Free Indoxyl Sulfate Research Articles

#2377 Association of uremic toxins with morbidity and mortality in patients on hemodialysis

Abstract Background and Aims Morbidity and mortality are increased in patients on hemodialysis (HD), and the possible impact of protein bound uremic toxins (PBUT) is of great interest. We assessed the association between PBUT serum levels and clinical events including cardiovascular (CVE), severe infections (hospitalization due to infection, respiratory infection) and all-cause mortality. Method In this prospective observational study, PBUT plasma levels [hippuric acid (HA), indoxyl sulfate (IxS), p-cresyl sulfate (pCS), p-cresyl glycuronide (pCG), indole-3-acetic acid (IAA), 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF)] were quantified by ultra-performance liquid chromatography in 54 patients on HD (Age 51.3±16.91 years) and the association with future clinical events (CVE, hospitalization due to severe infection, respiratory infection and all-cause mortality) during 24 months follow up (FU), was assessed. Results In patients who died during FU (n=6), increased levels of total HA 6.42 (4.54) vs 2.75 (3.03)mg/dl (p=0.007), free HA 4.79 (3.93) vs 1.363 (1.85)mg/dl (p=0.004); free IxS 0.29 (0.29) vs 0.13 (0.17)mg/dl (p=0.045); total pCG 0.36 (0.5) vs 0.19 (0.28)mg/dl (p=0.045), free pCG 0.33 (0.43) vs 0.18 (0.26)mg/dl (p=0.042) were found. Moreover, in patients who experienced a CVE (n=11), a higher concentration of CMPF [0.39 (0.67) vs 0.15 (0.19)mg/dl (p=0.015)] was observed. No significant correlations were found between pCS and IAA levels with morbidity and mortality in this study. A logistic regression model, describing the effect of each PBUT on death probability could explain 44% (Nagelkerke R2) of the variance in death and correctly classified 90.7% of cases. Patients with increased levels of free IxS, total and free HA were more likely to die within a 2-year FU period (p=0.047, p=0.013, p=0.003, respectively). PBUT levels were not different among HD patients with and without severe infections. Conclusion Increased levels of PBUT (HA and free IxS) in patients on HD, associate with increased risk of death and CMPF with cardiovascular events.

Predictive value of protein-bound uremic toxins for heart failure in patients with chronic kidney disease.

This retrospective cohort study aimed to be the first to evaluate the association between plasma protein-bound uremic toxins (PBUTs) concentrations, echocardiographic parameters of heart failure (HF), and incident HF events in patients with chronic kidney disease (CKD) not on dialysis. Retrospective, single-centre, cohort study at the Ghent University Hospital, Belgium. Adults with CKD stages G1-G5, not on dialysis, could be included. Exclusion criteria were ongoing pregnancy, age <18years, active acute infection, active malignancy, history of transplantation, or a cardiovascular event within 3months prior to inclusion. Free and total concentrations of five PBUTs were quantified at baseline: indoxyl sulfate (IxS), p-cresyl sulfate (pCS), p-cresyl glucuronide (pCG), indole-3 acetic acid (IAA), and hippuric acid (HA). Patients were grouped into three echocardiographic categories: normal left ventricular ejection fraction (LVEF) and normal left ventricular end-diastolic pressure (LVEDP), normal LVEF and increased LVEDP, and reduced LVEF, based on available echocardiographic data in a time interval of ±6months around the plasma sample collection. A total of 523 patients were included between January 2011 and January 2014. Echocardiographic data within the predefined timeframe were available for 210 patients (40% of patients). Levels of pCG and pCS were significantly higher in patients with reduced (<50%) versus normal LVEF (P<0.05). After a median follow-up 5.5years, 43 (8.4%) patients reached the composite endpoint of hospitalization or mortality due to HF. Free fractions of IxS, pCS, and pCG showed the strongest association with clinical outcome: free IxS: HR 1.71 (95% CI 1.11-2.63; P=0.015), free pCS: HR 1.82 (95% CI 1.11-3.01; P=0.019), and free pCG: HR 1.67 (95% CI 1.08-2.58; P=0.020), and these results were independent of age, gender, body mass index, diabetes, and systolic blood pressure. In models that were also adjusted for serum creatinine, the free fractions of these PBUTs remained significant. Elevated free concentrations of IxS, pCG, and pCS were independently associated with an increased risk of HF events in non-dialysed CKD patients. Further research is necessary to confirm these findings and investigate the potential impact of PBUT-lowering interventions on HF events in this patient group.

