle cells through OAT3/AhR/NF-κB pathway.

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The uremic toxin 3-indoxyl sulfate is a potent endogenous agonist for the human aryl hydrocarbon receptor.

Jennifer Schroeder, Brett C Dinatale, Iain Murray … (2010)

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in the regulation of multiple cellular pathways, such as xenobiotic metabolism and Th17 cell differentiation. Identification of key physiologically relevant ligands that regulate AHR function remains to be accomplished. Screening of indole metabolites has identified indoxyl 3-sulfate (I3S) as a potent endogenous ligand that selectively activates the human AHR at nanomolar concentrations in primary human hepatocytes, regulating transcription of multiple genes, including CYP1A1, CYP1A2, CYP1B1, UGT1A1, UGT1A6, IL6, and SAA1. Furthermore, I3S exhibits an approximately 500-fold greater potency in terms of transcriptional activation of the human AHR relative to the mouse AHR in cell lines. Structure-function studies reveal that the sulfate group is an important determinant for efficient AHR activation. This is the first phase II enzymatic product identified that can significantly activate the AHR, and ligand competition binding assays indicate that I3S is a direct AHR ligand. I3S failed to activate either CAR or PXR. The physiological importance of I3S lies in the fact that it is a key uremic toxin that accumulates to high micromolar concentrations in kidney dialysis patients, but its mechanism of action is unknown. I3S represents the first identified relatively high potency endogenous AHR ligand that plays a key role in human disease progression. These studies provide evidence that the production of I3S can lead to AHR activation and altered drug metabolism. Our results also suggest that prolonged activation of the AHR by I3S may contribute to toxicity observed in kidney dialysis patients and thus represent a possible therapeutic target.

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Indolic uremic solutes increase tissue factor production in endothelial cells by the aryl hydrocarbon receptor pathway.

Laurent Arnaud, Claire Cerini, Anneleen Pletinck … (2013)

In chronic kidney disease (CKD), uremic solutes accumulate in blood and tissues. These compounds probably contribute to the marked increase in cardiovascular risk during the progression of CKD. The uremic solutes indoxyl sulfate and indole-3-acetic acid (IAA) are particularly deleterious for endothelial cells. Here we performed microarray and comparative PCR analyses to identify genes in endothelial cells targeted by these two uremic solutes. We found an increase in endothelial expression of tissue factor in response to indoxyl sulfate and IAA and upregulation of eight genes regulated by the transcription factor aryl hydrocarbon receptor (AHR). The suggestion by microarray analysis of an involvement of AHR in tissue factor production was confirmed by siRNA inhibition and the indirect AHR inhibitor geldanamycin. These observations were extended to peripheral blood mononuclear cells. Tissue factor expression and activity were also increased by AHR agonist dioxin. Finally, we measured circulating tissue factor concentration and activity in healthy control subjects and in patients with CKD (stages 3-5d), and found that each was elevated in patients with CKD. Circulating tissue factor levels were positively correlated with plasma indoxyl sulfate and IAA. Thus, indolic uremic solutes increase tissue factor production in endothelial and peripheral blood mononuclear cells by AHR activation, evoking a 'dioxin-like' effect. This newly described mechanism of uremic solute toxicity may help understand the high cardiovascular risk of CKD patients.

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Indoxyl Sulfate Upregulates Expression of ICAM-1 and MCP-1 by Oxidative Stress-Induced NF-ĸB Activation

Zohra Tumur, Hidehisa Shimizu, Atsushi Enomoto … (2010)

Background/Aim: Indoxyl sulfate, a uremic toxin, is considered a risk factor for cardiovascular disease (CVD) in chronic kidney disease (CKD). The present study aimed to determine whether indoxyl sulfate increases the expression of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemotactic protein-1 (MCP-1) by reactive oxygen species (ROS)-induced activation of nuclear factor-ĸB (NF-ĸB) in vascular endothelial cells. Methods: Human umbilical vein endothelial cells (HUVEC) were incubated with indoxyl sulfate. The expression of ICAM-1 and MCP-1 in HUVEC was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Phospho-NF-ĸB p65 (Ser 536), an active form of the NF-ĸB subunit, was determined by Western blotting. Results: Indoxyl sulfate significantly increased the mRNA expression of ICAM-1 and MCP-1 in HUVEC in a time- and concentration-dependent manner. Inhibitors of NF-ĸB (ammonium pyrrolidinedithiocarbamate and isohelenin) and an antioxidant (N-acetyl- L -cysteine) suppressed the indoxyl sulfate-induced expression of ICAM-1 and MCP-1 in HUVEC. Indoxyl sulfate increased phospho- NF-ĸB p65 in HUVEC, and N-acetyl- L -cysteine suppressed it. Conclusions: Indoxyl sulfate upregulates the expression of ICAM-1 and MCP-1 by ROS-induced activation of NF-ĸB in vascular endothelial cells. Thus, indoxyl sulfate may play an important role in the development of CVD in CKD by increasing the endothelial expression of ICAM-1 and MCP-1.

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Author and article information

Journal

Title: Nephron Experimental Nephrology

Abbreviated Title: Nephron Exp Nephrol

Publisher: S. Karger AG

ISSN (Electronic): 1660-2129

Publication date Created: December 16 2014

Publication date Created: December 1 2014

Publication date (Electronic): November 5 2014

Volume: 128

Issue: 1-2

Pages: 1-8

Article

DOI: 10.1159/000365217

SO-VID: e57cd56d-3594-4dea-9d89-1d0b973c17f4

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