Effect of sucroferric oxyhydroxide on gastrointestinal microbiome and uremic toxins in patients with chronic kidney disease undergoing hemodialysis.

BACKGROUND: In patients with chronic kidney disease (CKD), dysbiosis in the gastrointestinal microbiome is thought to be associated with increased production of uremic toxins, such as indoxyl sulfate (IS) and p-cresyl sulfate (PCS). Sucroferric oxyhydroxide (SFO), an iron-based phosphate binder, may affect the gastrointestinal microbiome and the production of uremic toxins. We aimed to examine whether SFO administration affected distribution of gastrointestinal microbiome and serum uremic toxin levels in CKD patients undergoing hemodialysis. METHODS: In this single-center, open-label, interventional study, 18 maintenance hemodialysis patients with hyperphosphatemia were prescribed with SFO. We collected serum samples before and after 3 months of administration, and serum levels of IS and PCS were measured. A control group of 20 hemodialysis patients without SFO was evaluated. We evaluated gastrointestinal microbiome of patients pre- and post-SFO administration by 16S rDNA sequencing and bioinformatics analysis. RESULTS: Serum IS and PCS levels were significantly elevated after administration of SFO (IS before 2.52 ± 1.60 mg/dl vs. after 3.13 ± 1.51 mg/dl, P = 0.008; PCS before 2.32 ± 2.44 mg/dl vs. after 3.45 ± 2.11 mg/dl, P = 0.002), while serum IS and PCS levels did not change in the control group. Microbiome analysis in the SFO group showed no significant change in diversity and major components in phylum, class, order, family, gene, and species. CONCLUSION: Administration of SFO increased the serum levels of IS and PCS with no change of major components of gastrointestinal microbiome.

Factors of microinflammation in non-diabetic chronic kidney disease: a pilot study.

BACKGROUND: The relationships between digestive bacterial translocation, uremic toxins, oxidative stress and microinflammation in a population of chronic kidney disease (CKD) patients without metabolic nor inflammatory disease are unknown. METHODS: Bacterial translocation, uremic toxins, oxidative stress, and inflammation were assessed by measuring plasma levels of 16S ribosomal DNA (16S rDNA), p-cresyl sulfate (PCS), indoxyl sulfate (IS), indole acetic acid (IAA), F2-isoprostanes, hsCRP and receptor I of TNFα (RITNFα) in patients without metabolic nor inflammatory disease. 44 patients with CKD from stage IIIB to V and 14 controls with normal kidney function were included from the nephrology outpatients. 11 patients under hemodialysis (HD) were also included. Correlations between each factor and microinflammation markers were studied. RESULTS: 16S rDNA levels were not increased in CKD patients compared to controls but were decreased in HD compared to non-HD stage V patients (4.7 (3.9-5.3) vs 8.6 (5.9-9.7) copies/µl, p = 0.002). IS, PCS and IAA levels increased in HD compared to controls (106.3 (73.3-130.4) vs 3.17 (2.4-5.1) µmol/l, p < 0.0001 for IS; 174.2 (125-227.5) vs 23.7 (13.9-52.6) µmol/l, p = 0.006 for PCS; and 3.7 (2.6-4.6) vs 1.3 (1.0-1.9) µmol/l, p = 0.0002 for IAA). Urea increased in non-HD stage V patients compared to controls (27.6 (22.7-30.9) vs 5.4 (4.8-6.4) mmol/l, p < 0.0001) and was similar in HD and in non-HD stage V (19.3 (14.0-24.0) vs 27.6 (22.7-30.9) mmol/l, p = 0.7). RITNFα levels increased in HD patients compared to controls (12.6 (9.6-13.3) vs 1.1 (1.0-1.4) ng/ml, p < 0.0001); hsCRP levels increased in non-HD stage V patients compared to controls (2.9 (1.4-8.5) vs 0.8 (0.5-1.7) mg/l, p = 0.01) and remained stable in HD patients (2.9 (1.4-8.5) vs 5.1 (0.9-11.5) mg/l, p = 1). F2-isoprostanes did not differ in CKD patients compared to controls. Among uremic toxins, IS and urea were correlated to RITNFα (r = 0.8, p < 0.0001 for both). PCS, IS and urea were higher in patients with hsCRP≧5 mg/l (p = 0.01, 0.04 and 0.001 respectively). 16S rDNA, F2-isoprostanes were not correlated to microinflammation markers in our study. CONCLUSIONS: In CKD patients without any associated metabolic nor inflammatory disease, only PCS, IS, and urea were correlated with microinflammation. Bacterial translocation was decreased in patients under HD and was not correlated to microinflammation.

