The Effect Of Inflammation On Outcome Of Peritoneal Dialysis Patients And The Primary Observation Of Potential Detection And Intervention

BackgroundPeritoneal dialysis(PD)is one of the major renal replacement therapy of end stage renal disease patients.Peritoneal dialysis associated peritonitis(PDAP)is the severe complication of PD therapy.Recently,the newly defined short-term adverse outcome of PDAP is recognized as direct threat to PD patients’ survival and technical survival,however,very few domestic studies focus on it.Intranasal mupirocin could reduce nasal carriage and benefit PD catheter associated infection,but it is not widely used as antibiotic resistance,while,no new intervention is available.The pathological research of the effect of PDAP on the structure and function of peritoneum is restricted by peritoneal biopsy,exosomes are membranous vesicles of endocytic origin that are released by various types of living cells,therefore with the advantage of non-invasive biopsy.however,the research on peritoneal dialysate is almost absent.Inflammation play a critical role on cell function and fibrogenesis,yet the mechanisms remain poorly defined.The glycolysis programs has been detected in fibroblasts activation and kidney interstitial fibrosis,whether it works on mesothelial transition is unknown.This study retrospectively analyzed the short-term outcome of PDAP and risk factors for adverse outcome,and to observe whether nasal culture combined saline wash works for PDAP incidence rate.We established dialysate exosomes extraction method and use it to detect inflammation state of PD patients.Finally,we observed glycolysis level in the mesothelial transition in vitro.Objectives1.To observe clinical characteristics and risk factors for PDAP,and to analyze its effect on PD patients’ survival and technical survival;2.To analyze PDAP short-term adverse outcome and the predictors;to find out the risk factors for high peritonitis rate and its long-term survival and technical survival;3.To establish dialysate exosomes extraction methods,to detect inflammation state of PD patients and finally primarily observe glycolysis program in IL1β induced mesothelial transition.MethodsPart Ⅰ.Risk Factors and Outcome Analysis of PDAPAs a retrospective study,adult PD patients in Peking Union Medical College Hospital between Jan.1,2004 and Mar.31,2017 were enrolled.Baseline information including demographic information,clinical comorbid disease burden,laboratory examination and PD associated parameters were collected.For PDAP patients,clinical data at 0-3month before first peritonitis episode were collected.1.All the patients were followed until Mar.31.2018.Primary outcome were death and secondary outcome is technical failure.The survival rate was calculated by Kaplan-Meier method and the risk factors of outcome were analyzed by the Cox’s regression model.2.PDAP patients were followed up to 4 weeks after the onset.These patients were divided into 2 groups according to the short-term clinical outcome,namely poor outcome group and good outcome group.Characteristics at baseline and before peritonitis were compared.Risk factors associated with short-term outcomes were analyzed by Logistic regression model.3.Those PDAP patients who survived in first peritonitis episode and maintain PD treatment were divided into 2 groups according to peritonitis rate during follow-up period,namely high frequency PDAP patients(PDAP rate higher than 0.5 patients year)and low PDAP patients.To observe the effect of nasal carriage detection and intervention on the incidence rate of PDAP,and the long-term survival and technical survival were analy sized in high frequency PDAP patients.Part Ⅱ.The evaluation of inflammation state of PD patients by dialysate exosomes1.Dialysate exosome extraction by ultracentrifugationWith the reference of cavity effusion and urine exosome extraction protocol,we tried to extract dialysate exosomes with ultracentrifugation.Using Negative stain and transmission electron microscopy(TEM)for morphology identification.Particle concentration and size distribution was measured using Nanoparticle Tracking Analysis(NTA).Western Blot analysis was performed to detect exosome protein marker TSG101 and CD63.2.Primary observation of peritoneal inflammation in peritoneal dialysis patientsPeritoneal macrophage cells were isolated from dialysate effluent with centrifugation and identified with immunofluorescence detection of CD68.Detecting NLRP3,Caspase1 and IL1β on peritoneal macrophage.Prospectively collected peritoneal dialysate effluent of PDAP and normal PD patients(age and PD duration matched)for IL1β detection using ELISA kit and exosomes extraction,followed by NLRP3,Caspase1,IL1β and glycolysis enzyme detection using Western Blot.The protein levels were compared between the 2group,as well as clinical characteristics.3.The primary observation study of Glycolysis program in human peritoneal mesothelial transition induced by IL1βHuman peritoneal mesothelial cell line(HMsSV5)were cultured with 10%fetal serum containing DMEM/F12 medium,the cultured cells were identified by morphology and immunofluorescence detection of pan-Cytokeratin.Using CCK-8 kit and LDH cytotoxicity assay kit to find the proper concentration of IL1β.Detect lactic acid and ATP level by assay kit.Immunofluorescence and Western Blot method to analyze glycolysis enzyme,alpha-SMA,vimentin,Claudinl and fibronectin