#3386 PROTON-PUMP INHIBITORS AND SERUM CONCENTRATIONS OF UREMIC TOXINS IN PATIENTS WITH CHRONIC KIDNEY DISEASE

Abstract Background and Aims Use of proton-pump inhibitors (PPIs) is common in patients with chronic kidney disease (CKD). PPIs and many uremic toxins (UTs) are eliminated by a kidney tubular organic anion transporter system. In a cross-sectional study, we sought to evaluate the association between PPI prescription and serum concentrations of various UTs. Method We studied a randomly selected subgroup of participants in the CKD-REIN prospective cohort (adult patients with a confirmed diagnosis of CKD and estimated glomerular filtration rate (eGFR) &amp;lt;60ml/min/1.73m²) with available frozen samples collected at baseline. PPI prescription was recorded at baseline. Serum concentrations of 10 UTs were measured using a validated liquid chromatography tandem mass spectrometry technique. Multiple linear regression was performed, with the log UT concentration as the dependent variable. Results Of the 680 included patients [median age: 68 years; median eGFR: 32 ml/min/1.73 m2], 31% had PPI prescriptions at baseline. Patients using PPIs had higher levels of certain UTs in comparison to other patients, including total and free indoxyl sulfate (IS), total and free p-cresylsulfate, total and free p-cresylglucuronide (PCG), phenylacetylglutamine (PAG), free kynurenine, and free hippuric acid. After adjustment for baseline comorbidities, number of co-prescribed drugs and laboratory data including eGFR, associations between PPI prescription and elevated serum concentrations of free and total IS, free and total PCG, and PAG remained significant. Conclusion Our results indicate that PPI prescription is independently associated with serum UT retention. These findings indicate a potential mechanism for side effect of PPIs in CKD patients that will need to be confirmed by longitudinal studies.

Proton-Pump Inhibitors and Serum Concentrations of Uremic Toxins in Patients with Chronic Kidney Disease.

Use of proton-pump inhibitors (PPIs) is common in patients with chronic kidney disease (CKD). PPIs and many uremic toxins (UTs) are eliminated by the kidney's tubular organic anion transporter system. In a cross-sectional study, we sought to evaluate the association between PPI prescription and serum concentrations of various UTs. We studied a randomly selected sub-group of participants in the CKD-REIN cohort (adult patients with a confirmed diagnosis of CKD and estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2) with available frozen samples collected at baseline. PPI prescription was recorded at baseline. Serum concentrations of 10 UTs were measured using a validated liquid chromatography tandem mass spectrometry technique. Multiple linear regression was performed, with the log UT concentration as the dependent variable. Of the 680 included patients (median age: 68 years; median eGFR: 32 mL/min/1.73 m2), 31% had PPI prescriptions at baseline. Patients using PPIs had higher levels of certain UTs in comparison to other patients, including total and free indoxyl sulfate (IS), total and free p-cresylsulfate, total and free p-cresylglucuronide (PCG), phenylacetylglutamine (PAG), free kynurenine, and free hippuric acid. After adjustment for baseline co-morbidities, number of co-prescribed drugs, and laboratory data, including eGFR, associations between PPI prescription and elevated serum concentrations of free and total IS, free and total PCG, and PAG remained significant. Our results indicate that PPI prescription is independently associated with serum UT retention. These findings are interesting to better understand the factors that may modulate serum UT concentration in CKD patients, however, they will need to be confirmed by longitudinal studies.

