Identification of molecular partners responding to the activation of Aryl hydrocarbon Receptor by uremic toxins

La maladie rénale chronique a pour principale complication les maladies cardiovasculaires. L’accumulation des toxines urémiques dérivées de la voie indolique du métabolisme du tryptophane, l’indole-3-acétique acide (IAA) et l’Indoxyl sulfate (IS), sont impliquées dans un phénomène clé des événements cardiovasculaires ; la thrombose. Ces toxines induisent l'expression et l'activité pro coagulante du facteur tissulaire (FT), principal initiateur cellulaire de la coagulation sanguine. Le facteur de transcription, Aryl Hydrocarbon Receptor (AHR), un récepteur cellulaire pour les toxines indoliques, est impliqué dans l'induction du FT endothélial. Les mécanismes par lesquels AhR contrôle l’expression du FT dans les cellules endothéliales sont peu connus.L’objectif principal de cette thèse était de déterminer les voies de signalisation et les facteurs de transcription impliqués dans l'expression du FT médiée par AhR et induit par l’IAA et l’IS. L’expression du FT induite par l’IAA et l’IS au niveau endothélial est contrôlée principalement par la transcription. L’IAA et l’IS activent la voie génomique classique d’AhR. Cependant, l’expression du FT n'est pas médiée par cette voie. L’activation du FT en réponse à l’IAA passe par une voie AhR-p38MAPK-NF-kB. En conclusion, l’induction par l'IAA du FT dans les cellules endothéliales humaines implique la voie non génomique d’AhR-p38MAPK/NF-KB. L’identification de cette voie de signalisation suggère que nous puissions dissocier l’effet détoxifiant d’AhR dépendant de la voie génomique, de l’effet prothrombotique.The main complication of chronic kidney disease is cardiovascular disease. Accumulation of uremic toxins derived from the tryptophan metabolism indole-3-acetic acid (IAA) and Indoxyl sulfate (IS) pathways, are involved in a key phenomenon of cardiovascular events; thrombosis. These toxins induce the expression and pro-coagulant activity of tissue factor (TF), the main cellular initiator of blood coagulation. The transcription factor, Aryl Hydrocarbon Receptor (AHR), a cellular receptor for indole toxins, is involved in the induction of endothelial TF. The mechanisms by which AhR controls the expression of TF in endothelial cells are poorly understood. The main objective of this thesis was to determine the signaling pathways and transcription factors involved in the expression of AhR mediated TF and induced by the IAA and the SI.The expression of IAA-mediated TF and endothelial IS is controlled primarily by transcription. The IAA and the IS are activating the classical genomic AhR pathway. However, the expression of TF is not mediated by this way. Activation of TF in response to the IAA passes through an AhR-p38MAPK-NF-kB pathway.In conclusion, induction by IAA of TF in human endothelial cells involves the non-genomic pathway of AhR-p38MAPK / NF-KB. The identification of this signaling pathway suggests that we can dissociate the detrimental effect of AhR depending on the genomic pathway, the prothrombotic effect

Tryptophan-Derived Uremic Toxins and Thrombosis in Chronic Kidney Disease

Patients with chronic kidney disease (CKD) display an elevated risk of thrombosis. Thrombosis occurs in cardiovascular events, such as venous thromboembolism, stroke, and acute coronary syndrome, and is a cause of hemodialysis vascular access dysfunction. CKD leads to the accumulation of uremic toxins, which exerts toxic effects on blood and the vessel wall. Some uremic toxins result from tryptophan metabolization in the gut through the indolic and the kynurenine pathways. An increasing number of studies are highlighting the link between such uremic toxins and thrombosis in CKD. In this review, we describe the thrombotic mechanisms induced by tryptophan-derived uremic toxins (TDUT). These mechanisms include an increase in plasma levels of procoagulant factors, induction of platelet hyperactivity, induction of endothelial dysfunction/ impairment of endothelial healing, decrease in nitric oxide (NO) bioavailability, and production of procoagulant microparticles. We focus on one important prothrombotic mechanism: The induction of tissue factor (TF), the initiator of the extrinsic pathway of the blood coagulation. This induction occurs via a new pathway, dependent on the transcription factor Aryl hydrocarbon receptor (AhR), the receptor of TDUT in cells. A better understanding of the prothrombotic mechanisms of uremic toxins could help to find novel therapeutic targets to prevent thrombosis in CKD

Aryl Hydrocarbon Receptor Activation and Tissue Factor Induction by Fluid Shear Stress and Indoxyl Sulfate in Endothelial Cells

International audienceEndogenous agonists of the transcription factor aryl hydrocarbon receptor (AHR) such as the indolic uremic toxin, indoxyl sulfate (IS), accumulate in patients with chronic kidney disease. AHR activation by indolic toxins has prothrombotic effects on the endothelium, especially via tissue factor (TF) induction. In contrast, physiological AHR activation by laminar shear stress (SS) is atheroprotective. We studied the activation of AHR and the regulation of TF by IS in cultured human umbilical vein endothelial cells subjected to laminar fluid SS (5 dynes/cm2). SS and IS markedly increased the expression of AHR target genes PTGS2 (encoding for COX2), AHRR, CYP1A1, and CYP1B1, as well as F3 (encoding for TF), in an AHR-dependent way. IS amplified SS-induced TF mRNA and protein expression and upregulation of AHR target genes. Interestingly, tyrosine kinase inhibition by genistein decreased SS-but not IS-induced TF expression. Finally, the increase in TF expression induced by laminar SS was not associated with increased TF activity. In contrast, IS increased TF activity, even under antithrombotic SS conditions. In conclusion, IS and SS induce AHR activation and AHR-dependent TF upregulation by different mechanisms. Impairment of the antithrombotic properties of shear stressed endothelium by toxic AHR agonists could favor cardiovascular diseases in CKD

Aryl hydrocarbon receptor is activated in patients and mice with chronic kidney disease

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Mechanisms of tissue factor induction by the uremic toxin indole-3 acetic acid through aryl hydrocarbon receptor/nuclear factor-kappa B signaling pathway in human endothelial cells

International audienceChronic kidney disease (CKD) is associated with high risk of thrombosis. Indole-3 acetic acid (IAA), an indolic uremic toxin, induces the expression of tissue factor (TF) in human umbilical vein endothelial cells (HUVEC) via the transcription factor aryl hydrocarbon receptor (AhR). This study aimed to understand the signaling pathways involved in AhR-mediated TF induction by IAA. We incubated human endothelial cells with IAA at 50 mu M, the maximal concentration found in patients with CKD. IAA induced TF expression in different types of human endothelial cells: umbilical vein (HUVEC), aortic (HAoEC), and cardiac-derived microvascular (HMVEC-C). Using AhR inhibition and chromatin immunoprecipitation experiments, we showed that TF induction by IAA in HUVEC was controlled by AhR and that AhR did not bind to the TF promoter. The analysis of TF promoter activity using luciferase reporter plasmids showed that the NF-B site was essential in TF induction by IAA. In addition, TF induction by IAA was drastically decreased by an inhibitor of the NF-B pathway. IAA induced the nuclear translocation of NF-B p50 subunit, which was decreased by AhR and p38MAPK inhibition. Finally, in a cohort of 92 CKD patients on hemodialysis, circulating TF was independently related to serum IAA in multivariate analysis. In conclusion, TF up-regulation by IAA in human endothelial cells involves a non-genomic AhR/p38 MAPK/NF-B pathway. The understanding of signal transduction pathways related to AhR thrombotic/inflammatory pathway is of interest to find therapeutic targets to reduce TF expression and thrombotic risk in patients with CKD