Associations Of Mitochondrial DNA Content In Peritoneal Dialysis Effluentwith Peritoneal Solute Transport Rate And Peritonitis Outcome In Peritoneal Dialysis

PartⅠ The Effects of Peritoneal Effluent Mitochondrial DNA onIntraperitoneal Inflammation and Peritoneal SoluteTransport Rate in Peritoneal DialysisObjectiveLocal chronic intraperitoneal inflammatory status commonly affects peritoneal dialysis(PD)patients.Mitochondrial DNA(mtDNA)released into extracellular subsequent to cell injury and death can promote inflammation in patients and animal models.However,the effects of peritoneal effluent mtDNA on intraperitoneal inflammation and peritoneal solute transport rate(PSTR)in PD patients remain unclear.We aimed to examine the peritoneal effluent mtDNA and elucidate their relationship with intraperitoneal inflammation and PSTR.MethodsWe select theincident patients who began PD therapy at the First Affiliated Hospital of Zhejiang University between January 1,2009,and December 30,2010.Peritoneal dialysis effluent was collected at the time of peritoneal equilibration test.The peritoneal effluent mtDNA was detected by quantitative real-time PCR assay,the concentrationsof dialysate IL-6,IL-17A,TNF-a and IFN-y were quantitated by ELISA assay.Theresults were compared with PSTR,patient survival and technique survival.Results:One hundred and eighty-nine patients were included in the study.The average age was 47.1 ± 13.5 years,55.6%of the patients were males.The median follow-up period was 41.9 months.The average PSTR was 0.66 ± 0.12,the median mtDNA level was 4325 copies/ul.The median concentrations of IL-6,IL-17A,TNF-a and IFN-y were 25.9,10.8,25.8 and 17.9 pg/ml,respectively.We found that peritoneal effluent mtDNA was significantly correlated with PSTR(r =0.461,P<0.001),IL-6(r = 0.568,P<0.001),TNF-α(r = 0.454,P<0.001)and IFN-y(r = 0.203,P = 0.005).After adjustment for multiple covariates,peritoneal effluent mtDNA was independently correlated with IL-6 and PSTR.Peritoneal effluent mtDNA was not associated with patient and technique survival.ConclusionWe found that theperitoneal effluent mtDNA level correlated with the degree of intraperitoneal inflammatory status in PD patients.Peritoneal effluent mtDNA was an independent determinant of PSTR but did not affect patient and technique survival.Part Ⅱ Associations of Elevated Peritoneal Effluent Mitochondrial DNA Levels with Clinical Outcome in Peritoneal Dialysis Related PeritonitisObjectivePeritonitis remains a leading complication of peritoneal dialysis(PD).Severe and prolonged peritonitis can lead to peritoneal membrane failure and peritonitis remains a major cause of patients discontinuing PD and switching to hemodialysis.Mitochondrial DNA(mtDNA)released into extracellular subsequent to cell injury and death can promote inflammation in patients and animal models.However,the role of peritoneal effluent mtDNAin peritonitis remains unclear.We aimed to examine the peritoneal effluent mtDNA and elucidate their relationship with the clinical outcome of peritonitis in PD patients.MethodWe performed a prospective observational cohort study of patients with peritonitis between June 1,2014,and September 30,2015.Peritoneal dialysis effluent was collected at day 1 and 3.The peritoneal effluent mtDNA and bacterial DNA(bDNA)was detected by quantitative real-time PCR assay,the concentrationsof dialysate IL-6 were quantitated by ELISA assay.Theresults were compared with dialysate cell counts,high-sensitivity C-reactive(hs-CRP),’and the outcomes of peritonitis.ResultsDuring the study period,ninety-four patients with peritonitis were included for analysis.The average age was 54.2 ± 12.7years,63.8%of the patients were males.The median duration of PD therapy was 24.9 months.83 patients had treatment success defined as complete resolution of peritonitis without the need for Tenckhoff catheter removal.Of the remaining 11 episodes(treatment failure),9 required Tenckhoff catheter removal and 2 had peritonitis-related death.Treatment failure group had a significantly higher level of peritoneal effluent mtDNA than