Plasma Levels Of Indoxyl Sulfate Research Articles

#4592 A HIGH PROTEIN DIET INDUCES DEPRESSION-LIKE BEHAVIOR IN CKD RODENT MODELS

Abstract Background and Aims The prevalence of depression among patients with Chronic Kidney Disease (CKD) is higher than the general population. Around 21-27% of CKD patients is estimated to be affected by depression. Several studies hypothesized that mood disorders may be due to reduced family support, older age, low educational status and low quality of life. However, adults with CKD are also known to exhibit altered tryptophan metabolism causing reduced plasma levels of tryptophan and elevated kynurenine and kynurenic acid. Moreover, CKD patients show increased levels of indoxyl sulfate and indole-3-acetic acid, two protein-bound uremic toxins deriving from the gut microbiome metabolism of tryptophan. We postulate that depression is not just due to reduced quality of life but also to a biological reason. In addition, we hypothesize that a high protein diet may influence tryptophan metabolism and therefore affect depression symptoms. Method Eighteen Sprague-Dawley male rats (Janvier, Le Genest- St Isle, France), 7-8 weeks old, were induced with CKD utilizing 5/6 nephrectomy and were randomly assigned to a low protein (n = 10) or a high protein (n = 8) diet. A sham-operated control group for each diet was used (n = 7 respectively). The splash test was adopted to assess depressive symptoms in rats. In short, the splash test is a behavioral test consisting in sprinkling a 10% sucrose solution on the dorsal coat of a rat. Latency (time before the initiation of grooming) and duration of grooming were recorded for 5 min and used as an indication of self-care behavior. Delayed and reduced grooming is considered as a depression-like behavior of rats. Blood and urine creatinine were determined with standard laboratory techniques. Total plasmatic concentrations of tryptophan, kynurenine, kynurenic acid and indoxyl sulfate were measured using LC-MS/MS. Results The latency before the starting of the grooming behavior was significantly higher (p = .0499) in CKD rats on a high protein diet compared to sham rats on the same diet while no difference was seen between the low protein groups. CKD rats reported significantly higher plasma levels of indoxyl sulfate and kynurenic acid and reduced tryptophan. Interestingly, kynurenic acid plasma levels were significantly reduced when administering a low protein diet to CKD rats (p = .0025). Among CKD rats, the latency to the grooming behavior was correlated with plasmatic levels of creatinine clearance (Spearman r = -.57, p value = .019), indoxyl sulfate (Spearman r = .64, p value = .007), and kynurenic acid (Spearman r = .63, p value = .009). The total grooming time was shorter in both CKD groups compared to the sham rats, but not significantly (p = .4881 and p = .4312, respectively). Conclusion These results suggest that biological factors contribute to depression onset and that the decline of kidney function and the accumulation of uremic toxins are potential risk factors for depression. Dietary interventions such as a low protein diet, are potential strategies to lower the plasmatic levels of uremic toxins which may alleviate depressive-like symptoms.

Efficacy and safety of switching from febuxostat to dotinurad, a novel selective urate reabsorption inhibitor, in hyperuricemic patients with type 2 diabetic kidney disease: Protocol for a single-arm, open-label, prospective, exploratory study.

Dotinurad is a novel uricosuric drug in Japan with selective and potent urate transporter 1 (URAT1) inhibitory activity. This study aims to evaluate the efficacy and safety of dotinurad in hyperuricemic patients with type 2 diabetic kidney disease by comparing serum levels of urate and plasma and urinary levels of indoxyl sulfate excreted via the urate excretion transporter ATP binding cassette subfamily G member 2 (ABCG2), as indices, with baseline levels after switching from febuxostat to dotinurad. This single-center, single-arm, open-label, prospective, exploratory study aims to evaluate the effect of switching from febuxostat to dotinurad on serum urate levels and its background factors. The study will include 50 hyperuricemic patients with type 2 diabetic kidney disease and urate levels exceeding 6 mg/dL despite administration of febuxostat 20 mg/day for at least 3 months. The primary outcome is the achievement rate of serum urate levels of ≤6 mg/dL after 24 weeks of treatment with dotinurad at 0.5 mg to a maximum of 4 mg once daily. Secondary outcomes include the changes in serum urate levels, plasma and urinary indoxyl sulfate levels, and renal injury-related markers from baseline to observation points at weeks 4, 12, and 24. The study hypothesizes that switching to dotinurad may reduce the plasma levels of indoxyl sulfate and increase its urinary levels in patients with hyperuricemia. These suggest that dotinurad can potently lower the serum urate level by inhibiting URAT1 without adversely affecting ABCG2. Thus, findings of this study are expected to provide useful insights into the treatment of hyperuricemia associated with type 2 diabetic kidney disease and the discovery of new possibilities for dotinurad. Prior to the study, its study protocol was scientifically and ethically reviewed and approved by the Japan Physicians Association Clinical Research Review Board (approval number: JPA007-2204-02). In addition, patients who provide written informed consent will participate in the study. The results of this study will be published through submission to a peer-reviewed scientific journal. https://jrct.niph.go.jp/en-latest-detail/jRCTs031220080, identifier jRCTs031220080.

