Indoxyl Sulfate Activates NLRP3 Inflammasome to Induce Cardiac Contractile Dysfunction Accompanied by Myocardial Fibrosis and Hypertrophy.

In patients with chronic kidney diseases (CKD), high serum indoxyl sulfate (IS) levels correlate with cardiac fibrosis and hypertrophy and thus a critical risk factor for heart failure. The aim of this study was to determine the effects of IS on cardiac function and inflammasome pathway in a rat model of CKD. We assessed the physiological and pathological changes and measured biomarkers of fibrosis and hypertrophy in the hearts of Dahl salt-sensitive (DS), DS hypertensive (DH), and DH IS-treated rats (DH + IS). Low left ventricular (LV) ejection fraction, LV dilatation, and advanced myocardial fibrosis and hypertrophy were observed in DH + IS, which resemble changes found in uremic cardiomyopathy. These changes were independent of renal function and blood pressure. RT-PCR and western blotting analysis showed upregulation of fibrosis and hypertrophy-related biomarkers and adhesion molecules in the hearts of DH + IS rats. IS activated aryl hydrocarbon receptor (AHR) pathway, nuclear factor kappa B p65 (NF-κB p65), and inflammasome in the myocardium of DH + IS rat. Moreover, IS upregulated the expression of critical NLRP3 inflammasome components (NLRP3, ASC, and procaspase-1) and increased production of IL-1ß and IL-18. Finally, IS upregulated various inflammatory cytokines, such as MCP-1, TNF-α, IL-6, and TGFß1, in the myocardium. Our results suggested that IS induced cardiac fibrosis and hypertrophy and impaired LV function through activation of cardiac NLRP3 inflammasome via the AHR/NF-κB pathway.

Indoxyl Sulfate-induced Vascular Calcification is mediated through Altered Notch Signaling Pathway in Vascular Smooth Muscle Cells.

Introduction: The aim of this study was to determine the role of Notch in indoxyl sulfate (IS)-induced vascular calcification (VC). Materials and methods: VC and expression of Notch-related and osteogenic molecules were examined in Dahl salt-sensitive (DS), DS hypertensive (DH), and DH IS-treated rats (DH+IS). The effects of IS on expression of Notch receptors, apoptotic activity, and calcification were examined in cultured aortic smooth muscle cells (SMCs). Results: Medial calcification was noted only in aortas and coronary arteries of DH+IS rats. Notch1, Notch3, and Hes-1 were expressed in aortic SMCs of all rats, but only weakly in the central areas of the media and around the calcified lesions in DH+IS rats. RT-PCR and western blotting of DH+IS rat aortas showed downregulation of Notch ligands, Notch1 and Notch3, downstream transcriptional factors, and SM22, and conversely, overexpression of osteogenic markers. Expression of Notch1 and Notch3 in aortic SMCs was highest in incubation under 500 µM IS for 24hrs, and then decreased time- and dose-dependently. Coupled with this decrease, IS increased caspase 3/7 activity and TUNEL-positive aortic SMCs. In addition, pharmacological Notch signal inhibition with DAPT induced apoptosis in aortic SMCs. ZVAD, a caspase inhibitor abrogated IS-induced and DAPT-induced in vitro vascular calcification. Knockdown of Notch1 and Notch3 cooperatively increased expression of osteogenic transcriptional factors and decreased expression of SM22. Conclusion: Our results suggested that IS-induced VC is mediated through suppression of Notch activity in aortic SMCs, induction of osteogenic differentiation and apoptosis.

Chronic Restraint Stress Induces Gastric Mucosal Inflammation with Enhanced Oxidative Stress in a Murine Model.