Bowel Habits and the Association With Uremic Toxins in Non-Dialysis-Dependent Chronic Kidney Disease Patients.

OBJECTIVE: The aim of this study is to evaluate the association between bowel habits and microbial-derived uremic toxins p-cresyl sulfate (PCS) and indoxyl sulfate (IS) in patients with non-dialysis-dependent chronic kidney disease (NDD-CKD). DESIGN AND METHODS: This is a cross-sectional analysis including 43 nondiabetic NDD-CKD patients (58% men; 59.0 ± 13.5 years; estimated glomerular filtration rate, 21.3 ± 7.9 mL/min/1.73 m2). Bowel habit was assessed by the Bristol Stool Scale (BSS <3, characterized by hard consistency of stools and/or low frequency of evacuation and BSS ≥3, representing a more regular bowel habit) and by the Rome III criteria. PCS and IS (serum, free and total; urinary, total) were determined by high-performance liquid chromatography. Dietary intake was assessed by the 3-day food records. RESULTS: The frequency of constipation assessed by BSS and Rome III criteria was 33% (n = 14/43) and 35% (n = 15/43), respectively. The BSS <3 exhibited higher PCS, independent of renal function and dietary protein-fiber ratio (ß [95% confidence interval {CI}]: serum, total PCS = 1.54 [1.06-2.23], P = .02; serum free PCS = 1.40 [1.00-1.97], P = .05; urinary PCS = 1.78 [1.10-2.90], P < .02). According to the Rome III criteria, a tendency for a higher serum total PCS (ß [95% CI]: 1.39 [0.95-2.03 µmol/L], P = .09) and a significantly higher urinary PCS (ß [95% CI]: 1.80 [1.11-2.94 µmol/24 h], P = .02) was found in constipated participants. No effect of a compromised bowel habit (Rome III criteria or BSS) was found on IS. CONCLUSION: Constipation may lead to production of PCS in nondiabetic NDD-CKD patients.

Quantification of Dehydroepiandrosterone, 17ß-Estradiol, Testosterone, and Their Sulfates in Mouse Tissues by LC-MS/MS.

We report a high-performance, liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay to quantify without derivatizaton dehyroepiandrosterone (DHEA), 17ß-estradiol (E2), testosterone (T), and their sulfates in serum and tissues. This assay functions well with multiple adipose depots, a previously unattained analysis. To delipidate and facilitate recovery, tissues were homogenized in acetonitrile, and the homogenate was frozen. The supernatant was evaporated, resuspended in an aqueous acetate buffer, and extracted with hexane to separate free (unconjugated) from sulfated steroids. Sulfated steroids in the aqueous medium were then hydrolyzed with sulfatase and extracted with hexane. Each extract was analyzed separately. HPLC resolution combined with the sensitivity and specificity of MS/MS allowed quantification of DHEA, E2, and T with 10, 10, and 5 fmol lower limits of quantification and linear ranges to 1 pmol. Application of the method to mouse serum and tissues reveals ranges of DHEA, E2, and T and their sulfates, and tissue-specific differences in steroid profile, especially white versus brown adipose. In addition, marginal decreases of T in all tissues and considerable increases in DHEA in male iWAT and eWAT in response to a high-fat diet further strengthen the inference regarding the role of steroid metabolism in adipogenesis. This assay permits detailed studies of interactions between adiposity and sex steroids in serum and tissues, including adipose.

Indoxyl sulfate associates with cardiovascular phenotype in children with chronic kidney disease.

BACKGROUND: Cardiovascular disease is the leading cause of death in children with chronic kidney disease (CKD). Serum levels of gut-derived uremic toxins increase with deterioration of kidney function and are associated with cardiac comorbidities in adult CKD patients. METHODS: Indoxyl sulfate (IS) and p-cresyl sulfate (pCS) were measured by high-performance liquid chromatography in serum of children participating in the Cardiovascular Comorbidity in Children with CKD (4C) Study. Results were correlated with measurements of the carotid intima-media thickness (cIMT), central pulse wave velocity (PWV), and left ventricular mass index (LVMI) in children aged 6-17 years with initial eGFR of 10-60 ml/min per 1.73 m2. RESULTS: The median serum levels of total IS and of pCS, measured in 609 patients, were 5.3 µmol/l (8.7) and 17.0 µmol/l (21.6), respectively. In a multivariable regression model, IS and pCS showed significant positive associations with urea and negative associations with eGFR and uric acid. Furthermore, positive associations of pCS with age, serum albumin, and non-Mediterranean residency and a negative association with glomerular disease were observed. By multivariable regression analysis, only IS was significantly associated with a higher cIMT SDS at baseline and progression of PWV SDS within 12 months, independent of other risk factors. CONCLUSIONS: Serum levels of gut-derived uremic toxins IS and pCS correlated inversely with eGFR in children. Only IS was significantly associated with surrogate markers of cardiovascular disease in this large pediatric CKD cohort.