protein.Results1.Risk factors for PDAP and its influence on long-term technical survival of PD patientsThere were 162 PDAP patients among 533 PD patients,and the incidence rate of 1/77 patient months.The average age was 58.5±15.7 years old at baseline.The main primary diagnosis was diabetic nephropathy(34.0%)and chronic glomerular nephritis(16.0%).The average age at first PDAP onset was 61.0±15.9 years old,with the median PD duration of 20.5 months.The culture positive percent was 56.8%.Using Tenckhoff catheter(OR=2.428,95%CI 1.162-5.074,P=0.018)and comofbidity of atherosclerotic vascular disease at baseline(OR= 1.827,95%CI 1.138-2.934,P=0.013)were the independent risk factors for PDAP in Logistic regression model.The median survival time was 67.0(60.1-73.9)months.PDAP patients’ technical survival was significantly lower than non-PDAP patients by Kaplan-Meier survival analysis and Log Rank test(χ2=10.753,P=0.003),no difference was observed in patient survival.Cox regression model showed that PDAP(HR=2.738,95%CI 1.552-4.829,P=0.001),high BMI(HR=1.089,95%CI 1.013-1.172,P=0.022)and non-elderly patients were the risk factors for technical failure.2.The effect of micro-inflammation state on short-term adverse outcome of PDAPAmong 162 PDAP patients,34.0%patients experienced adverse short-term outcome,and the first two were transfer to hemodialysis(30.9%)and death(29.1%),the main etiology was gram negative bacteria.At baseline,the proportion of clinical atherosclerotic vascular disease was significantly higher in adverse outcome group(49.1%Vs.31.8%,P=0.031).Significantly higher level of high-sensitivity C-reactive protein(hsCRP)(9.3mg/dl Vs.3.6 mg/dl,P=0.004)were observed in adverse outcome group at 0-3month before peritonitis.in this group,the proportion of polymicrobial PDAP was significantly higher(15.6%Vs.3.3%,P=0.039).Logistic regression model showed that PD duration longer than 1 year(OR=2.324,95%CI 1.080-5.003,P=0.031),high hsCRP before PDAP(OR=1.027,95%CI 1.002-1.052,P=0.031)could independently predict short-term adverse outcome of PDAP.3.Nasal carriage and clearance improve recurrence peritonitis and outcomeAmong 129 PDAP patients who survived in the first episode of PDAP,50 patients(38.8%)were identified as high frequency PDAP patients.The nasal carriage detection and intervention percentage was significantly lower than low frequency PDAP patients.The baseline information was almost comparable between these two groups except for higher serum uric acid and lower CA125.Till the end of follow-up,the 4-hour D/P Creatinine and proportion of high transport peritoneum function were all higher in high frequency PDAP patients,together with a significantly lower long-term survival and technical survival(P<0.001).Finally,Logistic regression model showed that high basal serum uric acid could independently predict high frequency PDAP(OR=1.040,95%CI 1.008-1.073,P=0.014),while nasal carriage detection and intervention could efficiently decrease PDAP incidence rate(OR=0.230,95%CI 0.086-0.617,P=0.004).4.The evaluation of inflammation state of PD patients by dialysate exosomesWe established ultracentrifugation protocol of dialysate exosomes.We observed classical cup-shaped membranous microvesicles by TEM,with an average diameter of 136.0±61.1nm by NTA,at a concentration of 4.3×1010/ml.Western Blot analysis could see exosome protein markers,TSG101 and CD63.In peritoneal macrophage cells(CD68 positive)we found that some of them were also NLRP3,Caspase1,IL1β positive under immunofluorescence detection.We also detected NLRP3,Caspase1,IL1β protein expression in dialysate exosomes,and the NLRP3 and Caspasel protein of PDAP patients’ dialysate was significant higher than normal control PD patients,accompanied by a significantly higher level of glycolysis enzyme.5.The primary observation study of Glycolysis program in human peritoneal mesothelial transition induced by IL1βWe chose lng/ml IL1β to co-culture with HMsSV5.Compared with the blank control group,the level of lactic acid decreased and ATP increased in IL1β to co-culture group.Immunofluorescence and Western Blot results both showed an increase of Hexokinase Ⅱ、LDHA in this group,accompanied by significantly higher level of alpha-SM,vimentin and fibronectin protein,and lower Claudinl protein.Conclusion1.PDAP was the independent risk factor for PD patients’ long-term technical survival,however,it didn’t affect patients’survival.Using Tenckhoff catheter and comofbidity of atherosclerotic vascular disease at baseline were the independent risk factor for PDAP.2.PD duration longer than 1 year and high hsCRP before peritonitis onset could independently predict short-term adverse outcome of PDAP.High frequency PDAP patients’ long-term survival and technical survival were significantly lower than low frequency PDAP patients,accompanied by high proportion of high transport.nasal carriage detection and intervention could efficiently decrease PDAP incidence rate.3.Dialysate exosomes could be extracted with ultracentrifugation.Peritoneal macrophage cells could express NLRP3,Caspasel and IL1β,they could also be detected in dialysate exosomes.The IL1β level was significantly higher in PDAP patients,with a higher level of NLRP3 and Caspasel proteins in PDAP dialysate exosome.Meanwhile,Glycolysis enzyme was significantly higher in PDAP dialysate exosomes and glycolysis may play a role in mesothelial transition induced by IL1β.