BACKGROUND: Although the underlying mechanisms of chronic stress are still unknown, this condition has been related to the pathophysiology of gastric mucosal inflammation, whose development is accelerated by oxidative stress. The present study investigates how chronic stress influences gastric mucosal oxidative stress and inflammation. METHODS: Eight-week-old C57BL/6J male mice were subjected to two-week intermittent restraint stress. The expressions of CD11b (a specific for monocyte/macrophage), monocyte/macrophage cell surface markers (CD68 and F4/80), NADPH oxidase-4 (Nox-4) and 8-hydroxy-2'-deoxyguanosine (8-OHdG, a sensitive biomarker of oxidative stress) were determined using immunohistochemistry, RT-PCR, and enzyme-linked immunosorbent assay, respectively. The expressions of antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase, were examined by RT-PCR and Western blotting. The expressions of proinflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α), were determined using immunohistochemistry and RT-PCR, respectively. RESULTS: Chronic stress increased the lymphocytic infiltration and inflammation within the gastric mucosa of mice. Stress remarkably increased the expression levels of CD11b and mRNA expression levels of CD68 and F4/80 in the mucosa of the stomach of stressed mice. Stress remarkably increased both mRNA and plasma concentrations of Nox-4 and 8-OHdG; and markedly reduced gastric mRNA and protein expression levels of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. The expressions of proinflammatory cytokines (MCP-1, IL-1ß, and TNF-α) were predominantly observed in the gastric mucosal layers of the stressed mice. Furthermore, stress remarkably elevated the gastric mucosal mRNA expression levels of MCP-1, IL-1ß, and TNF-α. CONCLUSION: Two weeks of restraint stress induced gastric inflammation in the murine model with enhanced oxidative stress and reduced anti-oxidative system.

Chronic restraint stress induces esophageal fibrosis with enhanced oxidative stress in a murine model.

Although the underlying mechanism of stress remains unknown, it has been associated with the pathophysiology of gastroesophageal reflux diseases, the development of which appear to be accelerated by oxidative stress and fibrosis. The aim of the current study was to investigate the effect of chronic restraint stress on esophageal oxidative stress and fibrosis, as well as the impact of oxidative stress in a murine model whereby 8-week old C57BL/6J male mice were subjected to intermittent chronic restraint stress for a two-week period. The current study demonstrated that chronic restraint stress significantly reduced the body weight of mice compared with the control group. Although chronic restraint stress did not significantly alter the levels of triglycerides or cholesterol, free fatty acid concentration was significantly increased compared with the control group. Furthermore, chronic restraint stress significantly upregulated the expression levels of several fibrotic biomarkers including collagen type I, transforming growth factor ß-1, α-smooth muscle actin and SMAD-3 compared with the control group. In addition, the expression levels of the reactive oxygen species (ROS) NADPH oxidase-4 and malondialdehyde were significantly increased, while the expression levels of nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 were significantly decreased in esophageal tissue from mice in the chronic restraint stress group compared with the control group. In conclusion, chronic restraint stress may induce esophageal fibrosis by accumulating ROS and increasing fibrotic gene expression in a murine model.

Chronic stress augments esophageal inflammation, and alters the expression of transient receptor potential vanilloid 1 and protease­activated receptor 2 in a murine model.

Stress is a pivotal factor for inflammation, reactive oxygen species (ROS) production and formation of visceral hypersensitivity (VH) in the process of gastroesophageal reflux disease (GERD). In the present study, the effects of stress on esophageal inflammation, oxidative stress and VH were investigated in a chronic restraint stress mouse model. C57BL/6J male mice were subjected to 2 weeks of intermittent restraint stress, and histopathological analysis revealed that stress induced esophageal inflammation and fibrosis, while no distinct changes were detected in non­stressed control mice. In addition, increased NADPH oxidase 4 expression was observed in the plasma and esophagus of stressed mice, indicating accumulation of ROS. The expression levels of antioxidants, including Mn­superoxide dismutase (MnSOD), Cu/Zn­SOD, catalase and glutathione peroxidase, were also analyzed using reverse transcription­quantitative polymerase chain reaction (RT­qPCR). In addition, transient receptor potential vanilloid 1 (TRPV­1) and protease­activated receptor 2 (PAR­2), which are crucial receptors for VH, were measured by immunohistochemistry and RT­qPCR. The results demonstrated that stress markedly reduced antioxidant expression, while it significantly upregulated TRPV­1 and PAR­2 expression levels in the mouse esophagus. Finally, 2 weeks of restraint stress significantly increased the esophageal and plasma levels of inflammatory cytokines, including interleukin (IL)­6, IL­8, interferon­Î³ and tumor necrosis factor­α. Taken together, the present study results indicated that stress­induced esophageal inflammation and ROS generation involves VH.

Double-chambered left ventricle: A rare case in a child.

Double-chambered left ventricle (DCLV) is an extremely rare congenital heart disease. In this condition, the left ventricle is divided into two chambers by a septum or muscle fiber with abnormal proliferation. A symptomatic boy was diagnosed with DCLV at our hospital. The patient was admitted with the major complaint of 8 years of cardiac murmur, which was discovered through physical examination, and 5 years of palpitations and shortness of breath. He has been followed up without treatment.