Increasing the removal of protein-bound uremic toxins by liposome-supported hemodialysis.

Protein-bound uremic toxins (PBUTs) accumulate at high plasma levels and cause various deleterious effects in end-stage renal disease patients because their removal by conventional hemodialysis is severely limited by their low free-fraction levels in plasma. Here, we assessed the extent to which solute removal can be increased by adding liposomes to the dialysate. The uptake of liposomes by direct incubation in vitro showed an obvious dose-response relationship for p-cresyl sulfate (PCS) and indoxyl sulfate (IS) but not for hippuric acid (HA). The percent removal of both PCS and IS but not of HA was gradually increased with the increased concentration of liposomes in a rapid equilibrium dialysis setup. In vitro closed circulation showed that adding liposomes to the dialysate markedly increased the dialysances of PBUTs without greatly altering that of urea and creatinine. In vivo experiments in uremic rats demonstrated that adding liposomes to the dialysate resulted in higher reduction ratios (RRs) and more total solute removal (TSR) for several PBUTs compared to the conventional dialysate, which was approximately similar to the addition of bovine serum albumin to the dialysate. These findings highlight that as an adjunct to conventional hemodialysis, addition of liposomes to the dialysate could significantly improve the removal of protein-bound uremic solutes without greatly altering the removal of small, water-soluble solutes.

Moderate Renal Impairment and Toxic Metabolites Produced by the Intestinal Microbiome: Dietary Implications.

OBJECTIVE: Toxic metabolites produced by the intestinal microbiome from animal proteins, carnitine (mainly from red meat), or phosphatidylcholine (mainly from egg yolk), have important adverse effects on cardiovascular disease. These are renally eliminated and may be termed gut-derived uremic toxins (GDUT). We hypothesized that even moderate renal impairment and intake of nutrient precursors would raise plasma levels of GDUT. DESIGN: A cohort study. SETTING: Academic medical center. SUBJECTS: Patients attending stroke prevention clinics at a university medical center were recruited. MAIN OUTCOME MEASURE: Nutrient intake was assessed by the 131-item Harvard Food Frequency Questionnaire; estimated glomerular filtration rate (eGFR) was caculated using the Chronic Kidney Disease-Epidemiology (EPI) equations. Plasma levels of trimethylamine n-oxide, p-cresyl sulfate, hippuric acid, p-cresyl glucuronide, pheny acetyl glutamine, and phenyl sulfate were measured by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. RESULTS: Among 316 patients recruited, the mean (standard deviation [SD]) age was 66.74 (10.42) years; 59.7% were men. Mean eGFR was 76.03 ± 20.01; 57 (18%) had eGFR<60 mL/min/1.73 m2. Plasma levels of all GDUT were significantly higher even with moderate reduction of eGFR. Nutrient intake affected plasma levels of some GDUT; the effects differed by eGFR above and below 60 mL/min/1.73 m2. Plasma levels were obtained fasting, so we probably underestimated the effect of nutrient intake. CONCLUSIONS: Even moderate impairment of renal function was associated with higher plasma levels of GDUT. This has dietary implications for patients at risk of atherosclerosis, particularly in those with impaired renal function (including the elderly): they should limit intake of animal protein, red meat, and egg yolk. It also points the way to novel approaches to vascular prevention, including more intensive dialysis, renal transplantation, and modification of the intestinal microbiome with probiotics or fecal transplantation.

Residual Function Effectively Controls Plasma Concentrations of Secreted Solutes in Patients on Twice Weekly Hemodialysis.