Coordination of thin-film nanofibrous composite dialysis membrane and reduced graphene oxide aerogel adsorbents for elimination of indoxyl sulfate

• A novel combination of PVA/PAN TNFC dialysis membrane with rGO aerogel was proposed. • The rGO aerogel exhibited good adsorption performance for indoxyl sulfate. • The coordination of TFNC membrane and rGO lightened conventional hemodialysis. The protein-bound uremic toxins, represented by indoxyl sulfate (IS), have been associated with the progression of chronic kidney disease and the development of cardiovascular disease in the presence of impaired renal function. Herein, we proposed a novel strategy of thin-film nanofibrous composite (TNFC) dialysis membrane combined with reduced graphene oxide (rGO) aerogel adsorbents for clinical removal of IS as well as high retention of proteins. The TFNC membrane was prepared by electrospinning in conjunction with coating-reaction method and proved to have good selectivity and permeability. To further improve the removal rate of toxins, we used a medium hydrothermal method following by freeze-drying treatment to obtain the rGO aerogel adsorbents. It exhibited excellent adsorption for IS with a maximum adsorption capacity of 69.40 mg∙g −1 through π−π interaction and hydrogen bonding interaction based on Langmuir isotherm models. Time-dependent absorption experiments showed that it reached adsorption equilibrium within 4 h, which was matched with the hemodialysis time. The coordination was significantly exhibited by introducing rGO aerogel blocks into the dialysate for absorbing the diffused free IS during hemodialysis. Taking the advantages of the TFNC dialysis membrane and the rGO aerogel, the volume of dialysate for hemodialysis was only one-tenth of that without adsorbent blocks but with very comparable dialysis performance (the clearance of IS at 51.8% and the retention of HSA over 98%), which could lighten conventional hemodialysis effectively and be benefit to realize the miniaturization of the hemodialysis equipment. Therefore, the coordination of the TFNC dialysis membrane and rGO aerogel adsorbents would open a new path for the development of portable artificial kidney.

Serum concentrations of free indoxyl and p-cresyl sulfate are associated with mineral metabolism variables and cardiovascular risk in hemodialysis patients.

Indoxyl sulfate (IS) and p-cresyl sulfate (PCS) are uremic toxins associated with cardiovascular outcome in CKD patients. The present work is an analysis of the association of serum free, total IS and PCS with cardiovascular events and calcium-phosphate metabolism variables in hemodialysis patients. Serum levels of total and free IS and PCS were measured in 139 hemodialysis patients. Their relationship with calcium-phosphate metabolism variables were tested in an observational cohort study. In addition, their association with cardiovascular events was investigated during a 4-year follow-up. Patients in the highest tertile (T3) of serum free IS showed lower serum 1,25(OH)2D compared to patients in the middle (T2) and lowest tertile (T1); in addition to this, T3 patients showed lower serum irisin than T1 patients and lower serum PTH than all the other subjects (T1 + T2) combined. Serum PTH was also measured during the two years after the baseline measurement and was higher in patients in the T1 than in those in the T3 of serum free IS. Cox regression analysis showed that cardiovascular risk was lower in T1 patients than in those in the T3 of serum free PCS, both using a univariate (OR 2.55, 95% CI 1.2-5.43; p = 0.015) or multivariate model (OR 2.48, 95% CI 1.12-5.51; p = 0.003). Serum free IS may be associated with PTH and 1,25(OH)2D secretion, whereas free PCS may predict cardiovascular risk in hemodialysis patients.

The Effect of ß-Glucan Prebiotic on Kidney Function, Uremic Toxins and Gut Microbiome in Stage 3 to 5 Chronic Kidney Disease (CKD) Predialysis Participants: A Randomized Controlled Trial.

There is growing evidence that gut dysbiosis contributes to the progression of chronic kidney disease (CKD) owing to several mechanisms, including microbiota-derived uremic toxins, diet and immune-mediated factors. The aim of this study was to investigate the effect of a ß-glucan prebiotic on kidney function, uremic toxins and the gut microbiome in stage 3 to 5 CKD participants. Fifty-nine participants were randomized to either the ß-glucan prebiotic intervention group (n = 30) or the control group (n = 29). The primary outcomes were to assess kidney function (urea, creatinine and glomerular filtration rate), plasma levels of total and free levels of uremic toxins (p-cresyl sulfate (pCS), indoxyl-sulfate (IxS), p-cresyl glucuronide (pCG) and indoxyl 3-acetic acid (IAA) and gut microbiota using 16S rRNA sequencing at baseline, week 8 and week 14. The intervention group (age 40.6 ± 11.4 y) and the control group (age 41.3 ± 12.0 y) did not differ in age or any other socio-demographic variables at baseline. There were no significant changes in kidney function over 14 weeks. There was a significant reduction in uremic toxin levels at different time points, in free IxS at 8 weeks (p = 0.003) and 14 weeks (p < 0.001), free pCS (p = 0.006) at 14 weeks and total and free pCG (p < 0.001, p < 0.001, respectively) and at 14 weeks. There were no differences in relative abundances of genera between groups. Enterotyping revealed that the population consisted of only two of the four enterotypes: Bacteroides 2 and Prevotella. The redundancy analysis showed a few factors significantly affected the gut microbiome: these included triglyceride levels (p < 0.001), body mass index (p = 0.002), high- density lipoprotein (p < 0.001) and the prebiotic intervention (p = 0.002). The ß-glucan prebiotic significantly altered uremic toxin levels of intestinal origin and favorably affected the gut microbiome.

