Preliminary Study On The Influence Of Peritoneal Glucose Exposure On Peritoneal Function And Prognosis Of Peritoneal Dialysis Patients

BackgroundPeritoneal dialysis(PD)is one of the major renal replacement therapy modality in end-stage renal disease patients(ESRD).Peritoneal function is a determining factor for toxin clearance and ultrafiltration in PD.Peritoneal function has been found to be an independent hazard factor for technical survival and long-term survival.However,no descriptive study on peritoneal function time course has been reported domestically.Glucose is the most commonly used dialysate permeabilizer.Long-term exposure of hyperosmolar glucose affects survival by induces peritoneal fibrosis,angiogenesis,and increases peritoneal transport status.Dialysate concentration was prescribed based on peritoneal transport states,masking the causal relationship between peritoneal glucose exposure and peritoneal function evolution.Secondly,peritoneal glucose exposure may cause fluctuation in blood glucose.Glycemic fluctuations increase the risk of diabetic complications.However,very few studies focused on blood glucose fluctuations in PD patients.Macrophages are the dominant cells in the dialysate.M2 subtype has been known for promoting fibrosis and neoangiogensis in various disease models.Yet,peritoneal macrophage subtypes and its clinical relevance in PD patients remained unknown.In this study,we 1)retrospectively described the characteristics of glucose exposure and long-term peritoneal function in PD patients;2)studied the impact of glucose exposure and peritoneal function on long-term prognosis in PD;3)analyzed the characteristics of blood glucose fluctuations in PD patients using Continuous Glucose Monitoring System(CGMS),and explored the impact of glycemic fluctuations and peritoneal function;4)initially analyzed the relationship between different subtypes of macrophage in dialysate and its correlation with dialysis vintages,and observed mesothelial cell transdifferentiation.Purposes1.To retrospectively describe the characteristics of peritoneal function and glucose exposure,and their effects on prognosis in peritoneal dialysis patients.To study the relationship between glucose exposure and peritoneal function;2.To prospectively observe the characteristics of blood glucose fluctuations and explore its relationship with peritoneal function in peritoneal dialysis patients;3.To observe the effects of glucose exposure on peritoneal mesothelial transdifferentiation and macrophage subtypesMethodsPart I Preliminary study on the relationship between glucose exposure and peritoneal function in patients on peritoneal dialysisA retrospective cohort study was established to include adult patients with end-stage renal disease on peritoneal dialysis from January 1,2004 to June 1,2015 at the Peritoneal Dialysis Center of Peking Union Medical College Hospital.Common baseline data and laboratory indices were collected.Demographic information:Baseline demographic data,clinical complications such as atherosclerotic vascular disease,and the Cherlson Comorbidity Index;Laboratory Index:All HbAlc and monthly FBG data from June 1st,2008 to December 1st,2017,biochemical indicators such as baseline blood routine,liver and kidney function,electrolytes,inflammatory markers such as hsCRP or NLR,ALB,UA,SI and other nutritional and metabolic indicators.Peritoneal dialysis-related indicators:raw data of patient peritoneal equilibration test(PET)and Cr 4h D/P,Glu 4h D/D0,Urea 4h D/P,Ultrafiltration(UF),Garred method for calculating mass transfer area coefficient(MTAC).4.25%peritoneal balance test additionally calculates Dip Na.Adequacy of dialysis,peritoneal transport status,standard protein breakdown rate,occurrence of peritoneal-dialysis related peritonitis,and its clinical features.Peritoneal Prescription:Patient’s monthly outpatient follow-up date,prescription(dialysis mode,dialysate and volume of each concentration,diarrhea,low calcium/high calcium,monthly median ultrafiltration,and urine volume).Annual glucose exposure intensity was calculated accordingly.Part Ⅱ Characteristics of glycemic fluctuation in patients undergoing peritoneal dialysis and its relationship with peritoneal functionCGMS was used to monitor the changes of blood glucose at 72h before and after peritoneal dialysis in peritoneal dialysis patients(n=27),and based on age,sex,and 72-hour mean blood glucose matched with 20 patients