Anxiety and Depression among Hypertensive Outpatients in Afghanistan: A Cross-Sectional Study in Andkhoy City.

There is a relationship between mental and physical health. Depression and anxiety are linked with the development of several chronic diseases. The purpose of the present study was to determine the prevalence and factors associated with anxiety and depression among adult hypertensive outpatients in Afghanistan. Methods. Two hundred thirty-four consecutive hypertensive patients from December 2015 to August 2016 were recruited to complete the Hospital Anxiety and Depression Scale (HADS) questionnaire, which has scores for classifying the participants having anxiety and depression symptoms. Results. Of the total 234 patients, 81 (34.6%) were males and 153 (65.4%) were females. The mean age was 54.6 ± 12.7 for the hypertensive patients with anxiety and 63.8 ± 15.0 for the hypertensive patients with depression while this figure was 49.5 ± 10.2 for the adult participants in general population in Kabul city (Saeed, 2013). The prevalence of anxiety and depression (42.3% vs. 58.1%) among hypertensive persons is compared with the same mental disorders among Afghan refugees (39.3% vs. 22.1%) in Dalakee Refugee Camp (in Iran) (Hosseini Divkolaye and Burkle, 2017). Of the total participants, 99 had anxiety (42.3%), 136 had depression (58.1%), and 66 had (28.2%) comorbid anxiety-depression. Multivariate analysis was used. For anxiety age, female gender, smoking, diabetes mellitus, and 2 or more chronic diseases had a significant association. For depression, age and diabetes mellitus had a significant association, and for comorbid anxiety, depression, age, diabetes mellitus, and 2 or more chronic diseases had a significant association. Conclusion. This study shows that anxiety and depression are highly prevalent among hypertensive patients in an outpatient clinic in Afghanistan. There was an association between some sociodemographic and clinical characteristics and anxiety and depression. More studies are needed on a national level to inform the development of strategies for the prevention and control of psychological distress among patients with chronic diseases in Afghanistan.

Left ventricular hypertrophy and proteinuria in patients with essential hypertension in Andkhoy, Afghanistan.

Left ventricular hypertrophy (LVH) and proteinuria are known as independent predictors of cardiovascular death in hypertension. However, LVH and its association with proteinuria have not been investigated in adult hypertensive patients in Afghanistan. The objective of this research was to determine the prevalence of LVH and the correlation between LVH and proteinuria among the Afghan adult hypertensive population visiting an outpatient clinic in Afghanistan. We retrospectively evaluated 789 hypertensive patients (mean age is 56 years and 46% were men) who visited the clinic between December 2014 and August 2016. Patient characteristics and laboratory and clinical findings were recorded. The rate of LVH among hypertensive patients was 54.4%. Patients with proteinuria had a significantly higher LVH percentage compared to those without proteinuria (73.2% versus 55.8%; P<0.001). There was a significant correlation between LVH and proteinuria among hypertensive patients (r=0.182, P<0.001). Based on a multivariate regression analysis, age (odds ratio [OR], 1.04; 95% confidence interval [CI], 1.02-1.05), proteinuria (OR, 1.69; 95% CI, 1.19-2.41), and female sex (OR, 0.09; 95% CI, 0.06-0.13) were significant factors. In conclusion, the prevalence of LVH was more than 50% in the Afghan adult hypertensive population. This study indicates that there is a significant relationship between LVH detected by ECG and the presence of proteinuria among such subjects.

Angiotensin receptor blocker irbesartan reduces stress-induced intestinal inflammation via AT1a signaling and ACE2-dependent mechanism in mice.