Syndecan-1 and Free Indoxyl Sulfate Levels Are Associated with miR-126 in Chronic Kidney Disease.

Chronic kidney disease (CKD) is a major cause of death worldwide and is associated with a high risk for cardiovascular and all-cause mortality. In CKD, endothelial dysfunction occurs and uremic toxins accumulate in the blood. miR-126 is a regulator of endothelial dysfunction and its blood level is decreased in CKD patients. In order to obtain a better understanding of the physiopathology of the disease, we correlated the levels of miR-126 with several markers of endothelial dysfunction, as well as the representative uremic toxins, in a large cohort of CKD patients at all stages of the disease. Using a univariate analysis, we found a correlation between eGFR and most markers of endothelial dysfunction markers evaluated in this study. An association of miR-126 with all the evaluated uremic toxins was also found, while uremic toxins were not associated with the internal control, specifically cel-miR-39. The correlation between the expression of endothelial dysfunction biomarker Syndecan-1, free indoxyl sulfate, and total p-cresyl glucuronide on one side, and miR-126 on the other side was confirmed using multivariate analysis. As CKD is associated with reduced endothelial glycocalyx (eGC), our results justify further evaluation of the role of correlated parameters in the pathophysiology of CKD.

A low aromatic amino-acid diet improves renal function and prevent kidney fibrosis in mice with chronic kidney disease

Despite decades of use of low protein diets (LPD) in the management of chronic kidney disease (CKD), their mechanisms of action are unclear. A reduced production of uremic toxins could contribute to the benefits of LPDs. Aromatic amino-acids (AA) are precursors of major uremic toxins such as p-cresyl sulfate (PCS) and indoxyl sulfate (IS). We hypothesize that a low aromatic amino acid diet (LA-AAD, namely a low intake of tyrosine, tryptophan and phenylalanine) while being normoproteic, could be as effective as a LPD, through the decreased production of uremic toxins. Kidney failure was chemically induced in mice with a diet containing 0.25% (w/w) of adenine. Mice received three different diets for six weeks: normoproteic diet (NPD: 14.7% proteins, aromatic AAs 0.019%), LPD (5% proteins, aromatic AAs 0.007%) and LA-AAD (14% proteins, aromatic AAs 0.007%). Both LPD and LA-AAD significantly reduced proteinuria, kidney fibrosis and inflammation. While LPD only slightly decreased plasma free PCS and free IS compared to NPD; free fractions of both compounds were significantly decreased by LA-AAD. These results suggest that a LA-AAD confers similar benefits of a LPD in delaying the progression of CKD through a reduction in some key uremic toxins production (such as PCS and IS), with a lower risk of malnutrition.

An Innovative Synbiotic Formulation Decreases Free Serum Indoxyl Sulfate, Small Intestine Permeability and Ameliorates Gastrointestinal Symptoms in a Randomized Pilot Trial in Stage IIIb-IV CKD Patients.

