thromboplastin and Renal-Insufficiency--Chronic

CKD, characterized by retained uremic solutes, is a strong and independent risk factor for thrombosis after vascular procedures . Urem ic solutes such as indoxyl sulfate (IS) and kynurenine (Kyn) mediate prothrombotic effect through tissue factor (TF). IS and Kyn biogenesis depends on multiple enzymes, with therapeutic implications unexplored. We examined the role of indoleamine 2,3-dioxygenase-1 (IDO-1), a rate-limiting enzyme of kynurenine biogenesis, in CKD-associated thrombosis after vascular injury.. IDO-1 expression in mice and human vessels was examined. IDO-1. Both global IDO-1. Leveraging genetic and pharmacologic manipulation in experimental models and data from human studies implicate IS as an inducer of IDO-1 and a perpetuator of the thrombotic milieu and supports IDO-1 as an antithrombotic target in CKD.

Topics: Animals; Aorta; Carotid Artery Injuries; Carotid Artery Thrombosis; Culture Media; Enzyme Induction; Feedback, Physiological; Female; HEK293 Cells; Humans; Indican; Indoleamine-Pyrrole 2,3,-Dioxygenase; Kynurenine; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Targeted Therapy; Myocytes, Smooth Muscle; Postoperative Complications; Renal Insufficiency, Chronic; Thromboplastin; Thrombosis; Tryptophan; Uremia; Vascular Surgical Procedures

Patients with chronic kidney disease (CKD) commonly exhibit hypercoagulability. Increased levels of uremic toxins cause thrombogenicity by increasing tissue factor (TF) expression and activating the extrinsic coagulation cascade. TF is induced in monocytes and macrophages under pathological conditions, such as inflammatory diseases. However, the role of monocyte myeloid cell TF in CKD progression remains unclear. We aimed to clarify this issue, and the present study found that patients with CKD had elevated levels of D-dimer, a marker of fibrin degradation, which was associated with decreased estimated glomerular filtration rate and increased serum levels of uremic toxins, such as indoxyl sulfate. In vitro studies showed that several uremic toxins increased cellular TF levels in monocytic THP-1 cells. Mice with TF specifically deleted in myeloid cells were fed an adenine diet to cause uremic kidney injury. Myeloid TF deletion reduced tubular injury and pro-inflammatory gene expression in the kidneys of adenine-induced CKD but did not improve renal function as measured by plasma creatinine or blood urea nitrogen. Collectively, our findings suggest a novel concept of pathogenesis of coagulation-mediated kidney injury, in which elevated TF levels in monocytes under uremic conditions is partly involved in the development of CKD.

Topics: Adenine; Animals; Fibrin Fibrinogen Degradation Products; Glomerular Filtration Rate; Humans; Kidney Tubules; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Cells; Renal Insufficiency, Chronic; Thromboplastin; Toxins, Biological; Uremia

Endogenous 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

Topics: Basic Helix-Loop-Helix Transcription Factors; Cyclooxygenase 2; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Gene Expression Regulation; Gene Knockdown Techniques; Human Umbilical Vein Endothelial Cells; Humans; Indican; Receptors, Aryl Hydrocarbon; Renal Insufficiency, Chronic; RNA, Messenger; Stress, Mechanical; Thromboplastin

Chronic 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 µM, 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-κB site was essential in TF induction by IAA. In addition, TF induction by IAA was drastically decreased by an inhibitor of the NF-κB pathway. IAA induced the nuclear translocation of NF-κB 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-κB 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.

Topics: Adult; Aged; Aged, 80 and over; Basic Helix-Loop-Helix Transcription Factors; Cells, Cultured; Female; Gene Knockdown Techniques; Human Umbilical Vein Endothelial Cells; Humans; Indoleacetic Acids; Male; Middle Aged; NF-kappa B; Promoter Regions, Genetic; Prospective Studies; Receptors, Aryl Hydrocarbon; Renal Insufficiency, Chronic; RNA, Small Interfering; Signal Transduction; Thromboplastin; Young Adult

Topics: Humans; Phenotype; Renal Insufficiency, Chronic; Thromboplastin; Thrombosis; Ubiquitin-Protein Ligases