with normal renal function,collected basic clinical information and compared peritoneal dialysis with diabetes and kidneys.Characteristics of blood glucose fluctuations in patients with normal and non-diabetic peritoneal dialysis and calculation of related blood glucose fluctuations(standard deviation for 72 hours of continuous blood glucose,blood glucose>7.8 mmoL/L and>11.1 mmol/L for 72 hours,blood glucose>7.8 mmoL/L And>11.1 mmol/L area under the curve,average intra-day range,average blood glucose volatility,intraday glucose standard deviation,daytime blood glucose mean deviation,daytime blood glucose mean absolute difference,and mean post-dialysis blood glucose increase),and According to the standard peritoneal balance experiment conducted during the monitoring period,the standard PET test glucose peritoneal absorption and peritoneal transport function during patient monitoring were calculated,and the relationship between the blood glucose fluctuation index and peritoneal function and glucose absorption was preliminary analyzed.Part III Preliminary Study on the Relationship between Macrophage Subtypes and Clinical Characteristics in Peritoneal DialysatePatients with new peritoneal dialysis in our center(after two weeks of catheterization)and long-term dialysis patients with regular dialysis for more than 3 months(3m-5y,greater than 5 years)were selected to collect overnight peritoneal dialysis fluid.Peritoneal peritoneal cells were extracted by centrifugation and M1(CD68+TLR4+ or CD68+CD86+)macrophages,M2a(CD68+CD206+),and M2c(CD68+CD163+)subtype macrophages were identified by immunofluorescence.Mesothelial cells(panCK+);T,B lymphocy-tes(CD3+,CD4+,CD19+),and mesothelial cells(Lin-1-E-Cadherin+)were identified by flow cytometry.The ratio of M2a(CD206+CD14+)and M2c(CD163+CD14+)and the ratio of activated macrophages(CD14+CD16+)were analyzed by flow cy-tometry,and the relationship between the ratio of the above cells and the age.peritoneal function,and age of the abdominal cavity was analyzed.At the same time,macrophages secreting pro-fibrogenic factors TGFβ1 and IL-1β were detected by immunofluorescence,and Western blotting was used to detect whether the primary cells had M2-differentiated IL-34 and the mesothelial transdifferentiation markers Vimentin,Claudin,Fibronectin.and E-Cadherin.Continuous culture of primary cells of peritoneal dialysis fluid was performed to observe the effects of acute and chronic glucose exposure on the continuous culture of mesothelial cells.ResultsPart I Preliminary study on the relationship between glucose exposure and peritoneal function in patients on peritoneal dialysis1.457 ESRD patients included in this study with a mean age of 58.4±16.0 years initiating peritoneal dialysis,of which 52.5%were male.Mean follow-up duration was 38(22,61)months.Mean annual glucose exposure intensity of the patients was 49.3±18.2 Kg,with a maximum of 141 Kg/year.2.With a combined criteria of Cr 4h D/P and Glu 4h D/D0,the baseline peritoneal function of patients in this center was high(HA 51.3%),high(H,19.5%),and low(LA,18.8%)and low transit(L,10.4%).The 4 h Cr D/P and 4 h Urea D/P classifications were in good agreement,but Twardowski’s Glu 4h D/DO and UF criteria significantly underestimated the peritoneal function classification.Hyperglycemia is a risk factor for peritoneal function classification in patients with Glu 4h D/DO underestimation.When the regression model suggested that blood glucose is equal to 15.0 mmol/L,the probability of Glu 4h D/DO underestimating peritoneal function was 65.8%,and the probability of underestimating more than 2 levels was 4.2%.3.Baseline Cr 4h D/P was a risk factor for long-term survival at 3 months of dialysis(HR 6.189,p=0.015,CI 1.415-27.069).For patients on peritoneal dialysis that had a surival time of more than 2 years,BMI,average glucose exposure intensity in the previous two years,and two-year mean Cr 4h D/P transport status were independent factors for long-term technical survival.4.The annual average of peritoneal transport function(high Cr 4h D/P.low Glu 4h D/DO and low UF)in CAPD patients was significantly positively correlated with annual glucose exposure.In patients on peritoneal dialysis who had a peritoneal dialysis rate of more than 5 years(N=76),6 indicators of peritoneal function transport showed reduced transport function year by year.Annual glucose exposure intensity can predict changes in Cr 4h D/P in