The Medium Cut-Off Membrane Does Not Lower Protein-Bound Uremic Toxins.

The accumulation of protein-bound uremic toxins (PBUT) is associated with increased cardiovascular outcomes in patients on dialysis. However, the efficacy of PBUT removal for a medium-cutoff (MCO) membrane has not been clarified. This study was designed to assess the efficacy of PBUT clearance according to dialysis modalities. In this prospective and cross-over study, we enrolled 22 patients who received maintenance hemodiafiltration (HDF) thrice weekly from three dialysis centers. The dialysis removal of uremic toxins, including urea, beta 2-microglobulin (B2MG), lambda free light chain (λ-FLC), indoxyl sulfate (IS), and p-cresyl sulfate (pCS), was measured in the 22 patients on high-flux HD (HF-HD), post-dilution online HDF (post-OL-HDF), and MCO-HD over 3 weeks. The average convection volume in post-OL-HDF was 21.4 ± 1.8 L per session. The reduction rate (RR) of B2MG was higher in post-OL-HDF than in MCO-HD and HF-HD. The RR of λ-FLC was the highest in MCO-HD, followed by post-OL-HDF and HF-HD. The dialysate albumin was highest in MCO-HD, followed by post-OL-HDF and HF-HD. Post-dialysis plasma levels of IS and pCS were not statistically different across dialysis modalities. The total solute removal and dialytic clearance of IS and pCS were not significantly different. The clearance of IS and pCS did not differ between the HF-HD, post-OL-HDF, and MCO-HD groups.

Can Resveratrol Supplementation Reduce Uremic Toxin Plasma Levels From the Gut Microbiota in Nondialyzed Patients With Chronic Kidney Disease?

Uremic toxins such as indoxyl sulfate (IS), p-cresyl sulfate (pCS), and indole-3-acetic acid (IAA) produced by the gut microbiota are recognized as risk factors for many comorbidities, including cardiovascular diseases. Patients with chronic kidney disease (CKD) have an accumulation of these toxins, and nutritional strategies have been proposed to mitigate gut dysbiosis and, consequently, reduce these toxins. This study aimed to evaluate the effects of resveratrol supplementation on the plasma levels of IS, pCS, and IAA in nondialyzed patients with CKD. In this placebo-controlled crossover study, twenty nondialyzed patients were randomly divided into two groups: they received either one capsule/day containing 500mg of trans-resveratrol (63±7.5years, glomerular filtration rate [GFR]: 34±14mL/min, body mass index: 26.8±5.6kg/m2) or a placebo containing 500mg wheat flour (62±8.4years, GFR: 34±13mL/min, body mass index: 28.6±4.4kg/m2) during 4weeks. After 8weeks of washout (no supplementation), another 4weeks of supplementation with crossover was initiated. IS, IAA, and pCS plasma levels were quantified by the reverse phase high-efficiency liquid chromatography method with fluorescent detection. The mRNA expression of nuclear factor erythroid 2-related factor 2 and nuclear factor kappa B in peripheral blood mononuclear cells was evaluated by polymerase chain reaction. C-reactive protein plasma levels were also evaluated. As expected, the uremic toxin levels were negatively correlated with the GFR, but no effect of trans-resveratrol supplementation was found on levels of IS, IAA, and pCS. There was a positive correlation between IS and nuclear factor erythroid 2-related factor 2 (r=0.24, P=.03) and also between IS and C-reactive protein (r=0.21, P=.05). Supplementation with trans-resveratrol did not reduce the plasma levels of IS, pCS, and IAA in nondialyzed patients with CKD. The interactions among uremic toxins and anti-inflammatory and proinflammatory pathways deserve more studies.