Background Most patients on hemodialysis are treated thrice weekly even if they have residual kidney function, in part because uncertainty remains as to how residual function should be valued and incorporated into the dialysis prescription. Recent guidelines, however, have increased the weight assigned to residual function and thus reduced the treatment time required when it is present. Increasing the weight assigned to residual function may be justified by knowledge that the native kidney performs functions not replicated by dialysis, including solute removal by secretion. This study tested whether plasma concentrations of secreted solutes are as well controlled in patients with residual function on twice weekly hemodialysis as in anuric patients on thrice weekly hemodialysis.Methods We measured the plasma concentration and residual clearance, dialytic clearance, and removal rates for urea and the secreted solutes hippurate, phenylacetylglutamine, indoxyl sulfate, and p-cresol sulfate in nine patients on twice weekly hemodialysis and nine patients on thrice weekly hemodialysis.Results Compared with anuric patients on thrice weekly dialysis with the same standard Kt/Vurea, patients on twice weekly hemodialysis had lower hippurate and phenylacetylglutamine concentrations and similar indoxyl sulfate and p-cresol sulfate concentrations. Mathematical modeling revealed that residual secretory function accounted for the observed pattern of solute concentrations.Conclusions Plasma concentrations of secreted solutes can be well controlled by twice weekly hemodialysis in patients with residual kidney function. This result supports further study of residual kidney function value and the inclusion of this function in dialysis adequacy measures.

Unambiguous Characterization of p-Cresyl Sulfate, a Protein-Bound Uremic Toxin, as Biomarker of Heart and Kidney Disease.

p-Cresyl sulfate is one of the bound uremic toxins whose level increases in the sera of patients with the severity of chronic kidney disease and is therefore used as a standard for clinical investigations. Our first attempts to obtain p-cresyl sulfate led exclusively to the product of sulfonation of the aromatic ring instead of sulfation on the OH moiety. Nevertheless, this initial discouraging result allowed us to handle both p-cresyl sulfate and 2-hydroxy-5-methylbenzenesulfonic acid obtained by different synthetic pathways. Interestingly, the comparison between the two isomers pointed out that the two molecules show the same fragmentation pattern and are indistinguishable by mass spectrometry. They cannot be separated on several commercially available columns. The only difference between the two compounds is a 10-fold higher ionization yield under negative ion electrospray ionization. NMR spectral studies definitely confirmed the different molecular structures. We present here an unambiguous biomimetic synthetic route for p-cresyl sulfate and the spectroscopic characterization of both the compounds by nuclear magnetic resonance and mass spectrometry.

Exploration of the Fecal Microbiota and Biomarker Discovery in Equine Grass Sickness.

Equine grass sickness (EGS) is a frequently fatal disease of horses, responsible for the death of 1 to 2% of the U.K. horse population annually. The etiology of this disease is currently uncharacterized, although there is evidence it is associated with Clostridium botulinum neurotoxin in the gut. Prevention is currently not possible, and ileal biopsy diagnosis is invasive. The aim of this study was to characterize the fecal microbiota and biofluid metabolic profiles of EGS horses, to further understand the mechanisms underlying this disease, and to identify metabolic biomarkers to aid in diagnosis. Urine, plasma, and feces were collected from horses with EGS, matched controls, and hospital controls. Sequencing the16S rRNA gene of the fecal bacterial population of the study horses found a severe dysbiosis in EGS horses, with an increase in Bacteroidetes and a decrease in Firmicutes bacteria. Metabolic profiling by 1H nuclear magnetic resonance spectroscopy found EGS to be associated with the lower urinary excretion of hippurate and 4-cresyl sulfate and higher excretion of O-acetyl carnitine and trimethylamine-N-oxide. The predictive ability of the complete urinary metabolic signature and using the four discriminatory urinary metabolites to classify horses by disease status was assessed using a second (test) set of horses. The urinary metabolome and a combination of the four candidate biomarkers showed promise in aiding the identification of horses with EGS. Characterization of the metabolic shifts associated with EGS offers the potential of a noninvasive test to aid premortem diagnosis.

Metabolic profiles in community-acquired pneumonia: developing assessment tools for disease severity.

