The Accumulation Of Gut-Derived Toxin Trimethylamine N-oxide In Peritoneal Dialysis Patients And The Regulatory Effect Of Prebiotics

Trimethylamine N-oxide（TMAO）is a small molecule uremic toxin.Dietary choline,carnitine,betaine,etc.,are catabolized by gut microbiota to produce trimethylamine（TMA）.TMA is further transformed into TMAO by hepatic monooxygenase,and the generated TMAO is mainly excreted by the kidney.A high level of TMAO can aggravate kidney injury and increase the risk of cardiovascular events.Peritoneal dialysis（PD）patients are prone to intestinal flora disorders due to disease,drug use,and diet,coupled with severe renal impairment and low dialysis clearance efficiency,all of which leads to a high level of TMAO in patients with PD.This phenomenon is highly consistent with the substantially increased cardiovascular mortality among PD patients.Therefore,exploring the pattern and causes of TMAO changes in PD patients is of research and application value.It also contributes to elucidating further the relationship between TMAO and cardiovascular diseases in population epidemiology.Based on the established cohort of peritoneal dialysis,this paper explores the change in TMAO level in patients with the dialysis process.It further investigates the causes of TMAO accumulation in PD patients according to "dietary ingredient intake-intestinal production-renal and dialysis clearance." Finally,it explores the possibility of regulating TMAO level by modulating gut microbiota through inulin-type fructans.Part 1.The dynamics of TMAO level in peritoneal dialysis patients and its association with cardiovascular disease risk factorsObjective: To investigate the change of TMAO level in peritoneal dialysis patients with dialysis treatment and the influencing factors and to explore the association between plasma TMAO and cardiovascular disease risk factors such as blood lipids.Method: The subjects were from the "A Single-Center Cohort Study Based on Continuous Ambulatory Peritoneal Dialysis patients in Central China".A total of 202 adult PD patients who received dialysis treatment for more than 3 months were enrolled,and fasting venous blood,24-hour urine,and 24-hour dialysate were collected every 3 to 6 months.Basic demographic information was collected,and TMAO concentrations in plasma,urine,and dialysate were measured by ultrahigh performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）.An automatic biochemical analyzer measured biochemical indicators such as albumin,creatinine,and urea nitrogen.Smoothing curve fitting and a generalized additive model were used to analyze the change of plasma TMAO and its clearance rate in PD patients with dialysis treatment.The association between plasma TMAO and cardiovascular disease risk factors such as blood lipids was investigated by multiple linear regression and a restricted cubic spline model using the latest follow-up data.Results: The mean follow-up time for this cohort of peritoneal dialysis patients was 13.6 months and the man number of visits was 3.Of the 202 PD patients,40.1% were male.The median age of patients at baseline was 43.88 years.The mean BMI was 21.17 kg/m2.And the median dialysis duration was 8.78 months.A generalized additive model showed that plasma TMAO level gradually increased（P < 0.001）,and its clearance rate gradually decreased（P = 0.023）in PD patients as treatment progressed.Stratified by sex,age,BMI,albumin,Kt/V,Ccr,urine volume and residual GFR,female group（P = 0.004）,low and intermediate age groups（P < 0.001 and P = 0.035）,low and normal BMI groups（P = 0.012 and P < 0.001）,intermediate and high albumin group（P = 0.014 and P = 0.002）and Kt/V more than 1.7 group（P = 0.001）still showed an increase of plasma TMAO with disease progression.Multiple linear regression showed that for every 1 mg/L increase in plasma TMAO concentration,blood phosphorus concentration increased by 0.036 mmol/L（P = 0.014）and HDL-C concentration decreased by 0.026 mmol/L（P = 0.020）.The restricted cubic spline results suggested a monotonically increasing dose-response relationship between plasma TMAO and blood phosphorus（Poverall = 0.001,Pnonlinearity = 0.817）and a monotonically decreasing dose-response relationship with HDL-C（Poverall = 0.012,Pnonlinearity = 0.328）.Conclusion: As the treatment progressed,plasma TMAO concentrations in PD patients continued to increase,and this trend was affected by gender,age,BMI,albumin and dialysis adequacy.The clearance rate of TMAO gradually decreased.Plasma TMAO was positively correlated with blood phosphorus and negatively correlated with HDL-C,suggesting that those two may play an important role in TMAO-causing cardiovascular injury.Part 2.Association of gut microbiota with plasma TMAO level in peritoneal dialysis patientsObjective: To compare the differences in the gut microbiota,fecal TMA,plasma TMAO,and its clearance rate between PD patients and healthy control,and to investigate the possible causes of TMAO accumulation in PD patients.Method: In this part,11 PD patients and 11 healthy controls who had not received antibiotics and gut regulators within the past month were recruited,matched 1:1 for age（± 5 years）and sex.A dietary investigation was performed using the three-day dietary weighing and recording method,and fasting venous blood,24-hour urine,24-hour dialysate,and stool samples were collected.UPLC-MS/MS was used to detect TMAO