Effect of cranberry supplementation on toxins produced by the gut microbiota in chronic kidney disease patients: A pilot randomized placebo-controlled trial.

Patients with Chronic Kidney Disease (CKD) have an imbalance in the gut microbiota that can lead to increase levels of lipopolysaccharides (LPS) and uremic toxins such as indoxyl sulfate (IS), p-cresyl sulfate (p-CS), and indole-3 acetic acid (IAA). Among the therapeutic options for modulating gut microbiota are the bioactive compounds such as polyphenols present in cranberry, fruit with potential antioxidant and anti-inflammatory effects. This clinical trial focuses on evaluating the effects of supplementation with a dry extract of cranberry on plasma levels of LPS and uremic toxins in non-dialysis CKD patients. It was a randomized, double-blind, placebo-controlled study. Patients were randomized into two groups: the cranberry group received 500mg of dry cranberry extract (2 times daily), and the placebo group received 500mg of corn starch (2 times daily) for two months. LPS plasma levels were evaluated by enzyme-linked immunosorbent assay (ELISA) and uremic toxins (IS, p-CS, and IAA) by high-performance liquid chromatography-fluorescence detection. Anthropometric measurements and food intake using the 24-h food recall technique were also evaluated before and after the intervention. Twenty-five participants completed two months of supplementation: 12 patients in the cranberry group (8 women, 56.7±7.5 years, estimated glomerular filtration rate (eGFR) of 39.2±21.9mL/min); 13 patients in the placebo group (9 women, 58.8±5.1 years, eGFR of 39.7±12.9mL/min). As expected, there was a negative association between glomerular filtration rate and p-CS and IS plasma levels at the baseline. No change was observed in the uremic toxins and LPS levels. Cranberry dry extract supplementation for two months did not reduce the LPS and uremic toxins plasma levels produced by the gut microbiota in non-dialysis CKD patients.

Indoxyl sulfate in uremia: an old idea with updated concepts.

Patients with end-stage kidney disease (ESKD) have increased vascular disease. While protein-bound molecules that escape hemodialysis may contribute to uremic toxicity, specific contributing toxins remain ambiguous. In this issue of the JCI, Arinze et al. explore the role of tryptophan metabolites in chronic kidney disease–associated (CKD-associated) peripheral arterial disease. The authors used mouse and zebrafish models to show that circulating indoxyl sulfate (IS) blocked endothelial Wnt signaling, which impaired angiogenesis. Plasma levels of IS and other tryptophan metabolites correlated with adverse peripheral vascular disease events in humans. These findings suggest that lowering IS may benefit individuals with CKD and ESKD.

Protein-Bound Uremic Toxins Induce Reactive Oxygen Species-Dependent and Inflammasome-Mediated IL-1β Production in Kidney Proximal Tubule Cells.

Protein bound-uremic toxins (PBUTs) are not efficiently removed by hemodialysis in chronic kidney disease (CKD) patients and their accumulation leads to various co-morbidities via cellular dysfunction, inflammation and oxidative stress. Moreover, it has been shown that increased intrarenal expression of the NLRP3 receptor and IL-1β are associated with reduced kidney function, suggesting a critical role for the NLRP3 inflammasome in CKD progression. Here, we evaluated the effect of PBUTs on inflammasome-mediated IL-1β production in vitro and in vivo. Exposure of human conditionally immortalized proximal tubule epithelial cells to indoxyl sulfate (IS) and a mixture of anionic PBUTs (UT mix) increased expression levels of NLRP3, caspase-1 and IL-1β, accompanied by a significant increase in IL-1β secretion and caspase-1 activity. Furthermore, IS and UT mix induced the production of intracellular reactive oxygen species, and caspase-1 activity and IL-1β secretion were reduced in the presence of antioxidant N-acetylcysteine. IS and UT mix also induced NF-κB activation as evidenced by p65 nuclear translocation and IL-1β production, which was counteracted by an IKK inhibitor. In vivo, using subtotal nephrectomy CKD rats, a significant increase in total plasma levels of IS and the PBUTs, kynurenic acid and hippuric acid, was found, as well as enhanced urinary malondialdehyde levels. CKD kidney tissue showed an increasing trend in expression of NLRP3 inflammasome components, and a decreasing trend in superoxide dismutase-1 levels. In conclusion, we showed that PBUTs induce inflammasome-mediated IL-1β production in proximal tubule cells via oxidative stress and NF-κB signaling, suggesting their involvement in disease-associated inflammatory processes.