Stress is associated with pathophysiology of both irritable bowel syndrome (IBS) and hypertension. Angiotensin receptor blockers (ARB) have anti-inflammatory properties via inhibition of angiotensin II (Ang II)/Ang II type I receptor axis (AT1). Inhibition of the classical RAS pathway is also involved in upregulation of angiotensin converting enzyme-2 (ACE2), which activates the Ang-(1-7)/Mas pathway to counteract inflammatory signaling and acts as a partner of the amino acid transporter, B0AT-1, to absorb tryptophan for regulation of microbiota-gut-brain axis. In this study, we determined the effects of ARB irbesartan on stress-induced intestinal inflammation. C57BL/6J mice were subjected to 2-week intermittent restraint stress. They were orally treated during the stress with either vehicle, 3 or 10 mg/kg/day irbesartan. Restraint stress resulted in colon inflammation with higher histological damage scores, increased expression of Nox4, TLR-4 and IL1-ß, accumulation of reactive oxygen species (ROS), and activation of the ACE-angiotensin II-AT1 receptor axis. Stress also downregulated intestinal amino acid transporter, ACE2/B0AT-1, and activity of intestinal mammalian target of rapamycin (mTOR) and p70 S6 kinase (p70S6K), resulting in decrease in α-defensins, changes in intestinal microbial contents, and perturbation of tryptophan metabolism with activation of the kynurenine pathway. Administration of irbesartan inhibited activation of stress-induced AT1 pathway to reduce intestinal ROS accumulation and inflammation, restored expression of ACE2/B0AT-1, activity of mTOR and p70S6K, dysbiosis and tryptophan metabolism. Our results suggest that AT1 is a potentially suitable therapeutic target in stress-induced intestinal inflammation, and that irbesartan could be beneficially suitable for the treatment of stressed patients with IBS.

Indole-3-propionic acid suppresses indoxyl sulfate-induced expression of fibrotic and inflammatory genes in proximal tubular cells.

Indoxyl sulfate (IS) induces fibrosis and inflammation in kidneys via oxidative stress through the induction of transforming growth factor-ß1 (TGF-ß1) and monocyte chemotactic protein-1 (MCP-1). Furthermore, IS is a potent endogenous agonist for aryl hydrocarbon receptor (AHR), which regulates the transcription of genes such as cytochrome P450 (CYP) 1A1. Indole-3-propionic acid (IPA) is an antioxidant and has been reported to be neuroprotective. We determined whether IPA suppresses IS-induced expression of AHR, CYP1A1, TGF-ß1, and MCP-1 in proximal tubular cells. The effects of IS on the expression of AHR, CYP1A1, TGF-ß1, and MCP-1 were studied using normotensive rats and hypertensive rats. The effects of IPA on IS-induced expression of AHR, CYP1A1, TGF-ß1, and MCP-1 were studied using proximal tubular cells (HK-2). Furthermore, the effects of IPA on IS-induced expression and phosphorylation of signal transducer and activator of transcription 3 (Stat3) were studied in HK-2 cells. Administration of IS induced the expression of AHR, CYP1A1, TGF-ß1, and MCP-1 in the tubular cells of rat kidneys. IPA significantly suppressed IS-induced mRNA and protein expression of AHR, CYP1A1, TGF-ß1, and MCP-1 in HK-2 cells. IPA suppressed the IS-induced expression and phosphorylation of Stat3 in HK-2 cells. Furthermore, knockdown of Stat3 inhibited the IS-induced mRNA and protein expression of AHR, CYP1A1, TGF-ß1, and MCP-1 in HK-2 cells. In conclusion, IPA suppressed the IS-induced expression of AHR, CYP1A1, TGF-ß1, and MCP-1 through suppression of Stat3 in proximal tubular cells. Thus, IPA suppresses IS-induced expression of fibrotic and inflammatory genes in proximal tubular cells.

Increased dipeptidyl peptidase-4 accelerates diet-related vascular aging and atherosclerosis in ApoE-deficient mice under chronic stress.

OBJECTIVES: Exposure to psychosocial stress is a risk factor for cardiovascular disease. Given that dipeptidyl peptidase-4 (DPP4) regulates several intracellular signaling pathways associated with glucagon-like peptide-1 (GLP-1) metabolism, we investigated the role of DPP4 in stress-related vascular senescence and atherosclerosis in apolipoprotein E-deficient (ApoE-/-) mice. METHODS AND RESULTS: ApoE-/- mice fed a high-fat (HF) diet were randomly assigned to one of non-stress and immobilized stress groups for 12weeks. Chronic stress accelerated vascular senescence and atherosclerotic plaque growth at the aortic roots. Stressed mice had increased levels of plasma DPP4 and decreased levels of plasma GLP-1 and adiponectin (APN) and adipose APN expression. Stress increased plaque macrophage infiltration, neovessel density, and elastin fragmentation, lessened the plaque collagen content, and increased the levels of toll-like receptor-2 (TLR2), TLR4, C-X-C chemokine receptor-4, cathepsins S and K, osteopontin, peroxisome proliferator-activated receptor-α, p16INK4A, p21, and gp91phox mRNAs and/or proteins. Stressed aortas had also increased matrix metalloproteinase-2 (MMP-2) and MMP-9 activities. DPP4 inhibition with anagliptin reversed stress-related atherosclerotic lesion formation, and this benefit was abrogated by APN blocking. In vitro, the GLP-1 receptor agonist exenatide stimulated APN expression in 3T3-L1 cells. CONCLUSIONS: These results indicate that the DPP4 inhibition-mediated benefits are likely attributable, at least in part, to attenuation of plaque inflammation, oxidative stress and proteolysis associated with GLP-1-mediated APN production in ApoE-/- mice under stress. Thus, DPP4 will be a novel therapeutic target for the treatment of stress-related cardiovascular disease.

