Serum total p-cresol and indoxyl sulfate correlated with stage of chronic kidney disease in renal transplant recipients

Background

Uremic toxins are considered cardiovascular and mortality risk factors in chronic kidney disease (CKD) patients. Both p-cresol and indoxyl sulfate have been shown to induce oxidative stress in vitro and subsequent endothelial dysfunction in uremic patients. Our study evaluated the levels of p-cresol and indoxyl sulfate, and whether they contribute to the progression of CKD in transplant recipients.

Methods

We retrospectively evaluated 95 patients who had received a transplant from February 1987 to June 2010 in our center; the recipients had a mean transplant duration of 5.3 ± 4.9 years and a mean age of 47.8 ± 14.1 years. Among them, 56.8% (54/95) were male. Patients with glomerular filtration rate (GFR) ≥ 60 mL/min/1.73 m² were selected for group 1 (n = 35), and those with GFR < 60 mL/min/1.73 m² were selected for group 2 (n = 60). Demographic and clinical data were compared between groups. Serum and urine levels of p-cresol and indoxyl sulfate were also obtained.

Results

Baseline serum p-cresol and indoxyl sulfate levels were significantly higher in advanced CKD stages (P = .001 and <.0001, respectively). Patients at advanced CKD stages (group 2) had lower serum levels of hemoglobin and albumin (P < .0001), but higher levels of total cholesterol, triglyceride, and uric acid levels (P = .04, .04 and .001, respectively). Body mass index, C-reactive protein, and serum calcium and phosphate levels showed no significant differences between groups. The cut-off value for serum p-cresol between groups was 1.28 umol/L (P = .01), and that for the indoxyl sulfate level was 0.98 umol/L (P = .0001).

Conclusion

The serum p-cresol and indoxyl sulfate levels were significantly higher in advanced CKD stages in transplant recipients. To evaluate the use of serum p-cresol and indoxyl sulfate levels as a predictive tool for survival, larger clinical studies are needed.     

Suppressed serum and urine levels of indoxyl sulfate by oral sorbent in experimental uremic rats.

In uremic patients, the serum concentration of indoxyl sulfate is markedly increased. To determine if oral sorbent (AST-120) suppresses the endogenous synthesis of indoxyl sulfate, it was administered to experimental uremic rats, and the serum concentration and urinary excretion of indoxyl sulfate were quantified by high-performance liquid chromatography. Oral sorbent decreased both the serum concentration and urinary excretion of indoxyl sulfate, suggesting that there was suppression of the endogenous synthesis of indoxyl sulfate by the oral sorbent. Oral sorbent did not decrease the serum concentration and urinary excretion of hippuric acid, but it did alleviate the deterioration of renal function in the experimental uremic rats.     

硫酸吲哚酚对大鼠主动脉平滑肌细胞增生影响的机制研究

目的：探讨硫酸吲哚酚（indoxyl sulfate,IS）对大鼠血管平滑肌细胞（vascular smooth muscle cell,VSMC）增生的影响以及这种变化和氧化应激的关系。方法实验均分为8组：正常组、IS 100μM组、IS 300μM组、IS 500μM组、辛伐他汀10μM＋正常组、辛伐他汀10μM＋IS 100μM组、辛伐他汀10μM＋IS 300μM组、辛伐他汀10μM＋IS 500μM组。采用WST-1法检测VSMC增殖情况;硫代巴比妥酸法检测培养基中丙二醛（malondialdehyde,MDA）含量;ELISA方法测定晚期氧化蛋白产物（advanced oxidation protein products,AOPP）。结果与正常组比较,IS 300μM组和 IS 500μM 组上清液的 WST-1（OD 值）[（1.55±0.27）比（1.18±0.25）与（1.73±0.30）比（1.18±0.25）,P〈0.01]含量、MDA含量[（2.60±0.47）μg/ml比（1.59±0.21）μg/ml与（2.82±0.54）μg/ml比（1.59±0.21）μg/ml,P〈0.01]和 AOPP 含量[（67.94±8.58）μmol/L 比（54.97±8.46）μmol/L与（72.09±9.49）μmol/L比（54.97±8.46）μmol/L,P〈0.01]均明显增高。与同浓度 IS 组相比,辛伐他汀10μM＋IS 300μM 组和辛伐他汀10μM＋IS 500μM 组上清液 WST-1（OD 值）[（1.22±0.24）比（1.55±0.27）与（1.32±0.30）比（1.73±0.30）,P〈0.05]、MDA[（1.64±0.38）μg/mL 比（2.60±0.47）μg/ml 与（1.70±0.40）μg/ml 比（2.82±0.54）μg/ml,P〈0.01]含量和 AOPP 含量[（55.56±7.41）μmol/L比（67.94±8.58）μmol/L 与（55.54±6.80）μmol/L 比（72.09±9.49）μmol/L,P〈0.05]均明显降低。大鼠 VSMC 上清液的 WST-1含量与 MDA 含量呈正相关（r=0.621,P〈0.01）,大鼠VSMC上清液的WST-1含量与AOPP含量呈正相关（r=0.581,P〈0.01）。结论 IS可浓度依赖性地促进大鼠 VSMC增生,其作用可能与 IS增加 VSMC的氧化应激有关;辛伐他汀可抑制此作用。     