Plasma indoxyl sulfate levels predict cardiovascular events in patients with mild chronic heart failure

Indoxyl sulfate (IS) is associated with either chronic kidney disease or renal failure, which may predict cardiovascular events via cardiorenal syndrome. The present study aimed to elucidate whether the plasma levels of IS can predict the occurrence of cardiovascular events in patients with chronic heart failure (CHF) and investigate which causes of CHF leading to cardiovascular events are highly influenced by plasma IS levels. We measured the plasma IS levels in 165 patients with CHF [valvular disease: 78, dilated cardiomyopathy: 29, hypertrophic cardiomyopathy (HCM): 25 and others: 33] admitted to our hospital in 2012, and we followed up these patients for more than 5 years (the median follow-up period: 5.3 years). We measured the plasma IS level in 165 patients with CHF, and Kaplan–Meier analyses showed that high plasma IS levels (≥ 0.79 µg/mL, the median value) could predict the occurrence of cardiovascular events, i.e., cardiovascular death or rehospitalization due to the worsening of CHF. The sub-analyses showed that the high IS level could predict cardiovascular events in patients with CHF due to HCM and that the plasma IS levels were closely associated with left ventricular (LV) dimension, LV systolic dysfunction, and plasma B-type natriuretic peptide levels, rather than LV diastolic dysfunction. Plasma IS level predicts cardiovascular events in patients with CHF, especially those with HCM along with cardiac dysfunction. Besides, IS may become a proper biomarker to predict cardiovascular events in patients with CHF.

Plasma metabolites associated with arterial stiffness in patients with type 2 diabetes

BackgroundAlthough an increased arterial stiffness has been associated with traditional coronary risk factors, the risk factors and pathology of arterial stiffness remain unclear. In this study, we aimed to identify the plasma metabolites associated with arterial stiffness in patients with type 2 diabetes mellitus.MethodsWe used the metabolomic data of 209 patients with type 2 diabetes as the first dataset for screening. To form the second dataset for validation, we enlisted an additional 31 individuals with type 2 diabetes. The non-targeted metabolome analysis of fasting plasma samples using gas chromatography coupled with mass spectrometry and the measurement of brachial-ankle pulse wave velocity (baPWV) were performed.ResultsA total of 65 annotated metabolites were detected. In the screening dataset, there were statistically significant associations between the baPWV and plasma levels of indoxyl sulfate (r = 0.226, p = 0.001), mannitol (r = 0.178, p = 0.010), mesoerythritol (r = 0.234, p = 0.001), and pyroglutamic acid (r = 0.182, p = 0.008). Multivariate regression analyses revealed that the plasma levels of mesoerythritol were significantly (β = 0.163, p = 0.025) and that of indoxyl sulfate were marginally (β = 0.124, p = 0.076) associated with baPWV, even after adjusting for traditional coronary risk factors. In the independent validation dataset, there was a statistically significant association between the baPWV and plasma levels of indoxyl sulfate (r = 0.430, p = 0.016). However, significant associations between the baPWV and plasma levels of the other three metabolites were not confirmed.Conclusions/interpretationThe plasma levels of indoxyl sulfate were associated with arterial stiffness in Japanese patients with type 2 diabetes. Although the plasma levels of mannitol, mesoerythritol, and pyroglutamic acid were also associated with arterial stiffness, further investigation is needed to verify the results.

Efficacy of Divinylbenzenic Resin in Removing Indoxyl Sulfate and P-Cresol Sulfate in Hemodialysis Patients: Results From an In Vitro Study and An In Vivo Pilot Trial (xuanro4-Nature 3.2).