BACKGROUND: This study aimed to determine whether community-acquired pneumonia (CAP) had a metabolic profile and whether this profile can be used for disease severity assessment. METHODS: A total of 175 individuals including 119 CAP patients and 56 controls were enrolled and divided into two cohorts. Serum samples from a discovery cohort (n = 102, including 38 non-severe CAP, 30 severe CAP, and 34 age and sex-matched controls) were determined by untargeted ultra-high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based metabolomics. Selected differential metabolites between CAP patients versus controls, and between the severe CAP group versus non-severe CAP group, were confirmed by targeted mass spectrometry assays in a validation cohort (n = 73, including 32 non-severe CAP, 19 severe CAP and 22 controls). Pearson's correlation analysis was performed to assess relationships between the identified metabolites and clinical severity of CAP. The area under the curve (AUC), sensitivity and specificity of the metabolites for predicting the severity of CAP were also investigated. RESULTS: The metabolic signature was markedly different between CAP patients and controls. Fifteen metabolites were found to be significantly dysregulated in CAP patients, which were mainly mapped to the metabolic pathways of sphingolipid, arginine, pyruvate and inositol phosphate. The alternation trends of five metabolites among the three groups including sphinganine, p-Cresol sulfate, dehydroepiandrosterone sulfate (DHEA-S), lactate and L-arginine in the validation cohort were consistent with those in the discovery cohort. Significantly lower concentrations of sphinganine, p-Cresol sulfate and DHEA-S were observed in CAP patients than in controls (p < 0.05). Serum lactate and sphinganine levels were positively correlated with confusion, urea level, respiratory rate, blood pressure, and age > 65 years (CURB-65), pneumonia severity index (PSI) and Acute Physiology and Chronic Health Evaluation II (APACHE II) scores, while DHEA-S inversely correlated with the three scoring systems. Combining lactate, sphinganine and DHEA-S as a metabolite panel for discriminating severe CAP from non-severe CAP exhibited a better AUC of 0.911 (95% confidence interval 0.825-0.998) than CURB-65, PSI and APACHE II scores. CONCLUSIONS: This study demonstrates that serum metabolomics approaches based on the LC-MS/MS platform can be applied as a tool to reveal metabolic changes during CAP and establish a metabolite signature related to disease severity. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03093220 . Registered retrospectively on 28 March 2017.

Efficacy of Probiotics Supplementation On Chronic Kidney Disease: a Systematic Review and Meta-Analysis.

BACKGROUND/AIMS: Dysbiosis of the intestinal microbiota may accelerate the progression of chronic kidney disease (CKD) by increasing the levels of urea toxins. In recent years, probiotics have been recognized to maintain the physiological balance of the intestinal microbiota. In this study, we aim to assess the therapeutic effects of probiotics on CKD patients with and without dialysis via meta-analysis. METHODS: We conducted a meta-analysis of randomized controlled trials (RCTs) by searching the databases of Pubmed, EMBASE and Cochrane Library (No. CRD42018093080). Studies on probiotics for treatment of CKD adults lasting for at least 4 weeks were selected. The primary outcomes were the levels of urea toxins, and the second outcomes were the levels of interleukin (IL)-6, C-reactive protein (CRP) and hemoglobin (Hb). The risk of bias was assessed by Cochrane Collaboration' tool, and the quality of evidence was appraised with the Grading of Recommendation Assessment. Means and standard deviations were analyzed by random effects analysis. Stratified analysis was done and sensitivity analysis was performed when appropriate. RESULTS: Totally, eight studies with 261 patients at CKD stage 3 to 5 with and without dialysis were included. We found a decrease of p-cresyl sulfate (PCS) of 3 studies with 125 subjects (P = 0.01, SMD -0.57, 95% CI, -0.99 to -0.14, I2 = 25%) and an increase of IL-6 in 3 studies with 134 subjects (P = 0.03, 95% CI, SMD 0.37, 0.03 to 0.72, I2 = 0%) in the probiotics groups. Analysis of serum creatinine (P = 0.47), blood urine nitrogen (P = 0.73), CRP (P = 0.55) and Hb (P = 0.49) yielded insignificant difference. CONCLUSION: Limited number of studies and small sample size are limitations of our study. Probiotics supplementation may reduce the levels of PCS and elevate the levels of IL-6 whereby protecting the intestinal epithelial barrier of patients with CKD.

Generation and Characterization of Anti-phenyl Sulfate Monoclonal Antibodies and a Potential Use for Phenyl Sulfate Analysis in Human Blood.

