Wenhu Liu

The Inhibitory Effect of Quercetin on Asymmetric Dimethylarginine-Induced Apoptosis Is Mediated by the Endoplasmic Reticulum Stress Pathway in Glomerular Endothelial Cells

Asymmetric dimethylarginine (ADMA) is considered an independent mortality and cardiovascular risk factor in chronic kidney disease (CKD) patients, and contributes to the development of renal fibrosis. Quercetin (QC), a natural component of foods, protects against renal injury. Here, we explored the possible mechanisms that are responsible for ADMA-induced renal fibrosis and the protective effect of QC. We found that ADMA treatment activated the endoplasmic reticulum (ER) stress sensor proteins phosphorylated protein kinase RNA-activated-like ER kinase (PERK) and inositol requiring-1α (IRE1), which correspondingly induced C/EBP homologous protein (CHOP) expression and phosphorylated c-Jun N-terminal kinase (JNK) phosphorylation in glomerular endothelial cells (GEnCs). Following this, ADMA promoted ER stress-induced apoptosis and resulted in transforming growth factor β (TGF-β) expression in GEnCs. SP600125, an inhibitor of JNK, and CHOP siRNA protected against ADMA-induced cell apoptosis and TGF-β expression. QC prevented ADMA-induced PERK and IRE1 apoptotic ER stress pathway activation. Also, ADMA-induced GEnCs apoptosis and TGF-β expression was reduced by QC. Overexpression of CHOP blocked QC-mediated protection from apoptosis in ER stressed cells. Overall, these observations indicate that ADMA may induce GEnCs apoptosis and TGF-β expression by targeting the PERK-CHOP and IRE1-JNK pathway. In addition, drugs such as QC targeting ER stress may hold great promise for the development of novel therapies against ADMA-induced renal fibrosis.

Apelin-13 passes through the ADMA-damaged endothelial barrier and acts on vascular smooth muscle cells

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, is associated with vascular dysfunction. The polypeptide apelin mediates two major actions on blood vessels. However, their combined effects on vascular function are not fully understood. The present study aimed to determine the effect of apelin-13 on myosin light chain (MLC) phosphorylation in vascular smooth muscle cells (VSMCs) under ADMA-induced endothelial leakage conditions. To assess the increased permeability induced by ADMA, human umbilical vein endothelium cells (HUVECs) were plated in transwell dishes. The FITC-dextran flux and FITC-apelin-13 flux through the endothelial monolayer were measured. To examine the effect of leakage of apelin-13 on MLC phosphorylation in HUVSMCs, transwell dishes were used to establish a coculture system with HUVECs in upper chambers and HUVSMCs in lower chambers. Western blot was performed to assess the phospho-MLC levels. ADMA increased endothelial permeability in a concentration- and time-dependent manner, accompanied by actin stress fiber assembly and intercellular gap formation. When HUVECs were treated with ADMA, the permeability to both macromolecular dextran and micromolecular apelin-13 increased significantly. Both p38 MAPK inhibitor and NADPH oxidase inhibitor could prevent HUVECs from the increased permeability, and the changes of cytoskeleton and intercellular junction, which were induced by ADMA. Apelin-13 passed through the ADMA-stimulated endothelial monolayer and increased the expression of phospho-MLC in VSMCs. These results suggest that ADMA increases endothelial permeability, which may involve the p38 MAPK and NADPH oxidase pathway. Apelin-13 can pass through the damaged endothelial barrier, and acts directly on VSMCs to increase MLC phosphorylation.

