| Peritoneal dialysis(PD)is one of the commonly used renal replacement therapy for patients with end stage renal disease(ESRD).However,protein malnutrition is a very common complication of peritoneal dialysis,with an incidence of up to 60%.It is a strong risk factor for increasing patient mortality and infection rates.Peritoneal removal of protein is an important cause of protein malnutrition.Our previous study found that the mass of protein lost through daily peritoneal dialysis was about 4.04 ± 1.97 g / 24 h,which was enough to cause hypoproteinemia.However,the mechanism of protein loss through peritoneal dialysis has not yet been elucidated.It is generally believed to be related to increased peritoneal permeability.There are two ways to regulate permeability:the cell pathway and the intercellular pathway.The cellular pathway is endocytosis mediated by specific vesicles of caveolae or clathrin on the cell membrane,through which macromolecular substances such as albumin can pass through single endothelial cells.Caveolin-1 is the main marker of caveolae,which is related to the expression and function of caveolae.However,for peritoneal vascular endothelial cells(HUM-CELL-0116),whether the transport of albumin depends on caveolae / caveolin-1mediated endocytosis of macromolecules isunclear.In this study,under the condition of peritoneal dialysis,whether HUM-CELL-0116 cells express caveolae / caveolin-1 and involve in the transport of fluorescein-isothiocyanate bovine serum albumin(FITC-BSA).It aims to clarify the specific mechanism of protein loss through peritoneal dialysis and provide a theoretical basis for the prevention and treatment of hypoproteinemia in peritoneal dialysis.[Objective]1.Determine whether there is caveolin-1 expression in peritoneal vascular endothelial cells,and then evaluate whether there is a caveolae-mediated macromolecular transcellular transport pathway.2.To determine whether the peritoneal dialysate can promote the transport of albumin across endothelial cells.3.Toinvestigatewhether endocytosis mediated by caveolae / caveolin-1 is involved in the transport of albumin by peritoneal endothelial cells.[Method]1.Culture HUM-CELL-0116 cells in vitro and divide into 5 groups after passage:(1)Control group(DMEM-H+10%FBS medium,no intervention during the culture);(2)PDS group(1.5% Peritoneal dialysis solution co-culture with DMEM-H+10%FBS medium);(3)PDS+Filipin group(containin 2.5μg/ml or 5μg/ml Filipin+1.5% Peritoneal dialysis solution with DMEM-H+10%FBS);(4)PDS+DMSO group(containin 1‰DMSO+1.5% Peritoneal dialysis solution with DMEM-H+10%FBS);(5)PDS+caveolin-1 antibody group(containin 1‰ caveolin-1antibody +1.5% Peritoneal dialysis solution with DMEM-H+10%FBS).2.With 1.5% PDS as the positive control group,the cell survival rate was determined by a cell counting plate.After exploring the intervention 0h,4h,12 h,24h,48 h,the concentration of PDS in more than 80% of the cells survived to ensure the experimental significance of the subsequent drug intervention.3.The cells were treated with 10% fetal bovine serum(Fetal bovine serum,FBS)complete culture solution,and all groups except the control group were added with 1.5%peritoneal dialysate solution.After treatment at 0h,4h,12 h,24h and 48 h,the expression of caveolin-1 was detected by Western blot.4.Use the Transwell chamber to build an endothelial cell monolayer model,and use FITC-BSA as a tracer to determine the cell growth and fusion time in the Transwell chamber.After the cell growth and fusion,the monolayer cell permeability experiment was conducted.After treatment at 0h,4h,12 h,24h and 48 h,FITC-BSA was used as a tracer to detect the monolayer cell permeability of each group.5.Using FITC-BSA as a tracer,observe the endocytosis of FITC-BSA under fluorescent microscope,and further verify the role of caveolae / caveolin-1 in the transport of albumin in HUM-CELL-0116 cells.6.Replace cultured fetal bovine serum with 10% ESRD patient serum and repeat step 3 and step 4.[Result]1.Cells were treated with different concentrations of PDS medium.The concentration setting is as follows: with 10% as the concentration gradient,set 10 groups of PDS with different concentrations from 0% to 100%.And add 10% FBS complete medium to treat the cells.After treating cells for 0h,4h,12 h,24h and 48 h,the results show that at 50% PDS concentration,the cell survival rate of each group can still maintain more than 80%.The overall difference is significant and statistically significant(P<0.05),this concentration is used as the next experimental concentration.2.The expression of caveolin-1 in peritoneal endothelial cells: Compared with the control group,the expression of caveolin-1 in the PDS group began to decrease after 12 hours of intervention.The expression of caveolin-1 in the PDS + Filipin(2.5μg / ml)group,PDS + Filipin(5μg / ml)group,and PDS + caveolin-1 antibody group began to decrease after 4 hours of intervention.The decrease of caveolin-1 expression in the PDS+ Filipin(2.5μg / ml)group was less than that of the PDS + caveolin-1 antibody group,while the decrease of caveolin-1 expression in the PDS + Filipin(5μg / ml)group was greater than that of the PDS + caveolin-1 antibody group.The difference was significant and statistically significant(P<0.05).However,there was no significant difference between the PDS + DMSO group and the PDS group.3.Monolayer cell permeability:(1)10% FBS complete medium group:In the 0h group,there was no obvious change in the permeability of monolayer cells.In the 4h group,compared with the control group,the permeability of the monolayer cells in the PDS group and PDS + DMSO group was not significantly changed.The permeability of the monolayer cells in the PDS + Filipin(5μg / ml)group and the PDS + caveolin-1antibody group has increased.The difference was significant and statistically significant(P<0.05).In the 12 h,24h,and 48 h groups,compared with the control group,the monolayer cell permeability in the PDS group,PDS + Filipin(5μg / ml)group,and PDS+ caveolin-1 antibody group was significantly increased.And the increase of monolayer cell permeability in the PDS + Filipin(5μg / ml)group,and PDS + caveolin-1 antibody group was more obvious than that in the PDS group.The permeability of monolayer cells in the PDS + DMSO group was greater than that in the control group,but it was lower than that in the PDS group.(2)10% ESRD serum medium group:The overall trend of each group was basically consistent with the 10% FBS complete medium group.But from12 h,the monolayer cell permeability of the 10% ESRD serum medium group was greater than the 10% FBS complete medium group.The overall difference was significant and statistically significant(P<0.05).4.FITC-BSA internalization situation:Under the fluorescence microscope,there was no significant endocytosis of albumin by HUM-CELL-0116 cells in each group.It suggests that in the mechanism of increasing the albumin permeability of peritoneal endothelial cells,endocytosis may not participate in the transcellular transport of albumin by peritoneal endothelial cells.[Conclusion]1.Peritoneal vascular endothelial cells express caveolin-1,suggesting the presence of macromolecular endocytosis mediated by caveolae.2.Peritoneal dialysate increases the permeability of peritoneal vascular endothelial cells to albumin.It suggests that peritoneal dialysate can increase permeability of peritoneal endothelium and increase protein loss.3.There was no increase in albumin endocytosis under the fluorescence microscope.At the same time,Filipin inhibits caveolae / caveolin-1 dependent endocytosis,but the permeability of peritoneal endothelial cells to albumin increases.It suggests that caveolae / caveolin-1 dependent endocytosis plays no major role in albumin transport in peritoneal vascular endothelial cells.It is suggested that the transport of albumin across peritoneal endothelial cells across endothelial cells may depend on the increased permeability caused by the intercellular pathway,resulting in the loss of protein through peritoneal endothelial cells. |
PDF Full Text Request