Xanthine oxidase inhibition by febuxostat attenuates stress-induced hyperuricemia, glucose dysmetabolism, and prothrombotic state in mice.

Chronic stress is closely linked to the metabolic syndrome, diabetes, hyperuricemia and thromboembolism, but the mechanisms remain elusive. We reported recently that stress targets visceral adipose tissue (VAT), inducing lipolysis, low-grade inflammation with production of inflammatory adipokines, metabolic derangements such as insulin resistance, and prothrombotic state. In the present study, we hypothesized the involvement of VAT xanthine oxidoreductase (XOR), a source of reactive oxygen species (ROS) and uric acid (UA) in the above processes. Restraint stress in mice resulted in upregulation of XOR and xanthine oxidase activity, accumulation of ROS in VAT as well as liver and intestine, increase in serum UA levels, upregulation of NADPH oxidase subunits and downregulation of antioxidant enzymes. Immunohistochemistry and RT-PCR analysis also showed that restraint stress induced VAT monocyte accumulation and proinflammatory adipokine production, resulting in reduced insulin sensitivity and induction of plasminogen activator inhibitor-1 and tissue factor in VAT. Treatment with febuxostat, a potent XO inhibitor, suppressed stress-induced ROS production and VAT inflammation, resulting in improvement of serum UA levels, insulin sensitivity, and prothrombotic tendency. Our results suggest that stress perturbs glucose and UA metabolism, and promotes prothrombotic status, and that XO inhibition by febuxostat might be a potential therapy for stress-related disorders.

Associations between proteinuria and cardiovascular risk factors among hypertensive patients in Andkhoy, Afghanistan.

Proteinuria in hypertension is an early marker of renal disease and a predictor for the progression of end stage renal disease, and cardiovascular diseases. This study was designed to determine the prevalence of proteinuria and its association with cardiovascular risk factors among adult hypertensive patients in Afghanistan. Five hundred fifty-five patients with a high blood pressure recorded in an outpatient clinic in Andkhoy, Afghanistan from December 2014 to May 2015, were included in this study. Data obtained from each patient, included demographic characteristics, body mass index, blood pressure patterns, cardiovascular history, cardiovascular risk factors, comorbidity, and current drug-therapy. Dipstick screening for proteinuria was performed with reagent test strips. The mean age of the patients was 57.9 ± 13.3 years, and a female predominance was observed (n = 333, 60%). The prevalence of proteinuria was 67.2%. The predictors of proteinuria were found to be age ≥65 years (odds ratio [OR] 1.02, 95% confidence interval [CI] 1.00-1.04), smoking (OR 1.88, 95% CI 1.17-3.02), heart failure (OR 2.23, 95% CI 1.13-4.41), and diabetes mellitus (OR 3.41, 95% CI 1.49-7.81). In conclusion, this study shows that proteinuria is highly prevalent among hypertensive outpatients in an outpatient clinic in Andkhoy, Afghanistan, especially in those with high cardiovascular risk.

Dipeptidyl peptidase- IV inhibitor alogliptin improves stress-induced insulin resistance and prothrombotic state in a murine model.