Klotho alleviates toxic effect of indoxyl sulfate on vascular endothelial cells and its mechanism

Objective To investigate the effect of klotho on the human vein umbilical endothelial cells (HUVECs) injury induced by indoxyl sulfate (IS) and to explore its mechanism and the role of endoplasmic reticulum stress (ERS) in this process. Methods (1) The cell vitalities of HUVECs incubated with different concentration of IS (5, 25, 50 mg/L) for 48 h and with 50 mg/L IS for different time points (12, 24, 48 h) were measured by CCK-8 assay. (2) HUVECs were incubated with 50 mg/L IS and different concentration of klotho (0, 1, 10, 100 μg/L) for 48 h and their cell viabilities were measured by CCK-8 assay. (3) HUVECs were divided into four groups: control group, IS group (50 mg/L IS), klotho group (50 mg/L IS+100 μg/L klotho) and Compound C group (50 mg/L IS+100 μg/L klotho+10 μmol/L Compound C). The cell vitality and the apoptosis of HUVECs were evaluated by CCK-8 assay and flow cytometry, respectively. The mRNA and protein expressions of GRP78 and CHOP were measured by real-time PCR and Western blotting. The phosphorylation level of AMPK was tested by Western blotting. Results IS inhibited cell vitality in the time-dependent and concentration-dependent manner. The cell viability of HUVECs with 50 mg/L IS was lower than normal control (P＜0.05). The inhibited cell vitality induced by IS was partly restored by klotho in concentration-dependent manner. The cell viability was higher in 100 μg/L klotho+50 mg/L IS group than 50 mg/L IS group (P＜0.05). Compared with control group, the expressions of GRP78 and CHOP and cell apoptosis increased, however, the level of phosphorylated AMPK (p-AMPK) decreased in IS group (all P＜0.05). Compared with IS group, the expressions of GRP78 and CHOP and cell apoptosis decreased and the level of p-AMPK increased in klotho group (all P＜0.05). Furthermore, the above effects of klotho could be partly blocked by Compound C. The above indexes showed statistical differences between Compound C group and klotho group. Conclusions IS can inhibit the HUVECs cell vitality, and induce ERS and cell apoptosis. Klotho protein could antagonize the above effects, probably through activating AMPK pathway and reducing ERS-mediated cell apoptosis.     

Effect of oral sorbent, AST-120, on serum concentration of indoxyl sulfate in uremic rats