The regulatory peptide apelin: a novel inhibitor of renal interstitial fibrosis

Epithelial–mesenchymal transition (EMT) of tubular epithelial cells is a key event in renal interstitial fibrosis and the progression of chronic kidney disease (CKD). Apelin is a regulatory peptide involved in the regulation of normal renal hemodynamics and tubular functions, but its role in renal fibrosis remains unknown. In this study, we examined the inhibitory effects of apelin on transforming growth factor-β1 (TGF-β1)-induced EMT in HK-2 cells, and evaluated its therapeutic efficacy in mice with complete unilateral ureteral obstruction (UUO). In vitro, apelin inhibited TGF-β1-mediated upregulation of α-smooth muscle actin (α-SMA) and downregulation of E-cadherin. Increased levels of phosphorylated Smad-2/3 and decreased levels of Smad7 in TGF-β1-stimulated cells were reversed by apelin co-treatment. In the UUO model, administration of apelin significantly attenuated renal interstitial fibrosis, as evidenced by the maintenance of E-cadherin and laminin expression, and markedly suppressed expression of α-SMA, TGF-β1 and its type I receptor, as well as interstitial matrix components. Interestingly, in UUO mice, there was a reduction in the plasma level of apelin, which was compensated by upregulation of APJ expression in the injured kidney. Exogenous supplementation of apelin normalized the level of plasmatic apelin and renal APJ. In conclusion, our study provides the first evidence that apelin is able to ameliorate renal interstitial fibrosis by suppression of tubular EMT through a Smad-dependent mechanism. The apelinergic system itself may promote some compensatory response in the renal fibrotic process. These results suggest that apelin has potential renoprotective effects and may be an effective agent for retarding CKD progression.

Apelin-13 deteriorates hypertension in rats after damage of the vascular endothelium by ADMA.

Asymmetric dimethylarginine (ADMA) is a risk factor for endothelial dysfunction. The polypeptide apelin has biphasic effects on blood vessels in vivo and in vitro. We investigated the effect of apelin-13 on ADMA-damaged vessels. Rats were divided among ADMA-treated and control groups, which were treated with ADMA (10 mg·(kg body mass)(-1)·day(-1)) or saline, respectively, for 4 weeks. Systolic blood pressure (SBP) was measured before and after the injection of apelin-13. The ultrastructure of endothelial cells in caudal arteries was examined using transmission electron microscopy. The reactivities of isolated caudal artery rings were observed after exposure to apelin-13, and myosin light chain (MLC) phosphorylation was assessed by immunohistochemistry in rings treated with or without apelin-13. ADMA induced hypertension and endothelial dysfunction. After injection of apelin-13, SBP declined in the control group but was elevated in the ADMA-treated group. In vitro, apelin-13 caused relaxation in rings in the control group, but it contracted rings in the ADMA-treated group. Apelin-13 promoted MLC phosphorylation in vascular smooth muscle cells (VSMCs) in the ADMA group. These results indicate that apelin-13 might pass through ADMA-damaged endothelium and act on VSMCs to increase MLC phosphorylation, thus contributing to vasoconstriction and exacerbating hypertension.

Actin cytoskeleton modulates ADMA-induced NF-kappaB nuclear translocation and ICAM-1 expression in endothelial cells

Summary Background Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, increases the activity of NF-κB (NF-κB) and then induces the expression of intercellular adhesion molecule-1 (ICAM-1). However, the mechanisms regulating ADMA-induced NF-κB activation are unknown. This study investigated the function of actin cytoskeleton for ADMA-induced NF-κB activation and ICAM-1 expression in endothelial cells. Material/Methods Human umbilical vein endothelial cells (HUVEC) were cultured and left untreated or challenged for 24h with 100 μM ADMA in the absence and presence of 5 μM cytochalasin D (Cyt D), or 1 μM Jasplakinolide (Jas). The form of actin cytoskeleton, the translocation of NF-κB, NF-κB DNA binding activity, and the expression of ICAM-1 were determined. Results ADMA increased the formation of stress fiber in endothelial cells, and Cyt D clearly induced destabilization of the actin filaments. Either stabilizing or destabilizing the actin cytoskeleton prevented ADMA-induced NF-κB activation. It also showed that the inhibition of NF-κB activity was due to the impaired NF-κB nuclear translocation. Further, stabilizing or destabilizing the actin cytoskeleton inhibited the expression of the NF-κB target protein, ICAM-1. Conclusions Actin cytoskeleton may be engaged in modulated ADMA-induced NF-κB activation and thereby ICAM-1 expression in endothelial cells.