High serum levels of microbiota-derived uremic toxins, indoxyl sulfate (IS) and p-cresyl sulfate (PCS), are associated with chronic kidney disease (CKD) progression and cardiovascular complications. IS and PCS cannot be efficiently removed by conventional hemodialysis (HD), due to their high binding affinity for albumin. This study evaluates the efficacy of a divinylbenzene-polyvinylpyrrolidone (DVB-PVP) cartridge and a synbiotic to reduce uremic toxins in HD patients. First, the in vitro efficacy of DVB-PVP in adsorbing IS and PCS was evaluated. Second, a randomized, placebo-controlled pilot study in HD patients was carried out to establish whether the administration of a synbiotic, either individually and in association with DVB-PVP-HD, could reduce the production of uremic toxins. In vitro data showed that DVB-PVP resin removed a mean of 56% PCS and around 54% IS, after 6 h of perfusion. While, in the in vivo study, the DVB-PVP cartridge showed its adsorbing efficacy only for IS plasma levels. The combination of synbiotic treatment with DVB-PVP HD decreased IS and PCS both at pre- and post-dialysis levels. In conclusion, this study provides the first line of evidence on the synergistic action of gut microbiota modulation and an innovative absorption-based approach in HD patients, aimed at reducing plasma levels of IS and PCS.

Indoxyl Sulfate-Review of Toxicity and Therapeutic Strategies.

Indoxyl sulfate is an extensively studied uremic solute. It is a small molecule that is more than 90% bound to plasma proteins. Indoxyl sulfate is derived from the breakdown of tryptophan by colon microbes. The kidneys achieve high clearances of indoxyl sulfate by tubular secretion, a function not replicated by hemodialysis. Clearance by hemodialysis is limited by protein binding since only the free, unbound solute can diffuse across the membrane. Since the dialytic clearance is much lower than the kidney clearance, indoxyl sulfate accumulates to relatively high plasma levels in hemodialysis patients. Indoxyl sulfate has been most frequently implicated as a contributor to renal disease progression and vascular disease. Studies have suggested that indoxyl sulfate also has adverse effects on bones and the central nervous system. The majority of studies have assessed toxicity in cultured cells and animal models. The toxicity in humans has not yet been proven, as most data have been from association studies. Such toxicity data, albeit inconclusive, have prompted efforts to lower the plasma levels of indoxyl sulfate through dialytic and non-dialytic means. The largest randomized trial showed no benefit in renal disease progression with AST-120. No trials have yet tested cardiovascular or mortality benefit. Without such trials, the toxicity of indoxyl sulfate cannot be firmly established.

Correlations between Plasma Levels of Anionic Uremic Toxins and Clinical Parameters in Hemodialysis Patients.

When the kidney is seriously impaired, various uremic toxins (UTs) accumulate in the body, often exerting unfavorable effects on physiological functions and drug pharmacokinetics. To prevent this, it is important to determine plasma UT levels accurately in chronic kidney disease patients. Although attempts to predict plasma UT levels using biomarkers have been made, the correlation between UT levels and the markers is not yet fully understood. In this study, we assessed the correlations among plasma levels of indoxyl sulfate (IS), indoleacetic acid (IA), and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) in 20 hemodialysis patients and evaluated the relationship between the plasma levels of UTs and clinical parameters, such as serum creatinine (Scr), blood urea nitrogen, and estimated glomerular filtration rate (eGFR), with special focus on IS. There were no correlations among the plasma levels of the three UTs before and immediately after hemodialysis. However, a significant correlation was observed between plasma IS levels and Scr before hemodialysis (r=0.643, p=0.002), with the correlation becoming much stronger when using the data obtained immediately after hemodialysis (r=0.744, p<0.001). Further, plasma IS levels showed a significant negative correlation with eGFR (r=-0.558, p=0.011). However, no correlations were observed for IA or CMPF. The results obtained from this study suggest that plasma IS levels can be predicted from Scr values, although the precise mechanism behind the correlation remains to be clarified.

Indoxyl sulfate suppresses endothelial progenitor cell–mediated neovascularization