Indoxyl sulfate is a metabolite of tryptophan. Indole is synthesized in intestine from tryptophan by intestinal bacteria. The absorbed indole is converted to indoxyl sulfate through indoxyl in liver. Serum concentration of indoxyl sulfate is markedly increased as an inhibitor of drug-binding in uremic patients as compared with healthy subjects. Since indoxyl sulfate is bound to serum albumin, it cannot be removed efficiently by hemodialysis, and it tends to accumulate in uremic serum. To determine if oral sorbent, AST-120, could adsorb indole in intestine and then decrease serum concentration of indoxyl sulfate, it was administered to nephrectomized uremic rats. Serum concentration of indoxyl sulfate was markedly decreased in uremic rats fed with oral sorbent as compared with control uremic rats. However, serum concentrations of creatinine and urea nitrogen were not significantly decreased in the uremic rats fed with oral sorbent as compared with the control uremic rats. Serum concentration of tryptophan was not decreased but rather increased in the uremic rats fed with oral sorbent as compared with the control uremic rats. Concentration of indoxyl sulfate in bile of a uremic rat was much lower than that in the uremic serum, suggesting that the adsorption of indoxyl sulfate in intestine is not a major mechanism of decreasing the serum concentration of indoxyl sulfate. These results demonstrate that oral sorbent, AST-120, can decrease serum concentration of indoxyl sulfate in uremia due to adsorption of indole in intestine.     

Inhibitory effect of oral sorbent on accumulation of albumin-bound indoxyl sulfate in serum of experimental uremic rats

Serum indoxyl sulfate, which is markedly accumulated in uremic patients, cannot be removed efficiently by hemodialysis due to its albumin binding. To determine if oral adsorbent (AST-120) can decrease its serum concentration in uremic state, oral adsorbent was administered to experimental nephrectomized uremic rats. Uremic rats fed with oral adsorbent showed a significantly lower serum concentration of indoxyl sulfate compared to control uremic rats, even when serum concentrations of urea nitrogen and creatinine were not significantly decreased in the uremic rats fed with oral adsorbent. Indoxyl sulfate was detected only at a lower concentration in bile as compared with the serum of uremic rats. These results suggest that oral adsorbent adsorbs indole, a precursor of indoxyl sulfate, in the intestine and prevents the accumulation of indoxyl sulfate in uremic rats.     

尿毒症毒素硫酸吲哚酚在体外对人肾小球足细胞骨架的损伤及其机理研究

目的硫酸吲哚酚(IS)是一种公认的结合蛋白质的肠源性尿毒症毒素,在慢性肾脏病患者体内蓄积可导致和促进慢性肾脏病的发生和发展。本实验探索了IS是否能引起人肾小球足细胞骨架的损伤及其可能的机理。 方法以体外培养的人肾小球足细胞系作为研究对象。采用台盼蓝拒染法、MTT法检测足细胞活力;采用免疫荧光法检测足细胞骨架纤维状肌动蛋白(F－actin)、骨架蛋白Synaptopodin改变;Western印迹法分析骨架蛋白Synaptopodin蛋白水平;RT－qPCR法检测Synaptopodin mRNA表达;琼脂糖凝胶电泳法分析足细胞内蛋白激酶A(PKA)活性。 结果IS使足细胞F－actin、Synaptopodin蛋白荧光减弱;IS还下调Synaptopodin的mRNA (P<0.01)。IS刺激使PKA磷酸化增多;PKA通路阻断剂H89可以上调Synaptopodin的蛋白及mRNA表达(P＝0.002)。 结论IS可下调足细胞骨架F－actin、Synaptopodin表达,PKA信号通路可能部分参与了Synaptopodin表达的调节,本研究结果为防治和干预CKD发生发展提供了新的思路和潜在的治疗靶点。     

BCG-induced urinary cytokines inhibit microvascular endothelial cell proliferation

PURPOSE: Angiogenesis is thought to depend on a net balance of molecules that inhibit or stimulate microvascular endothelial cells. A variety of molecules that affect angiogenesis are induced locally by the administration of intravesical bacille Calmette-Guerin (BCG) for superficial bladder cancer. We sought to determine whether BCG-induced urinary cytokines alter the effects of patient urine on assays of angiogenic activity. MATERIALS AND METHODS: Patients undergoing BCG treatment provided urine samples before and at peak cytokine production times after BCG instillation. Fifty-four urine samples from 8 patients were analyzed by ELISA for a panel of molecules known to affect angiogenesis, and tested for angiogenic activity in human dermal microvascular endothelial cell (HDMEC) proliferation and migration assays. To assess the role of specific BCG-induced cytokines, urinary HDMEC proliferation assays were repeated in the presence of neutralizing antibodies to tumor necrosis factor-alpha (TNF-alpha), interferon-inducible protein-10 (IP-10), and/or interferon-gamma (IFN-gamma). RESULTS: Urinary IFN-gamma, IP-10, TNF-alpha, and vascular endothelial growth factor (VEGF) were induced to nanogram/ml amounts by BCG treatment. While pre-BCG treatment urine samples minimally stimulated microvascular endothelial cell proliferation (+ 9%), post-BCG treatment urine became progressively inhibitory to endothelial cells (to -85%, p = 0.005) during weekly treatment courses. Neutralizing antibodies to TNF-alpha or to IP-10, either alone or in combination, greatly reduced this inhibitory effect. CONCLUSIONS: Intravesical BCG induces a cytokine-rich urinary microenvironment that is inhibitory to human endothelial cells. Urinary cytokine profiles and assays of angiogenic inhibition may provide prognostically important information regarding BCG treatment outcomes.     

