Effect Of Protein-bound Uremic Toxins On Prognosis Of Peritoneal Dialysis Patients: A Cohort Study

BackgroundWith decreased renal function,chronic kidney disease(CKD)patients usually accompany by the accumulation of various of metabolites.Some of these affecting the biological functions,we call these performances uremic symptoms and related substances uremic toxins.The European uremic toxin Work Group(Eutox)has established a database,with a total of 146 uremic toxins by the year 2012.To CKD patients,dyfunctional kidneys can not clear these retention of uremic toxins,so renal replacement therapies-kidney transplantation,hemodialysis and peritoneal dialysis-are needed to play a role in scavenging.So far,many researchers have been working to identify more uremic toxins and try to confirm the mechanisms.Based on their molecules size,solubleness,there are three classification:(1)small water-soluble compounds,MW<500D,such as creatinine,urea nitrogen;(2)protein bound uremic toxins(PBUT),MW<500D;(3)middle molecules.including some polypeptides.According to places of toxins generated,we can divided them into gastro-intestinal toxins and non-gastro-intestinal ones.PBUT is a group of small molecular toxins which usually tightly combine with serum protein,for instance,indoxyl sulfate(IS)and p-cresol sulfate(PCS).Moreover,most of PBUT come from gastro-intestine.There are 10 major categories,in a total of 32 species.More and more evidences showed that PBUT were harmful to heart,brain,kidney and other organs.Through reading papers and using ultra high-performance liquid chromatography electrospray ionization tandem mass spectrometry(UHPLC-ESI-MS/MS)technology,we would like to detect the serum concentration of IS,Hippuric acid(HA),p-hydroxyhippuricacid(p-OHHA),Kynurenicacid(KYNA)and3-carboxy-4-methyl-5-propyl-2-furanpropionic acid(CMPF),confirm concentration of these PBUTs are abnormally high than healthy volunteers.In addition,we discuss the related factors which may affect the PBUT concentrations.Lastly,by following-up,we investigate whether PBUT could affect the long-term prognosis of PD patients.ObjectiveThe study is divided into three parts,the main purpose is:1.To confirm he serum concentration difference of PBUT(IS,HA,p-OH HA,KYNA,CMPF)in three groups of healthy volunteers,CKD4-5 patients and PD patients;2.To identify the related factors of serum protein binding toxins(IS,HA,p-OH HA,KYNA,CMPF)in patients who are undergoing peritoneal dialysis;3.To explore the effect of PBUT on the long-term prognosis of PD patients.Through these series of studies,we confirm that renal insufficiency leads to the accumulation of PBUT.And find out the related factors.Finally,to verify the effect of PBUT on the long-term prognosis of PD patients.MethodsPART 1:Confirm the serum concentration difference of PBUT in three groups of healthy volunteers,CKD4-5 patients and PD patientsWe recruited PD patients(n=50)who met the inclusion criteria in Pertional Dialysis center,Shanghai Changzheng Hospital,Naval Military Medical University,from January to December 2016.There were 25 men as well as 25 women.We excluded patients who were(1)older than 75 or younger than 18 year-old;(2)undergoing peritoneal dialysis less than 1 month;(3)with hemodialysis;(4)peritonitis or other infectious diseases in 1 month;(5)at the active stage of autoimmune disease;(6)with malignant tumors;(7)with cardiovascular events in 1 month.Meanwhile,we collected stable CKD patients(n=50)who went to nephrology department in Changzheng Hospital.And 50 healthy volunteers were recruited into this research.All subjects signed the consents.This research was approved by Biomedical Research Ethics Committee ofChangzheng Hospital.The number was 2014SL027.We chose UHPLC-ESI-MS/MS technology to detected the serum concentrations of PBUT and analysed the difference between these three subjects groups.Measurement data were shown as mean±standard or median with quartile.The differences were analyzed by LSD-t test and nemenyi test with software SPSS 21.0 version.Andwe chose the Kruskal-Wallis H test to verify the difference among three groups.Categorical datum,described by frequency and were analyzed by Fisher's exact method.P<0.05 determined statistical significance.PART 2:Identify the related factors of serum protein binding toxins in patients who are undergoing peritoneal dialysisWe recruited PD patients who met the inclusion criteria in Pertional Dialysis center,Shanghai Changzheng Hospital,from January 2016 to July 2017.We excluded patients who were(1)older than 75 or younger than 18 year-old;(2)undergoing peritoneal dialysis less than 1 month;(3)with hemodialysis;(4)peritonitis or other infectious diseases in 1month;(5)at the active stage of autoimmune disease;(6)suckling period or pregnant;(7)with malignant tumors;(8)with cardiovascular events in 1 month.All subjects signed the consents.We collected clinical datum and blood test results.UHPLC-ESI-MS/MS technology was used to detect the serum concentrations of IS,HA,p-OH HA,KYNA,CMPF.We tried to analyze correlation of the PBUT concentrations with sex,residual renal function,small soluble toxins,the adequacy of peritoneal dialysis,peritoneal equilibration test results,nutritional status and inflammation marker such as CRP.Measurement datum were shown as mean±standard or median with quartile.Spearman model and multiple linear regression analysis were used to analyze the correlation,if the datum were not normal distributed.P<0.05 determined a