Shunsuke Ito

Indoxyl Sulfate Induces Leukocyte-Endothelial Interactions through Up-regulation of E-selectin

Despite a positive correlation between chronic kidney disease and atherosclerosis, the causative role of uremic toxins in leukocyte-endothelial interactions has not been reported. We thus examined the effects of indoxyl sulfate, a uremic toxin, on leukocyte adhesion to activated endothelial cells and the underlying mechanisms. Pretreatment of human umbilical vein endothelial cells (HUVEC) with indoxyl sulfate significantly enhanced the adhesion of human monocytic cells (THP-1 cell line) to TNF-α-activated HUVEC under physiological flow conditions. Treatment with indoxyl sulfate enhanced the expression level of E-selectin, but not that of ICAM-1 or VCAM-1, in HUVEC. Indoxyl sulfate treatment enhanced the activation of JNK, p38 MAPK, and NF-κB in TNF-α-activated HUVEC. Inhibitors of JNK and NF-κB attenuated indoxyl sulfate-induced E-selectin expression in HUVEC and subsequent THP-1 adhesion. Furthermore, treatment with the NAD(P)H oxidase inhibitor apocynin and the glutathione donor N-acetylcysteine inhibited indoxyl sulfate-induced enhancement of THP-1 adhesion to HUVEC. Next, we examined the in vivo effect of indoxyl sulfate in nephrectomized chronic kidney disease model mice. Indoxyl sulfate-induced leukocyte adhesion to the femoral artery was significantly reduced by anti-E-selectin antibody treatment. These findings suggest that indoxyl sulfate enhances leukocyte-endothelial interactions through up-regulation of E-selectin, presumably via the JNK- and NF-κB-dependent pathway.

Protein-Bound Uremic Toxins: New Culprits of Cardiovascular Events in Chronic Kidney Disease Patients

Chronic kidney disease (CKD) has been considered a major risk factor for cardiovascular diseases. Although great advances have recently been made in the pathophysiology and treatment of cardiovascular diseases, CKD remains a major global health problem. Moreover, the occurrence rates of cardiovascular events among CKD patients increase even in cases in which patients undergo hemodialysis, and the mechanisms underlying the so-called “cardiorenal syndrome” are not clearly understood. Recently, small-molecule uremic toxins have been associated with cardiovascular mortality in CKD and/or dialysis patients. These toxins range from small uncharged solutes to large protein-bound structures. In this review, we focused on protein-bound uremic toxins, such as indoxyl sulfate and p-cresyl sulfate, which are poorly removed by current dialysis techniques. Several studies have demonstrated that protein-bound uremic toxins, especially indoxyl sulfate, induce vascular inflammation, endothelial dysfunction, and vascular calcification, which may explain the relatively poor prognosis of CKD and dialysis patients. The aim of this review is to provide novel insights into the effects of indoxyl sulfate and p-cresyl sulfate on the pathogenesis of atherosclerosis.

Reduction of indoxyl sulfate by AST-120 attenuates monocyte inflammation related to chronic kidney disease

Accelerated cardiovascular disease is a frequent complication of CKD. Monocyte‐mediated inflammation and adhesion of monocytes to vascular endothelium are key events in atherogenesis. An oral adsorbent, AST‐120, retards renal function deterioration by lowering IS, which is known to accumulate in CKD patients. However, the effect of AST‐120 on CKD‐related monocyte activation is unknown. We aimed to determine whether AST‐120 improves monocyte‐mediated inflammation through IS reduction. Flow cytometric analysis showed that Mac‐1 expression and ROS production were significantly higher in peripheral blood monocytes of subtotal Nx CKD mice than in sham‐operated mice. AST‐120 treatment significantly decreased Mac‐1 expression and ROS production in CKD model mice. Furthermore, administration of IS induced monocyte‐mediated inflammation and ROS generation. In vitro studies indicated that IS dose‐dependently increased THP‐1 monocytic cell adhesion to IL‐1β‐activated HUVECs under physiological flow conditions. IS also induced monocyte‐mediated inflammation and ROS production in THP‐1 cells. Phosphorylation of p38 MAPK and membrane translocation of NAD(P)H oxidase subunit p47phox in THP‐1 cells were induced by IS. Both SB203580 (p38 MAPK inhibitor) and apocynin [NAD(P)H oxidase inhibitor] reduced THP‐1 cell adhesion to HUVECs. Apocynin also inhibited IS‐induced ROS production in THP‐1 cells. IS induced monocyte‐driven inflammation through NAD(P)H oxidase‐ and p38 MAPK‐dependent pathways in monocytes. The main finding of this study was that AST‐120 inhibited monocyte activation by reducing IS in vivo. This provides new insights on how AST‐120 attenuates the progression of atherosclerosis in CKD.