Individuals with CKD are particularly predisposed to thrombosis after vascular injury. Using mouse models, we recently described indoxyl sulfate, a tryptophan metabolite retained in CKD and an activator of tissue factor (TF) through aryl hydrocarbon receptor (AHR) signaling, as an inducer of thrombosis across the CKD spectrum. However, the translation of findings from animal models to humans is often challenging. Here, we investigated the uremic solute-AHR-TF thrombosis axis in two human cohorts, using a targeted metabolomics approach to probe a set of tryptophan products and high-throughput assays to measure AHR and TF activity. Analysis of baseline serum samples was performed from 473 participants with advanced CKD from the Dialysis Access Consortium Clopidogrel Prevention of Early AV Fistula Thrombosis trial. Participants with subsequent arteriovenous thrombosis had significantly higher levels of indoxyl sulfate and kynurenine, another uremic solute, and greater activity of AHR and TF, than those without thrombosis. Pattern recognition analysis using the components of the thrombosis axis facilitated clustering of the thrombotic and nonthrombotic groups. We further validated these findings using 377 baseline samples from participants in the Thrombolysis in Myocardial Infarction II trial, many of whom had CKD stage 2-3. Mechanistic probing revealed that kynurenine enhances thrombosis after vascular injury in an animal model and regulates thrombosis in an AHR-dependent manner. This human validation of the solute-AHR-TF axis supports further studies probing its utility in risk stratification of patients with CKD and exploring its role in other diseases with heightened risk of thrombosis.

Topics: Adult; Aged; Clinical Trials as Topic; Female; Humans; Indican; Kynurenine; Male; Metabolomics; Middle Aged; Pattern Recognition, Automated; Receptors, Aryl Hydrocarbon; Renal Insufficiency, Chronic; Signal Transduction; Thromboplastin; Thrombosis; Uremia; Vascular System Injuries

Hypercoagulability is associated with chronic kidney disease (CKD). Tissue factor/factor VIIa complex and factor Xa in the coagulation cascade are known to activate protease-activated receptor 2 (PAR2), and to cause inflammation and tissue injury. Although PAR2 is highly expressed in the kidney, it is unclear whether PAR2 plays a pathogenic role in CKD. To test this, we fed the mice lacking Par2 (F2rl1

Topics: Adenine; Animals; Enzyme-Linked Immunosorbent Assay; Factor V; Factor Xa; Fibrin; Fibrosis; Gene Expression Regulation; Inflammation; Kidney; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Oxidative Stress; Receptor, PAR-2; Renal Insufficiency, Chronic; Thromboplastin

During chronic kidney disease progression, kidney-specific risk factors for cardiovascular disease come into play. The present study investigated the impact of indoxyl sulfate, dietary tryptophan-derived uremic toxin, accumulated in the blood of patients with chronic kidney disease on hemostatic parameters, markers of inflammation, oxidative stress and monocyte to macrophage transition.. Fifty-one CKD patients not undergoing hemodialysis were enrolled in the study. Coagulation factors, fibrinolytic parameters, adhesion molecules, endothelial dysfunction markers, oxidative stress as well as inflammation markers were examined using immune-enzymatic method. Indoxyl sulfate levels were assessed using high-performance liquid chromatography. Biochemical parameters were determined by routine laboratory techniques using an automated analyzers. All assessed parameters were compared with controls and subjected to cross-sectional statistical analysis.. Elevated concentrations of indoxyl sulfate, the vast majority of parameters affecting hemostasis, and markers of renal insufficiency conditions were observed. Part of hemostatic factors, namely tissue factor, von Willebrand factor, thrombomodulin, soluble urokinase-type plasminogen activator receptor, soluble intercellular adhesion molecule-1, soluble vascular cell adhesion protein were correlated with the fraction of indoxyl sulfate. A significant quantity of assessed parameters showed strong correlations with superoxide-dismutase, renal insufficiency rate, C-reactive protein, and neopterin. Levels of indoxyl sulfate were independently associated with markers of impaired endothelial function (thrombomodulin, adhesion molecules), oxidative stress (superoxide-dismutase) and monocytes activation determinant (neopterin), which indicate unconventional links between these systems and the role of indoxyl sulfate. Furthermore, parameters that correlated with the levels of indoxyl sulfate (von Willebrand factor, soluble urokinase-type plasminogen activator receptor, soluble intercellular adhesion molecule-1) were positively associated with the prevalence of cardiovascular disease in a CKD patients.. The study demonstrated that in conditions of chronic exposure to uremic toxins, indoxyl sulfate seems to be one of the "missing links" between impaired renal function and prevalence of cardiovascular events, especially hemostatic disorders. The main functions of the action appear to be altered monocytes activation, intensified inflammatory process, and augmented oxidative stress by this uremic toxin.