patients with PD over the next 2 years(p=0.006.Spearman).There was no significant difference in peritoneal function between different glucose-exposed groups during the first two years of regular peritoneal dialysis.After the fifth year,the peritoneal transport function of the high-glucose exposed group was significantly higher than that of the low-glucose exposure group;unlike in patients without peritonitis,dialysis occurred two years before dialysis.Peritonitis,peritoneal transport function increased,and continued until the 5th and 6th years.Part Ⅱ Characteristics of glycemic fluctuation in patients undergoing peritoneal dialysis and its relationship with peritoneal function1.Diabetic patients with diabetes mellitus(DMPD)have greater fluctuations in blood glucose than non-diabetics.They are characterized by the lowest morning rise,two peaks for breakfast and lunch,followed by a continuous increase,and peaks throughout the day after supper until bedtime.Different from diabetic patients with normal renal function,blood glucose fluctuations.When the mean blood glucose(MG)level was equivalent,many blood glucose fluctuations(SDT,CV%,MODD,LAGE,MAGE)were significantly lower in patients with DMPD than those with normal renal function(p<0.05).2.Peritoneal function(4h Cr D/P value)and 72h mean blood glucose(r=0.41,p=0.042),and various blood glucose fluctuation indicators:SD(Spearman,p=0.042),MIFPE(r=0.39),p=0.044),MAGE(Spearman,p=0.016)was significantly positively correlated;while glucose uptake was significantly correlated with mean minimum blood glucose(MIN,p=0.032)and MAGE(p=0.032).3.The correlation between peritoneal transport function and mean blood glucose and blood glucose fluctuations is better in nondiabetic PD patients.Part III Preliminary Study on the Relationship between Macrophage Subtypes and Clinical Characteristics in Peritoneal Dialysate1.Peritoneal dialysis patients can be separated from the new peritoneal dialysis fluid mesothelial cells,lymphocytes(CD4 + T cells,B cells)and macrophages Ml,M2a,M2c and other cells;2.The proportion of M2a(CD206+/CD14+)macrophages in patients with dialysis age less than 3 months was significantly higher than that in long-term patients and was negatively correlated with dialysis vintage(p=0.008).The proportion of CD14+,including CD 163+/CD14+ and CD 16+/,was not significantly associated with dialysis vintage.However,CD 16+/CD14+ cells was found to be associated with shorter dialysis age(p=0.009);3.The percent of lymphocyte-like hemocytes negatively associated with dialysis vintage(p=0.050)The percent of CD16+/CD14+ cells is positively correlated with the use of 4.25%PET ultrafiltration(p = 0.012),gender,4h Cr D/P,4h Glu D/D0,residual urine volume.4.Flow cytometry and immunoblotting showed Vimentin and Fibronectin positive and E-Cadherin negative revealed increased transdifferentiation of mesenchymal intermediate epithelial cells,in peritoneal dialysis patients with dialysis less than 3 months.Expression of IL-34 that promotes M2-type transformation.Immunofluorescence confirmed the presence of TGF-β1+ and IL-1β+ cells in the dialysate,but stimulating mesothelial cells with IL-1β did not affect IL-34 expression.Summary1.Annual glucose exposure in PD patients climbs annually and is positively associated with peritoneal transport function.The average annual glucose exposure,BMI,and peritoneal transport function in the first two years were independent predictors for long-term technical survival in PD patients;2.The peritoneal transport function showed a decline trend in the first five years of dialysis,and baseline peritoneal function(Cr 4h D/P)was a independent risk factor for overall survival,while glucose exposure could predict changes in peritoneal function in the next two years;3.With comparable mean glucose concentration,glycemic fluctuation index of diabetic PD patients is significantly lower than that of normal diabetic patients;the peritoneal transport function of peritoneal dialysis patients is significantly positively correlated with the amplitude of multiple fluctuation indexes;4.M2 appeared to be the dominant subtype of macrophage in peritoneal dialysis fluid,among which the proportion of M2a type decreased with the dialysis vintage.Mesothelial-mesenchymal transdifferentiation was found in patients on dialysis for less than 3 months.Macrophages were observed to secrete TGF-β1 and IL-1β,which may be involved in peritoneal fibrosis.