The Function Of Caveolin-1 In Peritoneal Dialysis And Its Associated Peritonitis

IntroductionPeritoneal dialysis(PD)is an effective renal replacement therapy world-widely used for the treatment of end-stage renal disease(ESRD).PD has not only the similar long-term effectiveness to hemodialysis(HD),but also possesses advantages over it, which HD could not compare.Therefore,to better understand the mechanism operating behind PD and further prevent PD-associated complications,are currently important prerequisites for PD developments.During the process of PD treatment,peritonitis is a significant source of technique failure,morbidity,and mortality in PD patients.Thus,understanding the pathophysiology and the molecular mechanisms operating under the acute peritonitis is a necessity to find feasible avenues of alleviating functional and structural damage associated with this situation,and pave a way for new therapeutic strategies.Previous studies have demonstrated that water and solute transport across the peritoneal membrane depends on the intrinsic permeability of the peritoneum and the effective peritoneal surface area(EPSA).Several lines of evidence,obtained both from the clinical patients and from the experimental animal models,have indicated that,during the episode of acute peritonitis,faster-than-normal dissipation of the osmotic gradient due to the increased EPSA and permeability,causes ultimately ultrafiltration failure (UFF).In these cases,infiltration of inflammatory cells and vascular proliferation within the peritoneum were usually observed.Caveolae are defined as 80～100nm flask-shaped invaginations of the plasma membrane.In the past few decades,caveolae have been long accredited a "portal organell" involved in multiple cellular functions.Caveolae are particularly abundant in endothelial cells and participate in many vascular functions.Two distinct pathways have been pictured in regard to caveolae for regulation of vascular permeability,which are①the direct transcellular pathway by endocytosis and②the indirect paracellular pathway by tonic inhibition of endothelial nitric oxide synthase(eNOS).Considering the vital location of caveolae,the endothelium monolayer of peritoneal microvessels,a major barrier for water and solute transport,we speculated that caveolae might play a critical role in the functional and structural maintainance or modifications of the peritoneum under certain circumstances,such as acute peritonitis,together with two other essential molecule regulators of peritoneal permeability in this barrier,eNOS and aquaporin-1(AQP-1).Caveolin-1(Cav-1)is an essential protein to drive the formation of caveolae.Cells deleted of Car1 failed to express cav-1,thus displaying the loss of caveolae.Recently, genetically modified mice have been attested to be advantageous and effective for investigating the molecular counterparts of PD mechanisms.Therefore,Cav1-deficient mice would undoubtedly provide a useful tool to study the putative function of caveolae in vivo setting.Lipopolysaccharide(LPS)or endotoxin,is a component of the outer membrane of gram-negative bacteria.It has been classically used to induce acute inflammation in rats. At present,we employed LPS-peritonitis mouse model to study the functional and structural impact induced by acute inflammation in Cav1-deficient(Car1-/-)mice,to further explore the roles played by cav-1 in the inflammatory conditions of PD.Methods(1)Animal groups and peritonitis modelExperimental animals were Car1-/- male mice and their wildtype(Car1+/+) counterparts,using C57BL/6J mice as the backgroud.Two groups of age-matched (9-12W)male mice were investigated respectively for basal PD(control)and inflammatory PD(peritonitis).Each group included 6 pairsof mice.Acute peritonitis was performed by intraperitoneal LPS injection(10mg/kg).18 hours after LPS injection,mice were used for peritoneal equilibration test(PET).2.5 ml of 7%glucose dialysate was exchanged for 2 hours to obtain the permeability parameters.