treatment success group on both day 1 and day 3.Peritoneal effluent mtDNA was significantly correlated withDNA,IL-6,dialysate white blood cell counts and hs-CRP.Peritoneal effluent mtDNA had a higher area under the receiver-operating characteristic(ROC)curves than dialysate white blood cell counts and bDNA on both day 1 and day 3.Using a peritoneal effluent mtDNA level cut point ≥ 16300 copies/ul,the sensitivity was 72.7%and the specificity was 95.9%for the prediction of treatment failure.In multiple logistic regression analysis,peritoneal effluent mtDNA level cut point ≥ 16300 copies/ul and bDNA ≥ 1700 copies/ul were independent prognostic markers for treatment failure after adjustment for multiple covariates(mtDNA,OR = 29.363,P=0.01;bDNA,OR = 12.151,P = 0.037).A summary risk scoring(SRS)model was developed according to the result of multiple logistic regression analysis,SRS = 3.38*mtDNA(0 or 1)+ 2.497*bDNA(0 or 1).The area under the ROC curves of the predictive model was 0.887(95%CI 0.742-1.000,P<0.001),the sensitivity was 72.7%and the specificity was 1000%for the prediction of treatment failure.ConclusionWe found that theperitoneal effluent mtDNA level correlated with the degree of inflammatory status in patients with peritonitis.Increased mtDNA levels were associated with increased risk of treatment failure.Peritoneal effluent mtDNA and bDNA appears to be useful biomarkers that correlate with peritonitis outcome in PD patients.Part ⅢInvestigation of effect and mechanism of Mitochondrial DNA in acute peritonitis in vivoObjectiveLocal intraperitoneal inflammatory status commonly affects peritoneal dialysis(PD)patients.Chronicinflammatory statusand acute peritonitis can lead to peritoneal embrane failure and remain the major causes of patients discontinuing PD and switching to hemodialysis.Mitochondrial DNA(mtDNA)released into extracellular subsequent to cell injury and death can promote inflammation through TLR9 and NLRP3 inflammasome activation.We found that theperitoneal effluent mtDNA level correlated with the degree of intraperitoneal inflammatory status in chronic PD patients and acute peritonitis.Increased mtDNA levels were associated with increased risk of treatment failure.Using in vivo study,we aimed to investigate the activation of TLR9 and NLRP3 inflammasome and its inflammation induced by mtDNA in the mouse peritoneum and to explore their roles in the damage of peritoneum.MethodsThe C57BL/6 mouse were divided in to 7 groups:the normal saline group(Control group),low LPS group(2mg/kg),high LPS group(10mg/kg),low mtDNA group(0.2mg/kg),high mtDNA group(1mg/kg),low bDNA group(0.2mg/kg),high bDNA group(1mg/kg).The animals were euthanized at 6 and 24 hours after intraperitoneal injection.NS was ip injected,and peritoneal fluid,parietal peritoneumand omentum were collected.Leukocytes of peritoneal fluid were counted.IL-1β and IL-6 levels were examined by ELISA.The mRNA expression of TLR9 and NLRP3 were determined by RT-PCR.Peritoneum tissue was stained using HE and Masson.The mitochondrial oxidative stress in peritoneum was detected by incorporation of MitoSOX fluorescence.The morphologic alterations of peritoneum were detected by electron microscopy.ResultsLPS significantly increased mtDNA levels in the peritoneal cavity at 6h after intraperitoneal injection and decreased at 24h,mtDNA levels were significantly higher in high LPS group than the low LPS group.The leukocyte count,IL-1β and IL-6 levelswere significantly correlated with the level of mtDNA.After mtDNA injection,the expression of TLR9 and NLRP3 mRNA was activated by mtDNA in a dose-dependent manner.mtDNA caused an immune response similar to that induced by bDNA,the leukocyte count,IL-1β and IL-6 levelswere significantly increased at 6h and decreased at 24h.mtDNA can lead to the morphologic changes,mitochondrial injury and dysfunction of peritoneum.ConclusionmtDNA level in peritoneal fluid was significantly increased in LPS-induced acute peritonitis.mtDNA can activate TLR9 and NLRP3 and contribute to cytockine production and peritoneum injury during peritonitis.