BACKGROUND: Stress evokes lipolytic release of free fatty acid (FFA) and low-grade inflammation in visceral adipose tissue, mediated by increased adipokine secretion, and contributes to glucose metabolism disorder and prothrombotic state. We tested the hypothesis that alogliptin, a dipeptidyl peptidase-4 inhibitor, can ameliorate the biological effects of chronic stress in mice. METHOD AND RESULTS: C57BL/6J mice were subjected to 2-week intermittent restraint stress and orally treated with vehicle or alogliptin (dose: 15 or 45mg/kg/day). Plasma levels of lipids, proinflammatory cytokines (monocyte chemoattractant protein-1, tumor necrosis factor-α, and interleukin-6), and 8-hydroxydeoxyguanosine were measured with enzyme-linked immunosorbent assay. Monocyte/macrophage accumulation in inguinal white adipose tissue (WAT) was examined by CD11b-positive cell count and mRNA expression of CD68 and F4/80 was examined by immunohistochemistry and RT-PCR, respectively. The mRNA levels of the above-mentioned proinflammatory cytokines, NADPH oxidase 4, adiponectin, and coagulation factors (plasminogen activation inhibitor-1 and tissue factor) in WAT were also assessed with RT-PCR. Glucose metabolism was assessed by glucose and insulin tolerance tests, plasma levels of DPP-4 activity, glucagon-like peptide-1, expression of DPP-4, insulin receptor substrate-1 and glucose transporter 4 in WAT and skeletal muscle. Alogliptin administration suppressed stress-induced FFA release, oxidative stress, adipose tissue inflammation, DPP-4 activation, and prothrombotic state in a dose-dependent manner, and improved insulin sensitivity in stressed mice. CONCLUSIONS: The results indicate that alogliptin improves stress-induced prothrombotic state and insulin resistance; suggesting that alogliptin could have beneficial therapeutic effects against cardiovascular complications in diabetic patients under stress.

Cathepsin S Activity Controls Injury-Related Vascular Repair in Mice via the TLR2-Mediated p38MAPK and PI3K-Akt/p-HDAC6 Signaling Pathway.

OBJECTIVE: Cathepsin S (CatS) participates in atherogenesis through several putative mechanisms. The ability of cathepsins to modify histone tail is likely to contribute to stem cell development. Histone deacetylase 6 (HDAC6) is required in modulating the proliferation and migration of various types of cancer cells. Here, we investigated the cross talk between CatS and HADC6 in injury-related vascular repair in mice. APPROACH AND RESULTS: Ligation injury to the carotid artery in mice increased the CatS expression, and CatS-deficient mice showed reduced neointimal formation in injured arteries. CatS deficiency decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and toll-like receptor 2 expression in ligated arteries. The genetic or pharmacological inhibition of CatS also alleviated the increased phosphorylation of p38 mitogen-activated protein kinase, Akt, and HDAC6 induced by platelet-derived growth factor BB in cultured vascular smooth muscle cells (VSMCs), and p38 mitogen-activated protein kinase inhibition and Akt inhibition decreased the phospho-HDAC6 levels. Moreover, CatS inhibition caused decrease in the levels of the HDAC6 activity in VSMCs in response to platelet-derived growth factor BB. The HDAC6 inhibitor tubastatin A downregulated platelet-derived growth factor-induced VSMC proliferation and migration, whereas HDAC6 overexpression exerted the opposite effect. Tubastatin A also decreased the intimal VSMC proliferation and neointimal hyperplasia in response to injury. Toll-like receptor 2 silencing decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and VSMC migration and proliferation. CONCLUSIONS: This is the first report detailing cross-interaction between toll-like receptor 2-mediated CatS and HDAC6 during injury-related vascular repair. These data suggest that CatS/HDAC6 could be a potential therapeutic target for the control of vascular diseases that are involved in neointimal lesion formation.

Indoxyl Sulfate Downregulates Mas Receptor via Aryl Hydrocarbon Receptor/Nuclear Factor-kappa B, and Induces Cell Proliferation and Tissue Factor Expression in Vascular Smooth Muscle Cells.

BACKGROUND/AIM: Angiotensin converting enzyme-related carboxypeptidase 2/angiotensin (Ang)-(1-7)/Mas receptor axis is protective in the development of chronic kidney disease and cardiovascular disease. This study is aimed at investigating whether indoxyl sulfate (IS) affects Mas receptor expression, cell proliferation and tissue factor expression in vascular smooth muscle cells, and if Ang-(1-7), an activator of Mas receptor, counteracts the IS-induced effects. METHODS: IS was administered to normotensive and hypertensive rats. Human aortic smooth muscle cells (HASMCs) were cultured with IS. RESULTS: IS reduced the expression of Mas receptor in the aorta of normotensive and hypertensive rats. IS downregulated the Mas receptor expression in a time- and dose-dependent manner in HASMCs. Knockdown of aryl hydrocarbon receptor (AhR) and nuclear factor-kappa B (NF-x03BA;B) inhibited IS-induced downregulation of Mas receptor. Further, IS stimulated cell proliferation and tissue factor expression in HASMCs. Ang-(1-7) attenuated IS-induced cell proliferation and tissue factor expression in HASMCs. Ang-(1-7) suppressed phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and NF-x03BA;B in HASMCs. CONCLUSION: IS downregulated the expression of Mas receptor via AhR/NF-x03BA;B, and induced cell proliferation and tissue factor expression in HASMCs. Ang-(1-7) inhibited IS-induced cell proliferation and tissue factor expression by suppressing the phosphorylation of ERK1/2 and NF-x03BA;B p65.