Topics: Adult; Aged; C-Reactive Protein; Cardiovascular Diseases; Case-Control Studies; Female; Hemostasis; Humans; Hydrogen Peroxide; Indican; Inflammation; Intercellular Adhesion Molecule-1; Lipoproteins; Male; Middle Aged; Neopterin; Oxidative Stress; Peptide Fragments; Plasminogen Activator Inhibitor 1; Prevalence; Prothrombin; Renal Insufficiency, Chronic; Thrombin; Thromboplastin; Tissue Plasminogen Activator; Urokinase-Type Plasminogen Activator; Vascular Cell Adhesion Molecule-1; von Willebrand Factor

Chronic kidney disease (CKD) is associated with an increased risk of cardiovascular disease (CVD). (Pro)renin receptor (PRR) is activated in the kidney of CKD. The present study aimed to determine the role of indoxyl sulfate (IS), a uremic toxin, in PRR activation in rat aorta and human aortic smooth muscle cells (HASMCs). We examined the expression of PRR and renin/prorenin in rat aorta using immunohistochemistry. Both CKD rats and IS-administrated rats showed elevated expression of PRR and renin/prorenin in aorta compared with normal rats. IS upregulated the expression of PRR and prorenin in HASMCs. N-acetylcysteine, an antioxidant, and diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, suppressed IS-induced expression of PRR and prorenin in HASMCs. Knock down of organic anion transporter 3 (OAT3), aryl hydrocarbon receptor (AhR) and nuclear factor-κB p65 (NF-κB p65) with small interfering RNAs inhibited IS-induced expression of PRR and prorenin in HASMCs. Knock down of PRR inhibited cell proliferation and tissue factor expression induced by not only prorenin but also IS in HASMCs.. IS stimulates aortic expression of PRR and renin/prorenin through OAT3-mediated uptake, production of reactive oxygen species, and activation of AhR and NF-κB p65 in vascular smooth muscle cells. IS-induced activation of PRR promotes cell proliferation and tissue factor expression in vascular smooth muscle cells.

Topics: Animals; Aorta; Cell Proliferation; Gene Expression Regulation; Humans; Indican; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Onium Compounds; Organic Anion Transporters, Sodium-Independent; Rats; Receptors, Cell Surface; Renal Insufficiency, Chronic; RNA, Small Interfering; Thromboplastin; Vacuolar Proton-Translocating ATPases

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.

Topics: Adult; Aged; Aged, 80 and over; Benzoquinones; Case-Control Studies; Cells, Cultured; Dioxins; Endothelium, Vascular; Female; Humans; In Vitro Techniques; Indican; Indoleacetic Acids; Lactams, Macrocyclic; Leukocytes, Mononuclear; Male; Middle Aged; Polymerase Chain Reaction; Receptors, Aryl Hydrocarbon; Renal Insufficiency, Chronic; RNA, Small Interfering; Signal Transduction; Thromboplastin; Tissue Array Analysis; Umbilical Veins; Up-Regulation

Atherosclerotic coronary arteries are more calcified in patients with than without chronic kidney disease (CKD). We addressed the potential for coronary microvascular obstruction in patients with and without CKD during stenting for saphenous vein aorto-coronary graft (SVG) stenosis under protection with a distal occlusion/aspiration device. In patients with and without CKD (n = 20/20), SVG plaque composition was analyzed from virtual histology using intravascular ultrasound analysis before stent implantation. There was more dense calcium and more necrotic core in patients with than without CKD (14 ± 3 vs. 3 ± 1 % and 21 ± 3 vs. 12 ± 2 % of plaque volume, respectively). Coronary aspirate was retrieved during stent implantation and divided into particulate debris and plasma. Patients with CKD had more particulate debris and calcium release than patients without CKD. In contrast, the release of serotonin was less in patients with than without CKD (0.4 ± 0.1 vs. 1.2 ± 0.3 μmol/L), whereas that of catecholamines, endothelin, tissue factor, thromboxane, tumor necrosis factor α, and C reactive protein was not significantly different. Confirming the biochemical results, aspirate plasma from patients with CKD induced less vasoconstriction of rat mesenteric arteries than that from patients without CKD (with endothelium (+E), 26 ± 7 %; without endothelium (-E): 28 ± 7 % vs. +E, 68 ± 12 %; -E: 95 ± 16 % of maximum KCl-induced vasoconstriction). Graft atherosclerosis of patients with CKD is more degenerated and releases more particulate debris and calcium, but the aspirate has surprisingly less serotonin and vasoconstrictor potential.

Topics: Adult; Aged; Aged, 80 and over; alpha-2-HS-Glycoprotein; Animals; Atherosclerosis; Blood Vessel Prosthesis Implantation; C-Reactive Protein; Calcium; Coronary Artery Disease; Graft Occlusion, Vascular; Humans; Male; Middle Aged; Necrosis; Plaque, Atherosclerotic; Rats; Renal Insufficiency, Chronic; Saphenous Vein; Stents; Thromboplastin; Tumor Necrosis Factor-alpha; Ultrasonography, Interventional; Vascular Calcification; Vasoconstriction