Crucial Role of the Aryl Hydrocarbon Receptor (AhR) in Indoxyl Sulfate-Induced Vascular Inflammation

AIM The aryl hydrocarbon receptor (AhR), a ligand-inducible transcription factor mediating toxic effects of dioxins and uremic toxins, has recently emerged as a pathophysiological regulator of immune-inflammatory conditions. Indoxyl sulfate, a uremic toxin, is associated with cardiovascular disease in patients with chronic kidney disease and has been shown to be a ligand for AhR. The aim of this study was to investigate the potential role of AhR in indoxyl sulfate-induced leukocyte-endothelial interactions. METHODS Endothelial cell-specific AhR knockout (eAhR KO) mice were produced by crossing AhR floxed mice with Tie2 Cre mice. Indoxyl sulfate was administered for 2 weeks, followed by injection of TNF-α. Leukocyte recruitment to the femoral artery was assessed by intravital microscopy. Vascular endothelial cells were transfected with siRNA specific to AhR (siAhR) and treated with indoxyl sulfate, followed by stimulation with TNF-α. RESULTS Indoxyl sulfate dramatically enhanced TNF-α-induced leukocyte recruitment to the vascular wall in control animals but not in eAhR KO mice. In endothelial cells, siAhR significantly reduced indoxyl sulfate-enhanced leukocyte adhesion as well as E-selectin expression, whereas the activation of JNK and nuclear factor-κB was not affected. A luciferase assay revealed that the region between －153 and －146 bps in the E-selectin promoter was responsible for indoxyl sulfate activity via AhR. Mutational analysis of this region revealed that activator protein-1 (AP-1) is responsible for indoxyl sulfate-triggered E-selectin expression via AhR. CONCLUSION AhR mediates indoxyl sulfate-enhanced leukocyte-endothelial interactions through AP-1 transcriptional activity, which may constitute a new mechanism of vascular inflammation in patients with renal disease.

Critical role of the C5a-activated neutrophils in high-fat diet-induced vascular inflammation

Exceed and chronic high-fat diet (HFD) contributes to the diagnosis and development of atherosclerosis, obesity, and metabolic syndrome. However, the key molecular component(s) triggered by HFD responsible for initiating vascular inflammation remain unknown. We observed that feeding HFD for 4 weeks is sufficient to induce leukocyte recruitment in the femoral artery of wild-type mice. Neutrophil- and monocyte-depletion analyses confirmed the preferential recruitment of neutrophils in these mice. Protein analysis of sera from HFD-fed mice revealed a marked elevation of complement component C5a levels. Exogenous C5a alone induced leukocyte recruitment, which was abolished by a C5a-receptor antagonist. We also examined the role of neutrophil-derived MCP-1 in accumulation of leukocytes in the artery. These results demonstrated a previously unrecognized role for C5a and neutrophils in the early onset of HFD-induced vascular inflammation. Further study may help in elucidating a novel regulatory pathway to control diet-induced inflammation such as that in case of atherosclerosis.

Impact of Indoxyl Sulfate on Coronary Plaques in Patients on Hemodialysis

Serum indoxyl sulfate (IS; a uremic toxin) levels, which are significantly higher in patients with chronic kidney disease, including those undergoing hemodialysis, than in the robust, are associated with both cardiovascular disease (CVD) and CVD-related mortality. Furthermore, coronary artery calcium (CAC) is an independent predictor of cardiovascular events in patients undergoing hemodialysis. This study aimed to interpret the association between serum IS levels and coronary plaque burden (CPB) or CAC.A total of 30 consecutive patients on hemodialysis, who underwent 320-row coronary multidetector computed tomography (MDCT) angiography for suspected coronary artery disease, were enrolled in this prospective study. Coronary artery percent atheroma volume (a CPB marker) and percent calcium volume (a CAC marker) assessed using MDCT were evaluated. Furthermore, various oxidative and inflammatory markers typified by serum IS levels at a dialysis-free day were measured. Using these data, we investigated correlation between the inflammatory marker IS and CPB or CAC.Multivariable analysis indicated that serum IS levels were positively correlated with CAC [partial regression coefficient, 2.89; 95% confidence interval (CI), 0.35-5.43; P = 0.03] but not with CPB, even after adjustment for cofounders. Composite cardiovascular events, namely, as all-cause death, non-fatal myocardial infarction, disabling stroke, and hospital admission for other cardiovascular events, were reported to be 50% in all patients (95% CI, 32.1-67.9).In patients undergoing hemodialysis, serum IS levels were significantly associated with CAC but not with CPB.