Indoxyl sulfate upregulates prorenin expression via nuclear factor-κB p65, signal transducer and activator of transcription 3, and reactive oxygen species in proximal tubular cells.

We have recently found that indoxyl sulfate induces prorenin expression in proximal tubular cells. The present study aimed to determine whether nuclear factor-κB (NF-κB) p65, signal transducer and activator of transcription 3 (Stat3), and reactive oxygen species are involved in indoxyl sulfate-induced prorenin expression in cultured human proximal tubular cells (HK-2 cells). Effects of indoxyl sulfate on prorenin expression were determined using HK-2 cells with small interfering RNAs (siRNAs) specific to NF-κB p65 and Stat3, N-acetylcysteine, an antioxidant, and diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase. Indoxyl sulfate increased prorenin expression in HK-2 cells. siRNAs specific to NF-κB p65 and Stat3 inhibited indoxyl sulfate-induced prorenin expression. Both N-acetylcysteine and diphenyleneiodonium suppressed indoxyl sulfate-induced prorenin expression. Indoxyl sulfate upregulates the expression of prorenin via NF-κB p65, Stat3, and reactive oxygen species in proximal tubular cells.

Indoxyl sulfate induces IL-6 expression in vascular endothelial and smooth muscle cells through OAT3-mediated uptake and activation of AhR/NF-κB pathway.

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.

Indoxyl sulfate-induced activation of (pro)renin receptor promotes cell proliferation and tissue factor expression in vascular smooth muscle cells.

UNLABELLED: 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. CONCLUSION: 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.

Indoxyl sulfate downregulates expression of Mas receptor via OAT3/AhR/Stat3 pathway in proximal tubular cells.

UNLABELLED: Renin-angiotensin system (RAS) plays a pivotal role in chronic kidney disease (CKD). Angiotensin converting enzyme-related carboxypeptidase 2 (ACE2)/angiotensin (Ang)-(1-7)/Mas receptor axis counteracts the deleterious actions of Ang II. ACE2 exerts its actions by cleaving Ang II into Ang-(1-7) which activates Mas receptor. This study aimed to determine if the expression of Mas receptor is altered in the kidneys of CKD rats, and if indoxyl sulfate (IS), a uremic toxin, affects the expression of Mas receptor in rat kidneys and cultured human proximal tubular cells (HK-2 cells). The expression of Mas receptor was examined in the kidneys of CKD and AST-120-treated CKD rats using immunohistochemistry. Further, the effects of IS on Mas receptor expression in the kidneys of normotensive and hypertensive rats were examined. The effects of IS on the expression of Mas receptor and phosphorylation of endothelial nitric oxide synthase (eNOS) in HK-2 cells were examined using immunoblotting. CKD rats showed reduced renal expression of Mas receptor, while AST-120 restored its expression. Administration of IS downregulated Mas receptor expression in the kidneys of normotensive and hypertensive rats. IS downregulated Mas receptor expression in HK-2 cells in a time- and dose-dependent manner. Knockdown of organic anion transporter 3 (OAT3), aryl hydrocarbon receptor (AhR), and signal transducer and activator of transcription 3 (Stat3) inhibited IS-induced downregulation of Mas receptor and phosphorylated eNOS. N-acetylcysteine, an antioxidant, also inhibited IS-induced downregulation of Mas receptor and phosphorylated eNOS. Ang-(1-7) attenuated IS-induced transforming growth factor-ß1 (TGF-ß1) expression. CONCLUSION: Mas receptor expression is reduced in the kidneys of CKD rats. IS downregulates renal expression of Mas receptor via OAT3/AhR/Stat3 pathway in proximal tubular cells. IS-induced downregulation of Mas receptor might be involved in upregulation of TGF-ß1 in proximal tubular cells.