Patients with chronic kidney disease (CKD) have increased blood levels of phenyl sulfate (PS), a circulating uremic toxin. In this study, we produced anti-PS monoclonal antibodies (mAbs) and characterized their cross-reactivity to structural PS analogs. To induce PS-specific mAbs, we synthesized 4-mercaptophenyl sulfate with a sulfhydryl group at the para-position of PS and conjugated it to carrier proteins via bifunctional linkers. Using these PS conjugates as immunogens and as antigens for enzyme-linked immunosorbent assay (ELISA) screening, we produced by a hybridoma method two novel mAbs (YK33.1 and YKS19.2) that react with PS conjugates independent of carrier and linker structures. Although all of the PS analogs tested, with the exception of indoxyl sulfate, were cross-reactive to both mAbs in phosphate buffered saline (PBS), PS specificity for YKS19.2 was enhanced in human plasma and serum. YKS19.2 mAb was cross-reactive only with o-cresyl sulfate, which is absent in human blood. PS sensitivity for YKS19.2 mAb increased to an IC50 of 10.4 µg/mL when 0.1% Tween 20 was added in a primary competitive reaction. To explore potential clinical applications, we determined concentrations of PS in serum samples from 19 CKD patients by inhibition ELISA using YKS19.2 mAb and compared them to those found using an LC-MS/MS method. A good correlation was observed between each value (R2=0.825). Therefore, the unique antigen specificity of YKS19.2 mAb could be useful for prescreening of patients with accumulated PS or for comprehensive analysis of uremic toxins that have a PS-like structure.

Adsorption of Protein-Bound Uremic Toxins Through Direct Hemoperfusion With Hexadecyl-Immobilized Cellulose Beads in Patients Undergoing Hemodialysis.

An accumulation of protein-bound uremic toxins (PBUTs) is one of major reasons for development of uremia-related complications. We examined the PBUT removal ability of a hexadecyl-immobilized cellulose bead (HICB)-containing column for patients undergoing hemodialysis. Adsorption of indoxyl sulfate (IS), a representative PBUT, to HICBs was examined in vitro. The HICB column was used in patients undergoing hemodialysis for direct hemoperfusion with a regular hemodialyzer. The serum IS, indole acetic acid (IAA), phenyl sulfate (PhS), and p-cresyl sulfate (PCS) levels were measured before and after passing the column. HICBs adsorbed protein-free (free) IS in a dose- and time-dependent manner in vitro (55.4 ± 1.4% adsorption of 1 millimolar, 251 µg/mL, IS for 1 h). In clinical studies, passing the HICB-containing column decreased the serum level of free IS, IAA, PhS, and PCS levels significantly (by 34.4 ± 30.0%, 34.8 ± 25.4%, 28.4 ± 18.0%, and 34.9 ± 22.1%, respectively), but not protein-bound toxins in maintenance hemodialysis patients. HICBs absorbed some amount of free PBUTs, but the clinical trial to use HICB column did not show effect to reduce serum PBUTs level in hemodialysis patients. Adsorption treatment by means of direct hemoperfusion with regular hemodialysis may become an attractive blood purification treatment to increase PBUT removal when more effective materials to adsorb PBUTs selectively will be developed.

Does Low-Protein Diet Influence the Uremic Toxin Serum Levels From the Gut Microbiota in Nondialysis Chronic Kidney Disease Patients?

OBJECTIVES: To evaluate the effects of low-protein diet (LPD) on uremic toxins and the gut microbiota profile in nondialysis chronic kidney disease (CKD) patients. DESIGN AND METHODS: Longitudinal study with 30 nondialysis CKD patients (stage 3-4) undergoing LPD for 6 months. Adherence to the diet was evaluated based on the calculation of protein equivalent of nitrogen appearance from the 24-hour urine analysis. Good adherence to LPD was considered when protein intake was from 90% to 110% of the prescribed amount (0.6 g/kg/day). Food intake was analyzed by the 24-hour recall method. The anthropometric, biochemical and lipid profile parameters were measured according to standard methods. Uremic toxin serum levels (indoxyl sulfate, p-cresyl sulfate, indole-3-acetic acid) were obtained by reversed-phase high-performance liquid chromatography (RP-HPLC). Fecal samples were collected to evaluate the gut microbiota profile through polymerase chain reaction and denaturing gradient gel electrophoresis. Statistical analysis was performed by the SPSS 23.0 program software. RESULTS: Patients who adhered to the diet (n = 14) (0.7 ± 0.2 g/kg/day) presented an improvement in renal function (nonsignificant) and reduction in total and low-density lipoprotein cholesterol (183.9 ± 48.5-155.7 ± 37.2 mg/dL, P = .01; 99.4 ± 41.3-76.4 ± 33.2 mg/dL, P = .01, respectively). After 6 months of nutricional intervention, p-cresyl sulfate serum levels were reduced significantly in patients who adhered to the LPD (19.3 [9.6-24.7] to 15.5 [9.8-24.1] mg/L, P = .03), and in contrast, the levels were increased in patients who did not adhere (13.9 [8.0-24.8] to 24.3 [8.1-39.2] mg/L, P = .004). In addition, using the denaturing gradient gel electrophoresis technique, it was observed change in the intestinal microbiota profile after LPD intervention in both groups, and the number of bands was positively associated with protein intake (r = 0.44, P = .04). CONCLUSION: LPD seems be a good strategy to reduce the uremic toxins production by the gut microbiota in nondialysis CKD patients.