Patients with chronic kidney disease have an increased prevalence of peripheral arterial disease. Endothelial progenitor cells (EPC) are pivotal in neovascularization, but their role in mediating peripheral arterial disease in chronic kidney disease is not fully known. Here we studied the impact of indoxyl sulfate, a protein-bound uremic toxin, on EPC function in response to tissue ischemia or cell hypoxia in mice that underwent subtotal nephrectomy or sham operation. At 16 weeks, unilateral hindlimb ischemia was induced in all. Four weeks later, subtotal nephrectomy mice had significantly increased plasma levels of indoxyl sulfate, reduced reperfusion, decreased EPC mobilization, and impaired neovascularization in ischemic hindlimbs compared with control mice. Treatment with AST-120, an oral adsorbent of uremic toxins, reversed these changes. Ischemia-induced protein expression including phospho-eNOS, phospho-STAT3, interleukin-10, and VEGF were significantly decreased in ischemic hindlimbs of subtotal nephrectomy mice versus control mice; all effects were reversed by AST-120. Subtotal nephrectomy mice fed a diet with indole for 12 weeks resulted in impaired neovascularization in ischemic hindlimbs; also reversed by AST-120. In cultured human EPCs, VEGF expression was increased in hypoxia through HIF-1α and interleukin-10/STAT3 signaling; effects suppressed by pretreatment with indoxyl sulfate. Moreover, indoxyl sulfate markedly attenuated hypoxia-induced EPC migration and tube formation. Thus, indoxyl sulfate may be a therapeutic target for EPC-rescue of impaired neovascularization in patients with chronic kidney disease and peripheral arterial disease.

Is there a relationship between tryptophan dietary intake and plasma levels of indoxyl sulfate in chronic kidney disease patients on hemodialysis?

Gut microbiota is involved in generation of uremic toxins in chronic kidney disease (CKD) patients on hemodialysis (HD), like indoxyl sulfate (IS) that is originated from tryptophan amino acid fermentation. To evaluate the tryptophan intake by chronic renal failure patients on HD and its possible relationship with IS plasma levels. Participated of the study 46 patients with CKD on HD regular program (56.5% men; 52.7 ± 10.3 years; 63 (32.2-118.2) months on HD; BMI 25.6 ± 4.9 kg/m2). The tryptophan intake was evaluated by a 24-hours dietary recall (R-24h) performed on 3 different days. Routine biochemical tests and anthropometric measurements were evaluated. IS plasma levels were determined by High Performance Liquid Chromatography (HPLC) with fluorescent detection and the interleukin-6 (IL-6) plasma levels by immunoenzymatic method (ELISA, Enzyme Linked Immunosorbent Assay). The average of tryptophan intake was according to recommendation, but IS plasma levels (35.0 ± 11.9 mg/L) were elevated, however according to the EUTox values for uremic individuals. There was no correlation between the tryptophan intake and IS plasma levels. However, there was positive correlation between protein intake and tryptophan and variables used to evaluate lean body mass, and moreover, IS levels were positively associated with IL-6 (r = 0.6: p = 0.01). The present study suggests that tryptophan dietary intake may not be a determinant factor to IS levels. However, it suggests that gut microbiota may play an important role in systemic inflammation in patients with CKD.

Abstract 12381: Ast-120, an Adsorbent of Uremic Toxins Normalized the Cardiac Hypertrophic/pro- Apoptotic And Cardioprotective/anti-apoptotic Signaling in Pacing-induced Heart Failure in Dogs

Background: Renal dysfunction increases the plasma levels of indoxyl sulfate (IS), a uremic toxin excreted from the kidney. Interestingly, IS is reported to directly stimulate both collagen synthesis and cardiomyocyte hypertrophy in vitro via the modulation of ERK and p38-MAPK. However, it is unclear whether the pathophysiology of heart failure (HF) changes such intracellular signaling via the IS-dependent pathways. AST-120 is known to inhibit the uptake of indole and decrease the plasma IS levels. Hypothesis: We tested the hypothesis that the removal of plasma IS by using AST-120 modulates the intracellular signaling for hypertrophy, collagen synthesis and apoptosis of the myocardium in the canine HF model in vivo. Method: A canine HF model was produced by continuous rapid ventricular pacing at 230 bpm for 6 weeks. We treated the canine with oral AST-120 (1g/kg/day) administration between 4th and 6th week following the onset of rapid pacing (HF with AST-120 group, HF without AST-120 group, n=7 in each). We also set a control (Control group, n=7) without pacing for 6 weeks. We extracted the protein from the hearts and analyzed it by Western blotting. Result: AST-120 treatment reduced both plasma IS elevated in the canine HF model. HF without AST-120 group showed the activation of ERK signaling and the deactivation of both Akt and p38-MAPK signaling compared with Control group. The treatment with AST-120 increased the phosphorylation levels of Akt and p38-MAPK, and suppressed the ERK phosphorylation. HF without AST-120 group also showed an increased expression of pro-apoptotic Bax protein, and a decreased expression of anti-apoptotic Bcl-2 protein. The increased Bcl-2/Bax ratio was significantly ameliorated by AST-120 treatment. Conclusion: The decreases in plasma levels of indoxyl sulfate by the treatment with AST-120 1) deactivates the cardiac hypertrophic signaling, 2) activates the cardiac survival and anti-apoptotic signals and 3) suppresses the pro-apoptotic signals in failing heart in vivo. Indoxyl sulfate may become a sensitive and novel biomarker/mediator of heart failure explaining the cardio-renal syndrome that links renal dysfunction to heart failure.