statistical significance.PART 3:Explore the effect of PBUT on the long-term prognosis of PD patientsAccording to the inclusion and exclusion criteria in the second part,242 PD patients were recruited.Collect serum samples and detect PBUT concentration.The patients were divided into two groups of high concentration and low one.We recorded the long-term clinicaloutcomes-cardio-cerebrovasculareventsandperitonitis.Theendof follow-up was February 10,2018,and we recorded this period of each patient.We analyzed datum by SPSS 21.0,and used the Kaplan Meier test.The difference between two groups analyzed by Log-rank test.P values less than 0.05 confirmed that the differences had statistical significance.ResultsPART 1:Confirm the serum concentration difference of PBUT in three groups of healthy volunteers,CKD4-5 patients and PD patientsAverage age of healthy volunteers was 54.38±8.95 year-old,58.22±10.30 year-old of CKD patients,and 55.14±13.09 year-old.These datum were not statistical significant.The serum concentrations of PBUT in healthy volunteers were:IS 170.88(79.17,398.06)ng/ml,HA101.19(52.99,266.68)ng/ml,p-OHHA5.94(0.00,36.72)ng/ml,KYNA1.15(0.00,4.34)ng/ml,CMPF 166.05(119.53,279.37)ng/ml.And in CKD patients:IS7922.58(2650.39,14589.39)ng/ml,HA1163.51(363.17,3200.91)ng/ml,p-OHHA222.47(100.43,714.56)ng/ml,KYNA75.04(36.48,180.32)ng/ml,CMPF1789.04(803.39,3557.57)ng/ml.RegardingPDpatients:IS26554.56(7282.30,55338.06)ng/ml,HA 4203.98(1350.10,17691.15)ng/ml,p-OH HA1730.12(478.52,3695.43)ng/ml,KYNA252.16(134.12,444.34)ng/ml,CMPF730.14(390.81,1448.12)ng/ml.Comparing serum concentration of healthy volunteers with CKD patients and PD patients respectively,the results were statistical significant.While,analyzing the datum between CKD patients and PD patients,the differences of p-OH HA,KYNA,HA,IS,CMPF were statistical significant.PART 2:Identify the related factors of serum protein binding toxins in patients who are undergoing peritoneal dialysisA total of 122 patients were enrolled in this study.Average age was 48.61±15.24year-old and 42 cases were female(34.4%).average duration of PD was 18.53±26.99months.The serum concentrations of PBUTs described that IS 21371.31(10875.88,46991.80)ng/mL,HA 4049.45(1350.10,16299.32)ng/mL,p-OH HA was 1607.74(657.72,3319.72)ng/mL,KYNA257.41(137.74,466.40)ng/mL,CMPF845.30(376.78,1588.37)ng/mL.There was a significantly positive correlation between the serum concentrations of IS with duration,peritoneal Kt/V,peritoneal Ccr,BUN,Scr,P and Ca×P,whereas negatively correlation between total Kt/V,residual renal Kt/V,total Ccr,residual renal Ccr,residual renal eGFR,AST,Na+and HDL.The serum concentration of HA had positive correlations with duration,peritoneal Kt/V,peritoneal Ccr,BUN,Scr,K+,P and Ca×P,but a negative correlation with total Kt/V,total Kt/V,total Ccr,residual renal Ccr,residual renal eGFR,Hb,ALT,AST and Na~+.There was a significantly positive correlation between the serum concentrations of p-OH HA and duration,peritoneal Kt/V,peritoneal Ccr,BUN,Scr,P,Ca×P,while,negatively related to total Kt/V,residual renal Kt/V,total Ccr,residual renal Ccr,residual renal eGFR,AST,Na+and HDL.Serum concentration of KYNA was positively related with duration,peritoneal Kt/V,peritoneal Ccr,BUN,Scr,P,Ca×P,and negatively related with age,total Kt/V,residual renal Kt/V,total Ccr,residual renal Ccr,residual renal eGFR,ALB,AST and TC.The last PBUT was CMPF,which was positively related to ALB,BUN,K,P and Ca×P,negatively with Na+.No significant correlation was shown between serum concentrations of PBUTs with WBC,PLT,CRP,Glu,UA,Ca,PTH and TG.PART 3:Explore the effect of PBUT on the long-term prognosis of PD patientsTheaveragefollow-upwas450.31±272.22days.ForKYNA,set cardio-cerebrovascular events and peritonitis as endpoint events,the difference between high and low concentration group was of no significance(P=0.100).What's more,regarding to cardio-cerebrovascular events,the datum of two groups were insignificant different(P=0.059).While for peritonitis,these two group difference was not statistically significant(P=0.817).In terms of HA,comparing the difference between high and low concentration group according to results of cardio-cerebrovascular events and peritonitis,the resultswasof no statistically significance(P=0.268).The outcomes of cardio-cerebrovascular events and peritonitis apart were insignificant(P=0.345 and P=0.828,respectively).In terms of other PBUTs(IS,p-OH HA,CMPF),there was no significance between two groups.Conclusions1.The serum PBUT Concentrations in patients with renal dysfunction were significantly higher than that of healthy volunteers.At the same time,there were different of PBUT concentrations between PD patients and CKD patients without dialysis.And the peritoneal dialysis was considered to have effect on clearance of PBUT.2.Serum concentrations of IS,HA,p-OH HA,and KYNA were positively with small soluble molecules(BUN,Scr)and phosphorus index(P,Ca×P),while negatively with residual renal function(Kt/V?Ccr?eGFR),total Kt/V and total Ccr.PBUTs had no significance correlations with WBC,PLT,CRP,Ca and PTH.However,CMPF were not related to peritoneal function,residual function.This results suggested that PBUTs mainly relied on clearance of residual function,and peritoneal function had supplementary clearance.Therefore,protection of residual renal function is conducive to the removal of serum PBUTs in PD patients.3.The PBUT of IS,HA,p-OH HA,KYNA and CMPF exert no influence on PD patients prognosis.