Proteolytic dysbiosis of the gut microbiota has been recognized as both a typical feature of chronic kidney disease (CKD) and a risk factor for its progression. Blood accumulation of gut-derived uremic toxins (UTs) like indoxyl sulfate (IS) and p-cresyl sulfate (PCS), intestinal permeability and constipation are typical features accompanying CKD progression and triggering chronic inflammation. In order to verify the efficacy of the innovative synbiotic formulation NATUREN G® in modulating the levels of circulating UTs, intestinal permeability and gastrointestinal symptoms, we set up a randomized, single-blind, placebo-controlled, pilot trial in stage IIIb-IV CKD patients and in healthy controls. Two-month administration of the synbiotic resulted in a decrease of free IS, as compared with the placebo-treated arm, only in the CKD group. The other UTs did not significantly change, although different trends in time (increase in the placebo arm and decrease in the synbiotic arm) were observed. Moreover, after supplementation, reduction of small intestinal permeability and amelioration of abdominal pain and constipation syndromes were observed only in the CKD group. The obtained results suggest the specificity of action of NATUREN G® in CKD and justify further validation in a wider study population.

Effect of a medium cut-off dialyzer on protein-bound uremic toxins and mineral metabolism markers in patients on hemodialysis.

Hemodialysis (HD) with medium cut-off (MCO) dialyzers may expand molecular clearance, predominantly larger middle molecules (molecular weight 25-60 kDa). However, the impact of MCO dialyzers on long-term clearance of various other components of the uremic milieu is unknown. The tRial Evaluating Mid cut-Off Value membrane clearance of Albumin and Light chains in HemoDialysis patients (REMOVAL-HD) provided an opportunity to assess the effect of MCO dialyzers on protein-bound uremic toxins and novel markers of mineral metabolism. This exploratory sub-study of REMOVAL-HD evaluated changes in protein-bound solutes (total and free indoxyl sulfate [IS] and p-cresyl sulfate [PCS]) and mineral metabolism markers (intact fibroblast growth factor-23 [iFGF23], fetuin-A and endogenous calciprotein particles [CPP-1 and CPP-2]). Mid-week, pre-HD serum samples were collected at baseline and after 12 and 24 weeks of MCO use in stable adult patients. Change from baseline to Week 12 and 24 was estimated using linear mixed effects models. Eighty-nine participants were studied (mean age 67 ± 15 years, 38% female, 51% diabetic, median urine output 200 ml/24 h). Serum iFGF23 was reduced at Week 12 compared to baseline (-26.8% [95%CI -39.7, -11.1], p = 0.001), which was sustained at Week 24 (-21.7% [95%CI -35.7, -4.5], p = 0.012). There was no significant change in serum IS, PCS, fetuin-A, CPP-1, or CPP-2. The use of a MCO dialyzer over 24 weeks was associated with a sustained reduction in FGF23, while other measured components of the uremic milieu were not significantly altered. Further studies are required to determine whether FGF23 reduction is associated with improved patient outcomes.

PH-Dependent Protein Binding Properties of Uremic Toxins In Vitro.

Protein-bound uremic toxins (PBUTs) are difficult to remove using conventional dialysis treatment owing to their high protein-binding affinity. As pH changes the conformation of proteins, it may be associated with the binding of uremic toxins. Albumin conformation at pH 2 to 13 was analyzed using circular dichroism. The protein binding behavior between indoxyl sulfate (IS) and albumin was examined using isothermal titration calorimetry. Albumin with IS, and serum with IS, p-cresyl sulfate, indole acetic acid or phenyl sulfate, as well as serum from hemodialysis patients, were adjusted pH of 3 to 11, and the concentration of the free PBUTs was measured using mass spectrometry. Albumin was unfolded at pH < 4 or >12, and weakened interaction with IS occurred at pH < 5 or >10. The concentration of free IS in the albumin solution was increased at pH 4.0 and pH 11.0. Addition of human serum to each toxin resulted in increased free forms at acidic and alkaline pH. The pH values of serums from patients undergoing hemodialysis adjusted to 3.4 and 11.3 resulted in increased concentrations of the free forms of PBUTs. In conclusion, acidic and alkaline pH conditions changed the albumin conformation and weakened the protein binding property of PBUTs in vitro.