Probiotic Supplementation in Chronic Kidney Disease: A Double-blind, Randomized, Placebo-controlled Trial.

OBJECTIVE: The objective of the study was to evaluate the effects of probiotic supplementation on the gut microbiota profile and inflammatory markers in chronic kidney disease patients undergoing maintenance hemodialysis (HD). DESIGN AND METHODS: This was a randomized, double-blind, placebo-controlled study. Forty-six HD patients were assigned to receive 1 of 2 treatments: probiotic (n = 23; Streptococcus thermophilus, Lactobacillus acidophilus e Bifidobacterialongum, 90 billion colony-forming units per day) or placebo (n = 23) daily for 3 months. Blood and feces were collected at baseline and after intervention. The inflammatory markers (C-reactive protein and interleukin-6) were analyzed by immunoenzymatic assay (enzyme-linked immunosorbent assay). Uremic toxins plasma levels (indoxyl sulfate, p-cresyl sulfate, and indole-3-acetic acid) were obtained by Reversed-Phase High-Performance Liquid Chromatography. Routine laboratory parameters were measured by standard techniques. Fecal pH was measured by the colorimetric method, and the gut microbiota profile was assessed by Denaturing Gradient Gel Electrophoresis analysis. RESULTS: Sixteen patients remained in the probiotic group (11 men, 53.6 ± 11.0 year old, 25.3 ± 4.6 kg/m2) and 17 in the placebo group (10 men, 50.3 ± 8.5 year old, 25.2 ± 5.7 kg/m2). After probiotic supplementation there was a significant increase in serum urea (from 149.6 ± 34.2 mg/dL to 172.6 ± 45.0 mg/dL, P = .02), potassium (from 4.4 ± 0.4 mmol/L to 4.8 ± 0.4 mmol/L, P = .02), and indoxyl sulfate (from 31.2 ± 15.9 to 36.5 ± 15.0 mg/dL, P = .02). The fecal pH was reduced from 7.2 ± 0.8 to 6.5 ± 0.5 (P = .01). These parameters did not change significantly in placebo group. Changes in the percentage delta (Δ) between groups were exhibited with no statistical differences observed. The inflammatory markers and gut profile were not altered by supplementation. CONCLUSIONS: Aprobiotic supplementation failed to reduce uremic toxins and inflammatory markers. Therefore, probiotic therapy should be chosen with caution in HD patients. Further studies addressing probiotic therapy in chronic kidney disease patients are needed.

Limited reduction in uremic solute concentrations with increased dialysis frequency and time in the Frequent Hemodialysis Network Daily Trial.

The Frequent Hemodialysis Network Daily Trial compared conventional three-times weekly treatment to more frequent treatment with a longer weekly treatment time in patients receiving in-center hemodialysis. Evaluation at one year showed favorable effects of more intensive treatment on left ventricular mass, blood pressure, and phosphate control, but modest or no effects on physical or cognitive performance. The current study compared plasma concentrations of uremic solutes in stored samples from 53 trial patients who received three-times weekly in-center hemodialysis for an average weekly time of 10.9 hours and 30 trial patients who received six-times weekly in-center hemodialysis for an average of 14.6 hours. Metabolomic analysis revealed that increased treatment frequency and time resulted in an average reduction of only 15 percent in the levels of 107 uremic solutes. Quantitative assays confirmed that increased treatment did not significantly reduce levels of the putative uremic toxins p-cresol sulfate or indoxyl sulfate. Kinetic modeling suggested that our ability to lower solute concentrations by increasing hemodialysis frequency and duration may be limited by the presence of non-dialytic solute clearances and/or changes in solute production. Thus, failure to achieve larger reductions in uremic solute concentrations may account, in part, for the limited benefits observed with increasing frequency and weekly treatment time in Frequent Hemodialysis Daily Trial participants.

Results of the HEMO Study suggest that p-cresol sulfate and indoxyl sulfate are not associated with cardiovascular outcomes.