Effect of increasing dietary fiber on plasma levels of colon-derived solutes in hemodialysis patients.

Numerous uremic solutes are derived from the action of colon microbes. Two such solutes, indoxyl sulfate and p-cresol sulfate, have been associated with adverse outcomes in renal failure. This study tested whether increasing dietary fiber in the form of resistant starch would lower the plasma levels of these solutes in patients on hemodialysis. Fifty-six patients on maintenance hemodialysis were randomly assigned to receive supplements containing resistant starch (n=28) or control starch (n=28) daily for 6 weeks in a study conducted between October 2010 and May 2013. Of these, 40 patients (20 in each group) completed the study and were included in the final analysis. Plasma indoxyl sulfate and p-cresol sulfate levels were measured at baseline and week 6. Increasing dietary fiber for 6 weeks significantly reduced the unbound, free plasma level of indoxyl sulfate (median -29% [25th percentile, 75th percentile, -56, -12] for fiber versus -0.4% [-20, 34] for control, P=0.02). The reduction in free plasma levels of indoxyl sulfate was accompanied by a reduction in free plasma levels of p-cresol sulfate (r=0.81, P<0.001). However, the reduction of p-cresol sulfate levels was of lesser magnitude and did not achieve significance (median -28% [-46, 5] for fiber versus 4% [-28, 36] for control, P=0.05). Increasing dietary fiber in hemodialysis patients may reduce the plasma levels of the colon-derived solutes indoxyl sulfate and possibly p-cresol sulfate without the need to intensify dialysis treatments. Further studies are required to determine whether such reduction provides clinical benefits.

Indoxyl Sulfate–Induced Endothelial Dysfunction in Patients with Chronic Kidney Disease via an Induction of Oxidative Stress

Recent data suggest indoxyl sulfate (IS), one of the uremic toxins that accelerate the progression of chronic kidney disease (CKD), may also be responsible for vascular disease via an induction of oxidative stress. The role of IS in endothelial dysfunction in CKD and potential mechanisms of IS-induced endothelial dysfunction were investigated. A prospective observational study in 40 CKD patients was performed. Flow-mediated endothelium-dependent vasodilatation (FMD) and its reaction time before and 24 weeks after an oral adsorbent of IS were evaluated. Plasma levels of IS and markers of oxidative stress were also measured. The proliferation, senescence, and production of nitric oxide and reactive oxygen species from human umbilical vein endothelial cells (HUVEC) were evaluated and the effect of antioxidants, N-acetylcysteine, rotenone, and apocynin was examined to explore the mechanism of IS-induced endothelial dysfunction. AST-120 treatment for 24 weeks resulted in a significant increase in FMD with a decrease in IS and oxidized/reduced glutathione ratio. The presence of diabetes and high-sensitivity C-reactive protein were the independent predictors for an improved FMD. IS induced a production of reactive oxygen species in HUVEC, and pretreatment with antioxidants ameliorated IS-induced inhibition of proliferation and nitric oxide production and inhibited a senescence of HUVEC. IS may play an important role in endothelial dysfunction via generation of oxidative stress with an induction of endothelial senescence. AST-120 improved endothelial dysfunction in patients with CKD associated with a decrease in IS and a restoration of antioxidant reserve.