P1059REMOVAL OF LOW MOLECULAR WEIGHT AND PROTEIN-BOUND UREMIC RETENTION SOLUTES WITH ALLO-HEMODIALYSIS: RESULTS FROM EX VIVO STUDY

Abstract Background and Aims It is projected that in 2030, 14.5 million people will have end stage kidney disease and need kidney replacement therapy, yet only 5.4 million will receive it due to economic, social, and political factors. We have proposed allo-hemodialysis (alloHD) as a simple and low-cost HD alternative (https://www.kidneynews.org/kidney-news/features/buddy-dialysis-probed-hemodialysis-alternative). In alloHD, the patient’s blood is dialyzed against the blood of a healthy subject (‘buddy’), who receives the excess fluid and uremic solutes and excretes them via his/her healthy kidneys. Method We conducted ex vivo experiment with bovine whole blood, 4L in a patient and buddy bucket, respectively. In this setup, buddy blood flows through the dialyzer fiber lumen, while patient blood flows in the dialysate space (Nipro Cellentia 17H dialyzer). The patient blood was spiked with urea, creatinine, potassium chloride, indoxyl sulfate (IS), and p-cresyl sulfate (pCS); solute levels on the buddy side were not altered. The alloHD session lasted for 3 hours, the ultrafiltration (UF) volume was 750 mL. The blood flow rates were kept constant at 150 and 200 mL/min on the patient and buddy side, respectively. Heparin (5000 IU/L) was added to either bucket. The alloHD machine prototype comprises 2 pumps on the buddy side and one on patient side (Figure 1A). UF is controlled by the blood flow rate differential between the buddy-sided arterial and venous pumps, respectively. Results The levels of small unbound solutes (urea, creatinine, potassium) equilibrate quickly between patient and buddy buckets (Fig. 1B, top panel). Of note, the buddy-sided potassium equilibrium concentration is lower, because ongoing UF dilutes albumin in the buddy bucket and concentrates it in the patient bucket, resulting in a Gibbs-Donnan potential. The protein-bound uremic solutes IS and pCS equilibrate towards lower total concentrations on the buddy side due to the earlier mentioned UF-induced albumin dilution. Of note, the levels of free IS and pCS, respectively, converge on both sides of the dialyzer membrane (Fig 1B, bottom panel). Conclusion Our ex vivo data suggest that alloHD can be used to treat hyperkalemia, a major cause of death in acute and chronic kidney failure. Both unbound and protein-bound low molecular weight uremic solutes are also removed. With elimination of “classical” dialysate, we simplify the HD procedure and reduce machine size and costs significantly, making it an affordable HD alternative. Feasibility and efficacy studies in animal models are the next step.

Protein-bound uremic toxins are associated with cognitive function among patients undergoing maintenance hemodialysis

Patients with chronic kidney disease have a greater risk of cognitive impairment. Cerebral uremic solute accumulation causes uremic encephalopathy; however, the association of protein-bound uremic toxins on cognitive function remains unclear. The present study aimed to investigate the association of two protein-bound uremic toxins, namely indoxyl sulfate (IS) and p-cresyl sulfate (PCS), on cognitive function in patients receiving hemodialysis (HD) for at least 90 days. Circulating free form IS and PCS were quantified by liquid chromatography/mass spectrometry. Mini-Mental State Examination (MMSE) and Cognitive Abilities Screening Instrument (CASI) were used to evaluate cognitive function. In total, 260 HD patients were recruited with a mean age of 58.1 ± 11.3 years, of which, 53.8% were men, 40% had diabetes, and 75.4% had hypertension. The analysis revealed that both free IS and free PCS were negatively associated with the CASI score and MMSE. After controlling for confounders, circulating free IS levels persisted to be negatively associated with MMSE scores [β = −0.62, 95% confidence interval (CI): −1.16 to −0.08] and CASI scores (β = −1.97, 95% CI: −3.78 to −0.16), mainly in the CASI domains of long-term memory, mental manipulation, language ability, and spatial construction. However, there was no correlation between free PCS and total MMSE or total CASI scores after controlling for confounders. In conclusion, circulating free form IS, but not PCS is associated with lower cognitive function test scores in HD patients. Thus, a further study is needed to evaluate whether a decrease in free IS levels can slow down cognitive decline in HD patients.

Nutritional Therapy Modulates Intestinal Microbiota and Reduces Serum Levels of Total and Free Indoxyl Sulfate and P-Cresyl Sulfate in Chronic Kidney Disease (Medika Study).