level in plasma,urine,dialysate,and TMA in feces.The automated biochemical analyzers were used to detect biochemical indexes such as albumin.Using 16 S r RNA technology detects the composition and structure of the gut microbiota.Wilcoxon test was used to compare the differences between groups,and Spearman rank correlation was used to analyze the correlation between gut microbiota and plasma TMAO and fecal TMA.Results: The median levels of plasma TMAO and fecal TMA in PD patients were 2.41（1.74-2.90）mg/L and 3.19（2.12-5.73）μg/g,respectively,which were higher than their levels in the control group,0.14（0.07-0.23）mg/L（P < 0.001）and 2.01（1.34-2.56）μg/g（P = 0.013）.The results of 16 S r RNA sequencing showed that there was a trend toward a decrease in the relative abundance of the Bacteroidetes（P = 0.056）and an increase in the relative abundance of the Proteobacteria（P = 0.056）in PD patients.The relative abundance of Roseburia（P = 0.018）,Ruminococcus（P = 0.036）,etc decreased in PD patients,while the relative abundance of Escherichia-Shigella（P = 0.018）,Pseudomona（P = 0.018）,etc.increased.Plasma TMAO level was negatively correlated with Roseburia（r =-0.711,P < 0.001）and Ruminococcus（r =-0.718,P < 0.001）and positively correlated with Escherichia-Shigella（r = 0.719,P < 0.001）and Pseudomona（r = 0.771,P < 0.001）.The abundance of TMAO reductase（EC 1.7.2.3）was increased in PD patients compared to healthy controls（P = 0.033）.Conclusion: Plasma TMAO level was significantly higher in PD patients compared to healthy controls.It was consistent with the results of changes in their fecal TMAO level,intestinal TMAO-producing bacteria,and related enzymes,suggesting that gut microbiota may play a vital role in plasma TMAO accumulation in PD patients.Part 3.The Effect of inulin-type fructans intervention on plasma TMAO in peritoneal dialysis patientsObjective: To investigate whether inulin-type fructans intervention can regulate plasma TMAO level by modulating gut microbiota in PD patients.Method: This part adopted a 2×2 crossover trial.PD patients who had not received antibiotics and gut regulators within the past month were randomly allocated to two groups,A and B.The intervention sequence of group A was to intake prebiotics（inulin-type fructans,10 g/d）in the first phase and take placebo（maltodextrin,10 g/d）in the second phase;the intervention sequence in group B was reversed.Each phase lasted three months,interspersed with a 3-month washout period.Blood,24-hour urine,24-hour dialysate,and stool samples were collected from subjects at the beginning and end of each intervention phase,and dietary surveys were performed at each stage using the three-day dietary weighing and recording method.UPLC-MS/MS was used to detect TMAO in plasma,24-hour urine,24-hour dialysate and stool TMA,and Gas Chromatography-Mass Spectrometer was used to detect fecal short-chain fatty acids.DNA from stool samples was extracted for metagenomics sequencing.Mixed linear models were used to compare the effects of inulintype fructans intervention on the structure and function of gut microbiota,fecal TMA and short-chain fatty acid levels,and plasma TMAO levels.Results: Twenty-two individuals completed all trial components and provided completed biological samples for each node.Among them,12 subjects were allocated to group A and 10 subjects were allocated to group B.There was no significant change in plasma TMAO level after inulin-type fructans intervention（P = 0.111）,with a median change of-2.44（-0.89-0.58）mg/L.There was no significant change in plasma TMAO concentrations after the placebo intervention（P = 0.844）,with a median change of-0.04（-0.80-0.70）mg/L.The between-group difference in change values（Δ inulin-type fructans vs.Δ placebo）was not statistically significant（P = 0.681）.Fecal TMA levels were 3.30（2.20-5.37）and 4.22（2.48-5.58）μg/g before and after the inulin-type fructans intervention,respectively,and 2.91（2.31-4.53）and 3.84（2.46-5.23）μg/g before and after the placebo intervention,respectively,with no statistically significant difference in the change values between the two groups（Δ inulin-type fructans vs.Δ placebo）（P = 0.510）.Fecal acetate and butyric acid levels were significantly increased in PD patients during the inulin-type fructans intervention,from 1.49 ± 0.69 to 1.67 ± 0.84 μg/mg（P = 0.023）and 0.59 ± 0.32 to 0.83 ± 0.42 μg/mg（P = 0.013）,respectively.Metagenomics sequencing results showed that inulin-type fructans intervention reduced the relative abundance of the Bilophila（P = 0.032）and increased the relative abundance of Anaerostipes（P = 0.026）.At the species level,inulin-type fructans intervention reduced the relative abundance of TMA-producing bacteria Bilophila_wadsworthensis（P = 0.036）,Dorea_formicigenerans（P = 0.035）,and Bilophila_sp.₄₁₃0（P = 0.021）.The inulin-type fructans intervention failed to significantly reduce the four intestinal TMA-producing enzymes such as choline-TMA lyase compared with placebo,all with P values greater than 0.05.Conclusion: Although this study did not observe a significant effect of inulin-type fructans intervention on fecal TMA and plasma TMAO levels,the inulin-type fructans intervention improved the composition and structure of the intestinal flora of PD patients and reduced the abundance of various TMA-producing bacteria.Modulating TMAO levels in vivo through gut microbiota is a pathway worth further exploration.