Protein-bound uraemic toxin removal in haemodialysis and post-dilution haemodiafiltration

The accumulation of larger and protein-bound toxins is involved in the uraemic syndrome but their elimination by dialysis therapy remains difficult. In the present study, the impact of the albumin permeability of recently introduced advanced high-flux dialysis membranes on the removal of such substances was tested in haemodialysis and online post-dilution haemodiafiltration. Two types of polyethersulfone membranes only differing in albumin permeability (referred as PU- and PU+) were compared in eight patients on maintenance dialysis in a prospective cross-over manner. Treatment settings were identical for individual patients: time 229 +/- 22 min; blood flow rate 378 +/- 33 mL/min; dialysate flow rate 500 mL/min; substitution flow rate in haemodiafiltration 94 +/- 9 mL/min. Removal of the protein-bound compounds p-cresyl sulfate (pCS) and indoxyl sulfate (IS) was determined by reduction ratios (RRs), dialytic clearances and mass in continuously collected dialysate. In addition, the elimination of the low-molecular weight (LMW) proteins beta(2)-microglobulin, cystatin c, myoglobin (myo), free retinol-binding protein (rbp) and albumin was measured. Plasma levels of the protein-bound toxins were significantly decreased by all treatment forms. However, the decreases were comparable between dialysis membranes and between haemodialysis and haemodiafiltration. The RRs of total pCS ranged between 40.4 +/- 25.3 and 47.8 +/- 10.3% and of total IS between 50.4 +/- 2.6 and 54.6 +/- 8.7%. Elimination of free protein-bound toxins as assessed by their mass in dialysate closely correlated positively with the pre-treatment plasma concentrations being r = 0.920 (P < 0.001) for total pCS and r = 0.906 (P < 0.001) for total IS, respectively. Compared to haemodialysis, much higher removal of all LMW proteins was found in haemodiafiltration. Dialysis membrane differences were only obvious in haemodialysis for the larger LMW proteins myo and rbp yielding significantly higher RRs for PU+ (myo 46 +/- 9 versus 37 +/- 9%; rbp 18 +/- 5 versus 15 +/- 5%; P < 0.05). Additionally, the albumin loss varied between membranes and treatment modes being undetectable with PU- in haemodialysis and highest with PU+ in haemodiafiltration (1430 +/- 566 mg). The elimination of protein-bound compounds into dialysate is predicted by the level of pre-treatment plasma concentrations and depends particularly on diffusion. Lacking enhanced removal in online post-dilution haemodiafiltration emphasizes the minor significance of convection for the clearance of these solutes. Compared to LMW proteins, the highly protein-bound toxins pCS and IS are less effectively eliminated with all treatment forms. For a sustained decrease of pCS and IS plasma levels, alternative strategies promise to be more efficient therapy forms.

Carbonic-adsorbent AST-120 reduces overload of indoxyl sulfate and the plasma level of TGF-β1 in patients with chronic renal failure

We previously reported a significant increase in plasma TGF-beta1 in patients with chronic renal failure (CRF). Progression of CRF may be caused by persistent renal production of TGF-beta1. In CRF rat models, an oral carbonic absorbent (AST-120) reduces the expression of the TGF-beta1 gene in the kidney, and delays the progression of CRF, in part by alleviating the overload of indoxyl sulfate. The aim of this study was to evaluate the effect of AST-120 on plasma levels of indoxyl sulfate and TGF-beta1 in CRF patients. Ten CRF patients (aged 59.3 +/- 9.5 years, 5 men, serum creatinine 4.37 +/- 1.72 mg/dl) were enrolled in this study. All patients maintained a regular dietary therapy and the same medication throughout the study. AST-120 was added at a dose of 6 g/day. Parameters including the slope of the reciprocal of the serum creatinine-time plot, plasma indoxyl sulfate level, and plasma and urinary levels of TGF-beta1 were compared before and after the treatment with AST-120. The mean observation periods before and after the treatment were 9.7 +/- 2.8 and 6.5 +/- 2.9 months, respectively. Administration of AST-120 significantly reduced the plasma levels of indoxyl sulfate (1.42 +/- 1.50 vs. 1.26 +/- 1.40 mg/dl, P < 0.05) and TGF-beta1 (17.9 +/- 7.2 vs. 10.6 +/- 4.7 ng/ml, P < 0.05) and improved the slope of the reciprocal of serum creatinine (-0.061 +/- 0.041 vs. -0.032 +/- 0.055 dl/mg/year, P < 0.05). These results support the notion that indoxyl sulfate and TGF-beta1 may be involved in the progression of CRF, and that the oral adsorbent AST-120 may suppress the progression, at least in part, by reducing overproduction of TGF-beta1.