(2)Peritoneal permeability measurement and samplingA considerately combination of ketamine and xylazine was subcutaneously injected into control mice or peritonitis mice 18 hours after LPS administration.Right common carotid artery was cannulated.30 minutes after operation,2.5 ml of 7% glucose dialysate was infused intothe peritoneal cavity via PD catheter.Blood or dialysate samples were collected through the catheter at minute 0,30,60 and 120, respectively.Hematocrit(HCT)was measured before PD.At the end of the dwell,all dialysate inside the cavity was recovered.Net ultrafiltration(UF)was calculated.The number of white blood cells in the dialysate was counted.Urea,glucose and sodium were assayed by biochemical analyzer.Total protein in the dialysate was measured by Bio-Rad method.After sacrifice of mice,a small part of visceral and parietal peritoneum were fixed in the paraformaldehyde,and the rest of the visceral peritoneum was snap-frozened and stored.(3)ImmunoblottingSodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)was done as described.Immunoblotting was repeated at least three times,followd by densitometry analysis for the scanned images.(4)Tissue staining and immunohistochemistryHemalun-eosine(HE)staining and immunohistochemistry were done as described. For immunohistochemistry,antigen retrieval was performed when necessary.Sections were viewed and photographed under the microscope coupled with camera.Cell counting was performed using a handy counter.(5)Determination of Nitrite/Nitrate(NO_x)The production of NO_x in the dialysate was measured using a colorimetric assay based on the Griess reaction following the manufacturer' s instructions.(6)Statistical AnalysesData are presented as mean±SEM.Comparisons between results from two groups were performed using t test,different groups were performed using ANOVA. Significance level was P＜0.05.Results(1)Effect of Cav1 deficiency on peritoneal transport in the basal conditionsHematocrit in Cav1-/- mice was apparently higher in comparison with Cav1+/+ mice.Although there was approximally 40%higher NOx in the Cav1-/- dialysate than in wildtype,this difference had no statistical significance.The transport status for small solutes(urea,glucose and sodium)and capacity of UF in Car1-/- mice were quite similar with Car1+/+ mice.However,macromolecule transport as reflected by total protein product in the final dialysate was significantly higher in Car1-/- mice.Tissue staining found no apparent vascular abnormalities or proliferation in Cav1-/-peritoneum.Immunoblotting demonstrated that the expression of AQP-1,eNOS and iNOS had almost no difference between Cav1-/- mice and Cav1+/+ ones.(2)Functional effect of LPS-induced acute peritonitis on Cav1-modifxed miceDistinguishably,there was a significant upheaval in plasma urea concerntration in endotoxic Cav1-modified mice.After 18-hour LPS treatment,P-Cav1-/- mice were not characterized by a multiple increase in dialysate WBC counts in comparison with control as wildtype.When compared with control,dialysate NO_x in the inflammatory mice was approximately 10-fold higher.LPS-induced inflammatory alterations were significantly attenuated in P-Cav1-/- mice,especially in the first-30-minute hypertonic dwell.Compared with P-Cav1+/+ mice,P-Car1-/- mice manifested a less increased permeability for urea and a relatively slower reabsorption of glucose from dialysate,a partial recovery of sodium sieving and a final alleviation of UF failure.Immunoblotting demonstrated that AQP-1 expression was significantly higher in P-Car1-/- mice than in P-Cav1+/+ ones.P-Cav1-/- peritoneum was less severely infiltrated by inflammatory leucocytes.Immunostaining showed that F4/80 expression was similar between two groups,whereas ICAM-1 expression lined inside the endothelium in P-Cav1-/-peritoneum was significantly diminished as compared to wildtype.ConclusionsOur study demonstrated①in the basal PD,caveolae do not influence the diffusive transport of small solutes,while slower the convective transport of macromolecule solutes;②in the inflammatory condition,the exist of caveolae facilitates small solute transport especially in the early dwell,but does not change the final clearance of small solutes;the effect of caveolae on macromolecule transport is disguised by peritonitis; also,the exist of caveolae leads to UFF by affecting AQP-1 expression as well as faster glucose reabsorption in the early dwell;③in the inflammatory condition,caveolae participate in the ICAM-1-mediated transendothelial cell migration of leukocytes,thus increasing leukocyte infiltration into peritoneal tissue.