The differences of asymmetric dimethylarginine removal by different dialysis treatments

Background: Asymmetric dimethylarginine (ADMA) as a uremia toxin is accumulated in end-stage renal disease (ESRD) patients. Elevated ADMA level has been shown to be predictive of cardiovascular diseases (CVDs) and all-cause mortality in ESRD. Therefore, we investigated the removal of ADMA by different dialysis treatments. Methods: There were 30 each of hemodialysis (HD), hemodiafiltration (HDF), peritoneal dialysis (PD) patients, and healthy volunteers enrolled. The ADMA concentrations in serum, urine, and spent dialysate samples were determined. The urine and spent dialysate volumes were recorded. The ADMA removals by urine and spent dialysate in 1 week were calculated and compared among four groups. It was also analyzed for the correlations between the total removal of ADMA in 1 week and the parameters of age, durance of dialysis, glomerular filtration rate, urine volume, urinal ADMA level, spent dialysate volume, and spent dialysate ADMA level. Results: The serum levels of ADMA in dialysis patients were much higher than in healthy subjects (0.32 ± 0.09 μmol/L), and their 1-week total removals of ADMA were much lower than healthy controls (249.21 ± 57.04 μmol/week) (p-values all were less than 0.01). Among dialysis groups, serum ADMA levels decreased significantly in PD patients compared with HD or HDF patients (1.38 ± 0.30 μmol/L vs. 1.82 ± 0.38 μmol/L and 1.63 ± 0.32 μmol/L, p < 0.01), and the total removal of ADMA diminished remarkably by turns of PD, HDF, and HD groups (47.79 ± 8.20 μmol/week, 31.79 ± 8.92 μmol/week, 14.63 ± 6.53 μmol/week, respectively, p < 0.01). The total removal of ADMA in 1 week was related directly with the spent dialysate concentrations of ADMA, the spent dialysate volume, and the urine volume. Conclusions: ADMA was mainly removed by dialysate in dialysis patients. Different dialysis models have different clearance capability on plasma ADMA. PD might be more effective on ADMA removal than HD and HDF, with HDF being more effective than HD.

Actin cytoskeleton-dependent pathways for ADMA-induced NF-κB activation and TGF-β high expression in human renal glomerular endothelial cells.

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, is considered to be an independent risk factor in the progression of chronic kidney diseases (CKD). It can induce kidney fibrosis by increasing transforming growth factor (TGF)-β1 expression, but its molecular mechanism is unclear. The aim of the present study was to investigate the role of actin cytoskeleton in ADMA-induced TGF-β1 high expression in human renal glomerular endothelial cells (HRGECs). The structure of stress fibers was visualized by immunofluorescence, nuclear factor-κB (NF-κB) DNA-binding activity was assessed by an electrophoretic mobility shift assay and TGF-β1 expression was assessed by western blot analysis. Results showed that ADMA induced the assembly of stress fibers, DNA binding of NF-κB, and increasing expression of TGF-β1. When the dynamics of actin cytoskeleton was perturbed by the actin-depolymerizing agent cytochalasin D and the actin-stabilizing agent jasplakinolide, or ablation of stress fiber bundles by the nicotineamide adenine dinucleotide phosphate oxidase inhibitor apocynin and p38 mitogen-activated protein kinase inhibitor SB203580, ADMA-induced DNA binding of NF-κB and TGF-β1 expression were inhibited. These results revealed an actin cytoskeleton-dependent mechanism in ADMA-induced NF-κB activation and TGF-β1 high expression in HRGECs. The specific targeting of the actin cytoskeleton may be a useful strategy to prevent ADMA-activated kidney fibrosis in CKD.

