The Role And Mechanism Of Autophagy On Peritoneal Fibrosis Associated With Peritoneal Dialysis

Context:Peritoneal dialysis(PD)is one of the most important renal replacement therapies for end stage renal disease(ESRD).It has many advantages,such as low cost,simple operation,effective removal of middle molecular solute and better protection of residual renal function.Currently,there are about more than 360,000 PD patients in the world,with an annual growth rate of 8%,During long-term peritoneal dialysis treatment,peritoneal fibrosis,as an important barrier to material exchange,occurs due to peritonitis,biocompatibility and other reasons,leading to ultrafiltration failure.At present,peritoneal fibrosis is still a worldwide problem,which is the most important factor that causes patients to be forced to suspend long-term peritoneal dialysis,and seriously affects the quality of life and long-term survival rate of patients with peritoneal dialysis.Moreover,there is no effective treatment method clinically.Recent studies have found that autophagy plays a key role in the occurrence of fibrosis.Therefore,it is of great scientific and practical significance to study the pathogenesis of peritoneal fibrosis from the perspective of autophagy to prevent and treat peritoneal fibrosis in the process of PD.Autophagy is a mechanism of self-digestion,which is the process of cells degrading their own damaged organelles and macromolecules,and plays an important role in maintaining cell homeostasis and cell life activities.The process of autophagy can be divided into diferent stages:initiation of autophagosome,nucleation,expansion and extension of autophagosome membrane,closure and fusion with lysosome,and degradation of intracavular products.The mechanisms of autophagy-related genes involved in different stages are also complex.Microtubule-associated protein 1 light chain 3(LC3)is a very important protein in the extension/closure stage of autophagosome membrane.However,when autophagosomes are fused to lysosomes,LC3-Ⅱ located on the autophagosome membrane is degraded in autophagy,while LC3-Ⅱ located on the cytoplasmic surface of autophagosomes is destroyed and recovered by Atg4.LC3-Ⅱ is widely considered as a good marker for autophagy research due to its specific binding with autophagosome membrane.By interacting with LC3,p62 protein can be localized on the autophagosome and continuously degraded by the autophagy-lysosome system.It also plays an important role in autophagy research.There’s evidence that says,enhancement of autophagy is usually beneficial for organ protection;however,in some cases,excessive autophagy may actually cause apoptosis and accumulation of extracellular matrix.Previous studies have shown that abnormal autophagy can lead to neurodegenerative diseases,tumor cell growth,cardiovascular diseases,and fibrosis of liver,kidney,lung,etc.At present,the mechanism of autophagy in peritoneal fibrosis has not been clearly studied,and further experimental studies are needed.Objectiv:To explore the role of cellular autophagy in peritoneal fibrosis associated with peritoneal dialysis and its potential molecular biological mechanism,so as to provide a new theoretical basis for the prevention and treatment of peritoneal fibrosis associated with peritoneal dialysis.Method:In this study,human peritoneal tissue,peritoneal dialysis fluid effluent cells from peritoneal dialysis patients and human peritoneal mesothelial cell line(HPMCs)were selected as the research objects.1.The parietal peritoneal tissue(about 1cm×lcm)was collected from patients with new peritoneal dialysis and PD ≥3year ultrafiltration failure terminated peritoneal dialysis,and the formation of autophagosomes and lysosomes in cells was observed by transmission electron microscopy.The expressions of fibrosis associated proteins(COL-I,FN),autophagy marker proteins LC3-Ⅱ and p62 were detected by Western blot.The mRNA expressions of COL-I,FN,LC3-Ⅱ and p62 in the cells were detected by real time PCR.J 2.The abdominal dialysate was collected from patients with PD<30 days and PD>1 year who were not in bed continuously,and the mesothelial cells of the peritoneal dialysate were isolated and cultured.The mRNA and protein expressions of Col-Ⅰ,FN,LC3-Ⅱ and p62 were detected.3.Cultivating HPMCs,divided into normal group(control),the high glucose group(HG,90 mM),HG+chloroquine group(chloroquine,CQ,90uM+HG),after stimulation 48h,The autophagosomes were observed by electron microscopy,realtime PCR and western blot test cells the expression of Col-Ⅰ,FN,LC3-Ⅱ and p62.Result:1.In the peritoneum of the patients with peritoneal dialysis termination,the expressions of fibrosis associated proteins FN,COL-Ⅰ and autophagy associated proteins LC3-Ⅱ were significantly higher than those of the initial dialysis patients,and the expression levels of p62 protein and mRNA were significantly lower than those of the initial dialysis patients(P<0.05).The number of autophagosomes was also significantly increased compared with the initial PD patients under transmission electron microscopy(P<0.05).2.The protein levels and mRNA expressions of Fn,Col-I,LC3-Ⅱ in the intermediate skin cells of dialysate in peritoneal dialysis patients with>1 years of dialysis age at night were significantly higher than those in dialysate patients with less than 30 days of dialysis age(P<0.05),and the expression level of p62 was significantly lower than that in patients with less than 30 days of dialysis age(P<0.05).3.The protein and mRNA expression levels of FN,COL-I,LC3-Ⅱ in HPMCS high glucose group were significantly higher than those in control group(VS control group)(P<0.05);The expression level of p62 was lower than that of control group(P<0.05).The number of autophagosomes under transmission electron microscope was significantly increased compared with the control group(P<0.05).High glucose+chloroquine group could effectively reduce the protein and mRNA expressions of FN,Col-I,LC3-Ⅱ in HPMCs(VS high glucose group)(P<0.05);The expression level of p62 in high glucose group was increased(P<0.05);Under transmission electron microscope,the number of autophagosomes in higher glucose group was significantly decreased(P<0.05).Conclusion1.Autophagy increased in patients with long-term peritoneal dialysis,which was positively correlated with peritoneal fibrosis.2.High glucose stimulated HPMCs,increased autophagy in peritoneal mesothelial cells and increased autophagosomes,which was positively correlated with peritoneal fibrosis.3.Chloroquine can inhibit autophagy and reverse peritoneal fibrosis.