Effects of p-cresol on the proliferation and cell cycle of human umbilical vein endothelial cells

Objective To investigate the effects of p-cresol on human umbilical vein endothelial cells. Methods The effects of p-cresol on endothelial cell growth, cell cycle, cell morphological change and p21 protein were detected by the CCK-8 assay, flow cytometry assay, inverted microscope and Western blotting. Results P-cresol could inhibit the growth of human umbilical vein endothelial cells in dose- and time-dependent manners (all P＜0.05). The human umbilical vein endothelial cells treated with p-cresol became elongated processes, cloudy cytoplasm, and irregular shapes. The p-cresol stopped human umbilical vein endothelial cells at cell cycle G1 and had no effect on cell apoptosis. The p-cresol could increase protein expression of p21 in a dose dependent manner (P＜0.05). Conclusion P-cresol can increase protein expression of p21, induce cell cycle arrest at G1 stage and inhibit the proliferation of human umbilical vein endothelial cells.     

The relationship between indoxyl sulfate and left ventricular hypertrophy in hemodialysis patients

Objective To investigate the relationship between indoxyl sulfate (IS) and left ventricular hypertrophy (LVH) in hemodialysis patients. Methods For the eligible patients (age ≥18 years, dialysis duration > 6 months, without history of congestive heart failure within 3 months and comorbidity of cardiac aneurysm), clinical data were collected, biochemical measurements were completed, and echocardiographic examinations were performed. Plasma IS concentration was determined by high performance liquid chromatography electrospray tandem spectrometry (HPLC-ESI-MS/MS). Linear and Logistic regression models were employed to assess the associations of plasma IS and left ventricular mass index (LVMI) and LVH, respectively. Results Two hundred and ten hemodialysis patients (117 males) with mean age of（57.2 ± 14.3）years were enrolled. The prevalence of LVH was up to 64.0%. Univariate linear regression showed that plasma IS was positively correlated with LVMI (β=7.09, P=0.02). The result persisted after adjustment for all kinds of risk factors (β=4.16, P=0.03). Patients were categorized into two groups: LVH and non-LVH group. Logistic regression models were employed to assess the relationship of plasma IS and LVH. The result showed that plasma IS was independently associated with LVH after adjustment for other confounding risk factors (β=6.54, OR=1.13, 95%CI 1.09-1.44, P=0.03). Conclusions LVH is prevalent in hemodialysis patients. Plasma IS is significantly correlated with LVMI and the independent risk factor for LVH.     

The impact of antihypertensive pharmacotherapy on interplay between protein-bound uremic toxin (indoxyl sulfate) and markers of inflammation in patients with chronic kidney disease

International Urology and Nephrology - Indoxyl sulfate (IS) is one of the most potent uremic toxins involved in chronic kidney disease (CKD) progression, induction of inflammation, oxidative...     