Cardiovascular disease, the leading cause of mortality in hemodialysis patients, is not fully explained by traditional risk factors. To help define non-traditional risk factors, we determined the association of predialysis total p-cresol sulfate, indoxyl sulfate, phenylacetylglutamine, and hippurate with cardiac death, sudden cardiac death, and first cardiovascular event in the 1,273 participants of the HEMO Study. The results were adjusted for potential demographic, clinical, and laboratory confounders. The mean age of the patients was 58 years, 63% were Black and 42% were male. Overall, there was no association between the solutes and outcomes. However, in sub-group analyses, among patients with lower serum albumin (under 3.6 g/dl), a twofold higher p-cresol sulfate was significantly associated with a 12% higher risk of cardiac death (hazard ratio 1.12; 95% confidence interval, 0.98-1.27) and 22% higher risk of sudden cardiac death (1.22, 1.06-1.41). Similar trends were also noted with indoxyl sulfate. Trial interventions did not modify the association between these solutes and outcomes. Routine clinical and lab data explained less than 22% of the variability in solute levels. Thus, in prevalent hemodialysis patients participating in a large U.S. hemodialysis trial, uremic solutes p-cresol sulfate, indoxyl sulfate, hippurate, and phenylacetylglutamine were not associated with cardiovascular outcomes. However, there were trends of toxicity among patients with lower serum albumin.

Diurnal and Long-term Variation in Plasma Concentrations and Renal Clearances of Circulating Markers of Kidney Proximal Tubular Secretion.

BACKGROUND: The renal proximal tubule is essential for removing organic solutes and exogenous medications from the circulation. We evaluated diurnal, prandial, and long-term biological variation of 4 candidate endogenous markers of proximal tubular secretion. METHODS: We used LC-MS to measure plasma and urine concentrations of hippurate (HA), cinnamoylglycine (CMG), indoxyl sulfate (IS), and p-cresol sulfate (PCS) in 25 healthy adults. We measured plasma concentrations of secreted solutes at 13 time points over a 24-h period, and again after 2 weeks and 14 weeks of follow-up. We further measured 24-h renal clearances of secreted solutes at baseline, 2 weeks, and 14 weeks. RESULTS: Plasma concentrations of secreted solutes varied over the 24-h baseline period. Diurnal variation was greatest for HA, followed by CMG, IS, and PCS. Plasma concentrations of HA (P = 0.002) and IS (P = 0.02), but not CMG and PCS, increased significantly following meals. Long-term intraindividual biological variation (CVI) in plasma concentrations of secreted solutes over 14 weeks varied from 21.8% for IS to 67.3% for PCS, and exceeded that for plasma creatinine (CVI, 7.1%). Variation in 24-h renal clearances was similar among the secreted solutes [intraindividual variation (CVA+I), 33.6%-47.3%] and was lower using pooled plasma samples from each study visit. CONCLUSIONS: Plasma concentrations of HA, CMG, IS, and PCS fluctuate within individuals throughout the day and over weeks. Renal clearances of these secreted solutes, which serve as estimates of renal proximal tubule secretion, are also subject to intraindividual biological variation that can be improved by additional plasma measurements.

Microbiome perturbation by oral vancomycin reduces plasma concentration of two gut-derived uremic solutes, indoxyl sulfate and p-cresyl sulfate, in end-stage renal disease.

BACKGROUND: Observational studies have suggested a relationship between the plasma concentration of indoxyl sulfate (IS) and p-cresyl sulfate (PCS), small gut-derived 'uremic solutes', and the high incidence of uremic cardiomyopathy in patients with end-stage renal disease (ESRD). IS and PCS are derived from the metabolism of dietary components (tryptophan and tyrosine) by gut bacteria. This pilot study was designed to examine the effects of a poorly absorbable antibiotic (vancomycin) on the plasma concentration of two gut-derived 'uremic solutes', IS and PCS, and on the composition of the gut microbiome. METHODS: Plasma concentrations of IS and PCS were measured by MS-HPLC. The gut microbiome was assessed in stool specimens sequenced for the 16S rRNA gene targeting the V4 region. RESULTS: The pre-dialysis mean plasma concentrations of both IS and PCS were markedly elevated. Following the administration of vancomycin (Day 0), the IS and PCS concentrations decreased at Day 2 or Day 5 and returned to baseline by Day 28. Following vancomycin administration, several changes in the gut microbiome were observed. Most striking was the decrease in diversity, a finding that was evident on Day 7 and was still evident at Day 28. There was little change at the phylum level but at the genus level, broad population changes were noted. Changes in the abundance of several genera appeared to parallel the concentration of IS and PCS. CONCLUSIONS: These findings suggest that alteration of the gut microbiome, by an antibiotic, might provide an important strategy in reducing the levels of IS and PCS in ESRD.