The Regulation Of Gut Microflora On Protein-bound Uremic Toxins Indoxyl Sulfate And P-Cresyl Sulfate In Peritoneal Dialysis Patients

Protein-bound uremic toxins are solutes with molecular weight smaller than 500Da but retained in CKD patients because of their high plasma protein binding.Indoxyl sulfate(IS)and p-cresyl sulfate(p CS)are two of the most important protein-bound uremic toxins,with?90%IS and 95%p CS binding to serum proteins.Indole and p-cresol,precursors of IS and p CS,are derived exclusively from tryptophan metabolism by bacterial tryptophanases and tyrosine and phenylalanine fermentation by bacterial tyrosine deaminase in the gut,respectively.The indole and p-cresol absorbed through the intestinal wall are metabolized into IS in the liver,and p CS in the mucosa of the colon or in the liver by sulfation metabolism pathway.Serum IS and p CS are excreted mainly by renal tubules secreting.For CKD patients,the enrichment of indole-producing bacteria and p-cresol-producing bacteria lead to high production,the reduced renal function lead to low excretion,and dialysis removal has limited effects.The present study was aimed to explore the change trend of serum IS and p CS by time in PD patients,the difference in serum IS and p CS,and their producing bacteria in intestine between PD patients and health control,and the effects of Inulin-type fructan on serum IS and p CS and their producing bacteria.Section 1Objective:With prospective study,we aimed to explore the change trend of serum IS and p CS by time in PD patients,and the correlation between serum IS and p CS and metabolic parameters associated with cardiovascular disease.Methods:This prospective study was conducted basing on 116 PD patients.Fecal microbiome was measured by PCR amplification and 16S r RNA gene Phylo Chip.Fecal indole and p-cresol were measured by HPLC-fluorescence detector.Total IS and p CS in serum,24h spent dialysate and urine were measured by ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).The generalized additive model was used to fit the time trend of IS and p CS in serum,24h urine and 24h dialysis.A cross-sectional design was used to explore the correlation between serum IS and p CS and metabolic parameters associated with cardiovascular disease,including serum phosphorus,K⁺,Ca²⁺,blood pressure,blood lipid and blood glucose.Results:1.With time forward,serum IS concentration(P<0.001)and its removal by24h spent dialysis(P=0.001)increased significantly,while the removal by 24h urine decreased(P=0.003).All the change trend was affected by the age at ESRD diagnosis,24h urine volume and residual GFR.2.The serum p CS concentration gradually decreased with the time forward,but was not significant(P=0.089),while its removal by 24h urine and 24h spent dialysis remained stable.The change trend of serum p CS concentration was influenced by 24h urine volume,serum albumin concentration and residual GFR.3.With age,sex,BMI,ESRD duration,PD duration and residual GFR adjusted,serum IS concentration was positively correlated with serum Ca²⁺(P=0.002)and daily ultrafiltration(P=0.046),but negatively correlated with plasma HDL-C(P=0.004).4.Serum p CS concentration was negatively correlated with serum K⁺(P=0.038),serum phosphorus(P=0.014)and serum HDL-C(P=0.023),while positively correlated with HBA1C(P=0.025).Conclusions:The serum IS concentration and its removal by 24h spent dialysis increased significantly with time forward,while the removal by 24h urine decreased.The trend for serum p CS concentration and its removal by 24h urine excretion and24h dialysis remained stable.Serum IS and p CS were negatively correlated with plasma HDL-C.Section 2Objective:With ESRD patients and health controls included,we aimed to explore the difference in serum IS and p CS and their producing bacteria in intestine.Methods:This was a case control study based on 15 PD patients and 15 healthy volunteers.Fecal microbiome was measured by PCR amplification and 16S r RNA gene Phylo Chip.Fecal indole and p-cresol were measured by HPLC-fluorescence detector.Total IS and p CS in serum,24h spent dialysate and 24h urine were measured by UPLC-MS/MS.The difference between PD patients and health controls was analyzed by student t-test or rank-sum test in gut microbiota,the relative abundance of indole-producing bacteria and p-cresol producing bacteria,fecal indole and p-cresol concentrations,and