Preservation of Residual Renal Function with Limited Water Removal in Hemodialysis Patients

Residual renal function (RRF) is of paramount importance for hemodialysis (HD) adequacy, morbidity, and mortality. Some studies have shown that overhydration is beneficial for preservation of RRF, but it can also increase the probability of adverse events such as hypertension and heart failure in HD patients. To determine the optimal amount of dehydration, we performed HD with limited water removal in HD patients. Eighteen HD patients included in this self-controlled study underwent HD with limited water removal. Water removal volume was determined by a previous volume as follows. Total water removal volume was divided into levels: ≤3.0, 3.0–9.0, and >9.0 L per week. Water removal was performed to obtain dry weight in the last dialysis, and was performed three times with a ratio of 1:1:2 and 2:2:3, respectively. Urine volume, endogenous creatinine clearance rate, Kt/V, hemoglobin, and serum albumin were recorded before and after the study at 3, 6, 9, and 12 months. The follow-up period was 12 months. Ten patients withdrew from the study because of adverse events including hypertension (n = 3), heart failure (n = 3), angina (n = 1), polycystic kidney rupture (n = 1), obvious edema (n = 1), and one patient had too much interdialytic weight gain to continue. As a result, we stopped this study after 1 month. Our data suggest that the preservation of RRF with limited water removal in HD patients must be interpreted with caution.

Differences in Bio-incompatibility among Four Biocompatible Dialyzer Membranes Using in Maintenance Hemodialysis Patients

Abstract Background: Following the introduction of modified cellulosic and then synthetic membrane dialyzers, it was realized that the dialyzer bio-incompatibility depends on the membrane composition. We designed a prospective, randomized, cohort study of 6 months to determine several parameters of biocompatibility in maintenance hemodialysis (MHD) patients treated with four different membrane dialyzers. Methods: There were 60 MHD patients enrolled in the study. In baseline, synthetic low-flux dialyzer, polysulfone (PS) membrane was used in all patients for at least 3 months. Then the patients were randomly divided into three groups according to different dialyzer membranes. Synthetic high-flux dialyzer group, ployethersulfone membrane, cellulose triacetate (CTA) high-flux membrane, and synthetic low-flux dialyzer, polymethylmethacrylate (PMMA) membrane were used in 6 months. A new dialyzer was used for each study treatment, and there was no dialyzer reuse. The biocompatibility markers and solutes removal markers were detected repeatedly at different time points. Results: The blood levels of highly sensitive C reactive protein, interleukin (IL)-1β, and interleukin (IL)-13 showed no difference among different groups at al time points. However, the blood complement levels and white blood cell counts were significantly different among three groups. When the dialyzers changed from PS to PMMA membrane, C3a levels and white blood cell counts changed significantly (p < 0.05). Moreover, the changes of C5a levels were significantly different between group CTA and group PMMA in month 3 (p < 0.05). Conclusion: There were much more differences on bio-incompatibility among different dialyzer membranes.

Indoxyl sulfate promotes vascular smooth muscle cell calcification via the JNK/Pit-1 pathway

Abstract We determined the effect of indoxyl sulfate (IS) on Pit-1 expression and the role of Pit-1 in IS-induced osteoblastic differentiation and calcification of vascular smooth muscle cells (VSMCs). To assess osteoblastic differentiation and Pit-1 expression, VSMCs were incubated with various concentrations of IS for different durations. Phosphonoformic acid (PFA), a competitive inhibitor of Pit-1, was used to verify the role of Pit-1. Western blot analysis and quantitative real-time polymerase chain reaction (PCR) were performed to assess Pit-1 protein and mRNA levels, respectively. To evaluate calcification, calcium content was measured. After IS treatment, we observed osteoblastic differentiation and calcification of VSMCs and up-regulation of Pit-1 expression. Moreover, the effect of IS on osteoblastic differentiation and Pit-1 expression was partly dose- and time-dependent. PFA abrogated the IS-induced osteoblastic differentiation and calcification of VSMCs to a certain extent. The c-Jun N-terminal kinase (JNK) pathway was activated after treatment with IS, whereas inhibition of the JNK pathway partially attenuated the effect of IS on both the stimulation of Pit-1 expression and calcium deposition. Our study is the first to demonstrate that IS promotes Pit-1 expression in part by activation of the JNK pathway that is involved in the mechanism of IS-induced osteoblastic differentiation and matrix mineralization.