In chronic kidney disease (CKD), the gut-microbiota metabolites indoxyl sulfate (IS) and p-cresyl sulfate (PCS) progressively accumulate due to their high albumin-binding capacity, leading to clinical complications. In a prospective crossover controlled trial, 60 patients with CKD grades 3B–4 (GFR = 21.6 ± 13.2 mL/min) were randomly assigned to two dietary regimens: (i) 3 months of free diet (FD) (FD is the diet usually used by the patient before being enrolled in the Medika study), 6 months of very low protein diet (VLPD), 3 months of FD and 6 months of Mediterranean diet (MD); (ii) 3 months of FD, 6 months of MD, 3 months of FD, and 6 months of VLPD. VLPD reduced inflammatory Proteobacteria and increased Actinobacteria phyla. MD and VLPD increased some butyrate-forming species of Lachnospiraceae, Ruminococcaceae, Prevotellaceae, Bifidobacteriaceae, and decrease the pathobionts Enterobacteriaceae. The increased level of potential anti-inflammatory Blautia and Faecalibacterium, as well as butyrate-forming Coprococcus and Roseburia species in VLPD was positively associated with dietary intakes and it was negatively correlated with IS and PCS. Compared to FD and MD, VLPD showed a lower amount of some Lactobacillus, Akkermansia, Streptococcus, and Escherichia species. MD and VLPD reduced both the total and free serum IS (MD −36%, −40% and VLPD −69%, −73%, respectively) and PCS (MD −38%, −44% and VLPD −58%, −71%, respectively) compared to FD. VLPD reduced serum D-lactate compared to MD and FD. MD and, to a greater extent, VLPD are effective in the beneficial modulation of gut microbiota, reducing IS and PCS serum levels, and restoring intestinal permeability in CKD patients.

Differences in Dialysis Efficacy Have Limited Effects on Protein-Bound Uremic Toxins Plasma Levels over Time.

The protein-bound uremic toxins para-cresyl sulfate (pCS) and indoxyl sulfate (IS) are associated with cardiovascular disease in chronic renal failure, but the effect of different dialysis procedures on their plasma levels over time is poorly studied. The present prospective, randomized, cross-over trial tested dialysis efficacy and monitored pre-treatment pCS and IS concentrations in 15 patients on low-flux and high-flux hemodialysis and high-convective volume postdilution hemodiafiltration over six weeks each. Although hemodiafiltration achieved by far the highest toxin removal, only the mean total IS level was decreased at week three (16.6 ± 12.1 mg/L) compared to baseline (18.9 ± 13.0 mg/L, p = 0.027) and to low-flux dialysis (20.0 ± 12.7 mg/L, p = 0.021). At week six, the total IS concentration in hemodiafiltration reached the initial values again. Concentrations of free IS and free and total pCS remained unaltered. Highest beta2-microglobulin elimination in hemodiafiltration (p < 0.001) led to a persistent decrease of the plasma levels at week three and six (each p < 0.001). In contrast, absent removal in low-flux dialysis resulted in rising beta2-microglobulin concentrations (p < 0.001). In conclusion, this trial demonstrated that even large differences in instantaneous protein-bound toxin removal by current extracorporeal dialysis techniques may have only limited impact on IS and pCS plasma levels in the longer term.

Measuring serum total and free indoxyl sulfate and p-cresyl sulfate in chronic kidney disease using UPLC-MS/MS

Chronic kidney disease (CKD) is a complex disorder that affects multiple organs and increases the risk of cardiovascular complications. CKD affects approximately 12% of the population in Taiwan. Loss of kidney function leads to accumulation of potentially toxic compounds such as indoxyl sulfate (IS) and p-cresyl sulfate (pCS), two protein-bound uremic solutes that can stimulate the progression of CKD. The aim of this study was to assess whether IS and pCS levels were correlated with CKD stage. We developed and validated a method for quantitating total and free IS and pCS in serum by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Serum samples were pretreated using protein precipitation with acetonitrile containing stable isotope-labeled IS and pCS as internal standards. After centrifugation, the supernatant was diluted and injected into a UPLC-MS/MS system. Analyte concentrations were calculated from the calibration curve and ion ratios between the analyte and the internal standard. The calibration curves were linear with a correlation coefficient of >0.999; the analytical measurement range was 0.05–5 mg/L. The limit of quantitation of this assay was 0.05 mg/L for both analytes. The reference interval was ≤0.05–1.15 mg/L for total-form IS, ≤0.05–5.33 mg/L for total-form pCS, ≤0.05 mg/L for free-form IS, and ≤0.12 mg/L for free-form pCS. A positive correlation was observed between analyte concentration and CKD stage. Our sensitive UPLC-MS/MS method for quantifying total and free-form IS and pCS in serum can be used to monitor the progression of CKD in clinical settings, identify patients at risk, and facilitate development of further therapies for this devastating disease.