serum IS and p CS concentrations.The analysis of covariance was conducted with age and sex adjusted.Results:1.The bacteria richness(Chao index)of OTUs level of gut microbiota in PD patients was significantly lower than that of healthy controls(P=0.024).2.For the phylum-level fecal microbiota,the relative abundance of Firmicutes(P=0.010),Proteobacteria(P=0.001),Actinobacteria(P=0.037)and Fusobacteria(P=0.008)were higher in PD patients than in healthy controls,whereas the relative abundance of Bacteroidetes was lower in PD patients than in healthy controls(P(27)0.001).3.For the genus-level bacteria with total population in the top 40,the relative abundance of Blautia(P(27)0.001),Escherichia-Shigella(P(27)0.001),Ruminococcus gnavus group(P(27)0.001),Lachnoclostridium(P(27)0.001),Anaerostipes(P=0.001),norank Lachnospiraceae(P=0.005)and Tyzzerella?4(P(27)0.001)were significantly higher than in the healthy controls.However,the relative abundance of Prevotella?9(P=0.004)and Megamonas(P=0.001)were lower.4.The relative abundance of indole-producing bacteria Escherichia coli(P<0.001),Bacteroides fragilis(P<0.001),Klebsiella pneumonia(P=0.001),Enterococcus faecalis(P=0.001)and Pseudomonas protegens(P<0.001)were significantly higher in ESRD patients than in healthy controls.5.PD patients have higher population of p-cresol-producing bacteria than healthy controls,including unclassified Ruminococcus gnavus(P<0.001),Bacteroides fragilis(P<0.001),Clostridium symbiosum(P=0.002),Clostridium scindens(P=0.002),Clostridium innocuum(P<0.001)and Anaerostipes caccae(P=0.006).6.Fecal indole concentrations and p-cresol concentrations in PD patients were not statistically different from those of healthy controls,but the daily intake of protein(P(27)0.001),tryptophan(P=0.026),phenylalanine(P=0.002)and tyrosine(P=0.003)in the healthy controls were significantly higher than that of PD patients.7.The serum IS concentration was 47 times higher in PD patients than in healthy controls(P<0.001),and serum p CS concentration was 18 times higher than in healthy controls(P<0.001).Conclusions:Compared to healthy controls,PD patients have higher relative abundance of indole-producing bacteria and p-cresol-producing bacteria in intestine,higher serum IS and p CS concentrations,while lower 24h urine excretion and 24h dialysis removal of IS and p CS.Section 3Objective:With randomized cross-over study conducted,we aimed to determine whether inulin-type fructans(ITF)reduce the production of indole and p-cresol by altering their producing bacteria in patients with peritoneal dialysis.Methods:Patients receiving peritoneal dialysis for longer than 3 months without diabetes and not using antibiotics were recruited to a randomized,double-blind,placebo-controlled,crossover trial of ITF intervention over 36 weeks(12-week washout).The primary outcomes were gut microbiome,fecal indole and p-cresol,their producing bacteria and serum IS and p CS.The secondary outcomes were fecal p H,24h urine and dialysate removal of IS and p CS.Results:1.There was no significant difference in the daily nutrients intake,including energy,protein,carbohydrate,fat,tryptophan,tyrosine and phenylalanine,during the ITF intervention,wash-out and placebo intervention.2.ITF intervention had no treatment,time or treatment-time interaction effects for the phylum-level fecal microbiota and the genus-level bacteria with total population in the top 40.3.The indole-producing bacteria Bacteroides thetaiotaomicron responded differently over time between the two treatments,with significant treatment and time interaction effect(P=0.047).4.There was a trend of treatment by time effect for fecal indole concentrations(P=0.052),with quantitatively reduction in the ITF treatment whilst increase in the control.The difference in the changes between the two treatments was significant(-10.07(?)7.48 vs.+13.35(?)7.66,P=0.040)?g/g.5.ITF intervention significantly reduced intestinal p H(P=0.010),which was positively correlated with fecal indoles concentration(rrm=0.51(95%CI:0.25,0.71),P=0.0003)and p-cresol concentration(rrm=0.35(95%CI:0.06,0.59),P=0.018).6.ITF intervention had no treatment,time or treatment-time interaction effects for serum IS and p CS concentrations and their removal.Conclusions:Our results suggested that inulin-type fructans restricted the increase of gut microbiome-generated indole in patients with peritoneal dialysis.