Removal of the uremic retention solute p-cresol using fractionated plasma separation and adsorption

Abstract:  Removal of protein-bound uremic retention solutes, including p -cresol, by peritoneal dialysis and hemodialysis (HD) is limited. p -Cresol, mainly circulating as sulfate conjugate ( p -cresyl sulfate [PCS]), is independently associated with mortality. Fractionated plasma separation and adsorption (FPSA) is a nonbiologic detoxification system for the treatment of liver failure. The FPSA clearance of uremic retention solutes is unknown. We studied PCS clearance by FPSA, using the Prometheus system. The neutral resin adsorbent and the anion exchange adsorbent bind PCS in vitro (reduction ratios [RRs] 37 and 70%). Ex vivo, the adsorbent mass removal (MR) (median 47.5 mg) contributes more than half to total MR (median 89.6 mg). In vivo, PCS RR during FPSA (50%) exceeded the RR during high flux HD (30%). We halted the study after four inclusions due to repeated thrombosis of the arterio-venous conduit. In conclusion, FPSA is a promising technique to improve clearance of protein-bound uremic retention solutes.     

Evolution of protein-bound uremic toxins indoxyl sulphate and p-cresyl sulphate in acute kidney injury

International Urology and Nephrology - There is a gradual increase in serum concentrations of protein-bound colon-derived uremic toxins indoxyl sulphate (IxS) and p-cresyl sulphate (pCS) as chronic...     

Association between blood indoxyl sulfate and carbonyl stress marker in hemodialysis patients

BACKGROUND: Indoxyl sulfate (IS) is a protein-bound solute, which is progressively retained in blood according to the decline of renal function. However, clinical relevance of excess IS in hemodialysis (HD) patients remains unknown. PATIENTS AND METHODS: We measured serum IS and clinical parameters including pentosidine, carboxymethyllysine (CML) and homocysteine levels in 55 HD patients (age: 67 +/- 2 years, time on HD: 67 +/- 11 months, male/female = 30/25), and examined the relationship between IS and these data. RESULTS: Serum IS was markedly increased in HD patients (80.49 +/- 6.17 microg/ml) compared to normal range (< 1.9 microg/ml). IS was significantly and positively correlated with time on HD (p < 0.01), blood urea nitrogen (p < 0.01), beta2-microglobulin (p < 0.03), and protein catabolic rate (PCR) (p < 0.01). The patients with increased IS needed a higher erythropoietin dosage. Blood IS was positively correlated with pentosidine (r = 0.505, p < 0.01) and CML (r = 0.275, p < 0.05). In contrast, blood IS was not associated with nutritional and inflammatory parameters. A stepwise multiple regression analysis revealed that time on HD, PCR and pentosidine were significant determinants of IS. CONCLUSIONS: Serum IS was related to time on HD and dietary protein intake. Accumulated IS may be associated with enhanced carbonyl stress in chronic HD patients.     

Platelet-rich fibrin matrix improves wound angiogenesis via inducing endothelial cell proliferation

The economic, social, and public health burden of chronic ulcers and other compromised wounds is enormous and rapidly increasing with the aging population. The growth factors derived from platelets play an important role in tissue remodeling including neovascularization. Platelet-rich plasma (PRP) has been utilized and studied for the last four decades. Platelet gel and fibrin sealant, derived from PRP mixed with thrombin and calcium chloride, have been exogenously applied to tissues to promote wound healing, bone growth, hemostasis, and tissue sealing. In this study, we first characterized recovery and viability of as well as growth factor release from platelets in a novel preparation of platelet gel and fibrin matrix, namely platelet-rich fibrin matrix (PRFM). Next, the effect of PRFM application in a delayed model of ischemic wound angiogenesis was investigated. The study, for the first time, shows the kinetics of the viability of platelet-embedded fibrin matrix. A slow and steady release of growth factors from PRFM was observed. The vascular endothelial growth factor released from PRFM was primarily responsible for endothelial mitogenic response via extracellular signal-regulated protein kinase activation pathway. Finally, this preparation of PRFM effectively induced endothelial cell proliferation and improved wound angiogenesis in chronic wounds, providing evidence of probable mechanisms of action of PRFM in healing of chronic ulcers.     

Zinc sulfate and wound healing.

The assumption that wounds heal at a constant rate over time, when measured by volume or area, was questioned. Data from two studies indicate that healing rate changes over time. When this bias was taken into consideration, the conflicting findings of studies of zinc sulfate and wound healing support the hypothesis that zinc therapy is effective for patients with low serum zinc. It was concluded that in order to study the effect of zinc therapy, lesion size and zinc intake should be controlled and serum zinc should be monitored.