The role of indoxyl sulfate in renal anemia in patients with chronic kidney disease.

Renal anemia is a common complication in patients with advanced chronic kidney disease. In vitro studies have shown that indoxyl sulfate decreases erythropoietin production. Whether this effect is seen in vivo remains unclear. Our goal was to explore the role of indoxyl sulfate in renal anemia. We found serum indoxyl sulfate levels are significantly and negatively associated with erythropoietin levels in human. A multiple stepwise linear regression analyses after adjustment for other independent parameters revealed that free indoxyl sulfate, and total indoxyl sulfate were significantly associated with erythropoietin levels. In animal studies, erythropoietin gene and protein expression were markedly inhibited in rats with chronic kidney disease; however, this effect was significantly reversed by lowering serum indoxyl sulfate with AST-120. Indoxyl sulfate may also inhibit erythropoietin expression in animal models with chronic kidney disease. These findings further support the role of indoxyl sulfate in the development of renal anemia.

Levels of Indoxyl Sulfate in Kidney Transplant Patients, and the Relationship With Hard Outcomes.

Indoxyl sulfate (IS) is a protein-bound uremic toxin that is known to be associated with the risk of cardiovascular (CV) disease and death in both predialysis and dialysis patients. Data on levels of protein-bound uremic toxins in kidney transplant patients are scarce. The study's objective was to evaluate the levels of IS in kidney transplant patients and the relationship with hard outcomes. In 311 kidney transplant patients, IS levels were measured immediately before transplantation (T0), and 1 month (M1) and 12 months (M12) afterwards. Over a mean±standard deviation follow-up period of 113±29 months, a total of 55 deaths, 70 CV events and 71 graft losses were recorded. We observed a rapid significant decrease (below or near the normal value) in IS levels after kidney transplantation. Total and free IS levels at M12 were significantly higher in non-transplant patients than in transplant patients (P=0.003 and <0.0001 respectively), despite having similar estimated glomerular filtration rates. Lastly, IS levels were not associated with overall mortality, CV events or graft loss at T0, M1 or M12. IS levels were significantly lower in kidney transplant receipts than in non-recipients suggesting that kidney transplantation protects against an increase in IS levels. IS levels were not associated with hard outcomes in kidney transplant patients. (Circ J 2016; 80: 722-730).

Indoxyl Sulfate Impairs Endothelial Progenitor Cells and Might Contribute to Vascular Dysfunction in Patients with Chronic Kidney Disease

Background/Aims: Indoxyl sulfate (IS) is a protein-bound uremic toxin that accumulates in patients with chronic kidney disease (CKD). We explored the effect of IS on human early endothelial progenitor cells (EPCs) and analyzed the correlation between serum IS levels and parameters of vascular function, including endothelial function in a CKD-based cohort. Methods: A cross-sectional study with 128 stable CKD patients was conducted. Flow-mediated dilation (FMD), pulse wave velocity (PWV), ankle brachial index, serum IS and other biochemical parameters were measured and analyzed. In parallel, the activity of early EPCs was also evaluated after exposure to IS. Results: In human EPCs, a concentration-dependent inhibitory effect of IS on chemotactic motility and colony formation was observed. Additionally, serum IS levels were significantly correlated with CKD stages. The total IS (T-IS) and free IS (F-IS) were strongly associated with age, hypertension, cardiovascular disease, blood pressure, PWV, blood urea nitrogen, creatine and phosphate but negatively correlated with FMD, the estimated glomerular filtration rate (eGFR), hemoglobin, hematocrit, and calcium. A multivariate linear regression analysis also showed that FMD was significantly associated with IS after adjusting for other confounding factors. Conclusions: In humans, IS impairs early EPCs and was strongly correlated with vascular dysfunction. Thus, we speculate that this adverse effect of IS may partly result from the inhibition of early EPCs.