The Effect And Mechanism Of Rapamvcin Inhibits Peritoneal Fibrosis

Peritoneal dialysis(PD)is now widely accepted as one of effective renal replace-ment therapies for patients with end-stage renal disease.However,long-term exposure of the peritoneal membrane to nonbiocompatible peritoneal dialysis fluid,including high concentrations of glucose and lactate,glucose degradation products(GDPs),and low pH,can result in functional and structural abnormalities of peritoneum,eventually leading to the PD failure.High glucose(HG)has been shown to cause fibrosis by up-regulating the expression of transforming growth factor-b1(TGF-b1),vascular endo-thelial growth factor(VEGF),and induction of epithelial-to-mesenchymal transition(EMT)of peritoneal mesothelium.EMT of mesothelial cells(MCs)is an early event during PD and has been identified as a key process and one pivotal mechanism in the onset and progression of peritoneal fibrosis.The major steps in EMT involve peritoneal mesothelial cells(PMCs)losing epithelial phenotype and acquiring fibroblast-like characteristics,tight junction and ad-herens junction dissociation,cytoskeletal rearrangement,higher migratory and invasive capacities,basement membrane degradation,and extracellular matrix(ECM)deposi-tion.All these events are a physiologic process of tissue repair but can also promote peritoneal fibrosis in nonphysiologic conditions.The mammalian target of rapamycin(mTOR),a member of the phosphoinositide 3-kinase(PI3K)-related kinase family,is a central regulator of cell proiferation,growth,survival,cytoskeletal organization,and cell motility.Rapamycin,as a specific inhibitor of mTOR,with its immunosuppressive and antiproliferative properties,is mainly used in antirejec-tion reactions and drug-eluting stents in clinics.When bound to its inter-cellular receptor,FKBP12(FK506-binding protein-12),rapamycin selectively inhibits the activity and function of the mTOR complex.It has been proven to have antifibrotic effects in a number of tissues,such as the kidney,liver,and skin.In the peritoneum,recent studies have reported that rapamycin is also effective in suppressing the EMT of PMCs and preventing peritoneal fibrosis.However,the underlying molecular mecha-nisms remain poorly understood.Small Rho GTPases are a subset of the Ras superfamily of GTPases,which can in-duce coordinated changes in the actin cytoskeleton dynamics and in gene transcription to drive a large variety of biological responses including cell polarity,adhesion,migra-tion,morphogenesis,proliferation,etc..The best characterized members of the Rho GTPase family are Rho A,Rac1,and Cdc42.Generally,RhoA stimulates formation of actin stress fibers and focal adhesions,Racl promotes formation of lamellipodia,and Cdc42 induces formation of filopodia.It has been shown that Rho GTPases play im-portant roles in EMT.Further studies investigated the relationship between mTOR and Rho GTPases(RhoA,Rac1,and Cdc42)during EMT.However,the clear relationship needs more substantial proofs:on one hand,the importance of each Rho GTPase family differs in different cell types;on the other hand,whether Rho GTPases function down-stream of mTOR in regulating EMT of peritoneal MCs has not been unveiled.In this study,our data show that HG activates the PI3K-Akt-mTOR pathway and induces EMT of PMCs,and rapamycin suppresses the process by inhibiting the activa-tion of Rho GTPases(RhoA,Racl,and Cdc42).Part IRapamycin inhibits peritoneal fibrosisAims:To define the effect of rapamycin treatment with peritoneal fibrosis rats.Methods:We classified 45 normal Sprague Dawley(SD)rats equally into 5 groups:the nomal ones(NC),the normal saline(NS)ones,the high glucose onesm(HG),the low-level rapamycin ones(L-RAPA),the high-level rapamycin ones(H-RAPA).After 6 weeks’ treatment,we executed all the rats to evaluate the peritoneal function,followed by taking the entire anterior abdominal wall which would be used for HE staining,im-munohistochemistry staining,We stern blot and realtime PCR of a-SMA and TGF-β1.Results:In vivo.Compared with the NC and NS group,the peritoneal function of HG group were significantly diminished,which were improved after thetreatment with ra-pamycin(L-RAPA and H-RAPA).The peritoneal pathology of the normal saline group was similar to that of group NC except a mildly thicker peritoneum.The group HG showed fibroproliferative response,with markedly increased peritoneal thickness,as well as higher expression of a-SMA and TGF-β1(Figs 2-4),compared with that of group NC and NS.Rapamycin clearly ameliorated those pathological changes in a dos-age-dependent manner.Conclusions:Rapamycin showed an obvious protective effect on high glucose in-duced peritoneal fibrosis.Rapamycin prevented EMT and fibrosis through reducing the secretion of TGF-β and inhibiting the formation of fibroblasts and extra cellular matrx(ECM).Part IIRapamycin inhibits EMT of peritoneal mesothlium cellsAims:To define the effect of rapamycin treatment with EMT of peritoneal mesotheli-um induced by high glucose.Methods:Rat peritoneal mesothelium cells(RPMCs)were isolated from normal rats’peritoneal.Some of the subconfluent RPMCs were stimulated with high glucose of dif-ferent concentration(30,60,120mM)to induce epithelial-mesenchymal transition(EMT),while others were co-administered with rpamycin(10,100nM).As makers of EMT we included a-SMA,E-cadherin,fibronectin and Collagen I by western blot or realtime PCR.Meanwhile,the cell motility were analyzed by the in vitro scratch assay and Transwell assay.Results:In vitro.We found that high concentration of glucose(30,60,120mM)in-duced EMT of RPMC,suggested by increased expression of a-SMA,fibronectin,Col-lagen I and decreased expression of E-cadherin and by increased cell migration in dos-age-dependent manner.Treatment with rapamycin resulted in a dosage-dependent pre-vention of high glucose-induced EMT.Conclusions:Rapamycin obviously inhibits the EMT of peritoneal mesothelium in-duced by high glucose.Part IIIRapamycin inhibits EMT of peritoneal mesothelium cells through reg-ulation of Rho GTPasesAims:To investigated the undefined molecular mechanisms by which rapamycin in-hibits EMT of peritoneal mesothelium cells.Methods:RPMCs were treated with high glucose(HG)(30,60,120mM)to induce EMT,and/or rapamycin(10,100nM)for indicated time.The phosphorylation(activa-tion)of PI3K-Akt-mTOR pathway and activation of RhoGTPases(RhoA,Racl,and Cdc42)were then examined by immunoblotting and RhoA/Rac1/Cdc42 Activation As-say.Furthermore,the cytoskeletal rearrangement was examined by immunofluorescence staining.Results:HG increased the phosphorylation of PI3K,Akt and mTOR.Importantly,ra-pamycin inhibits the Rho A,Racl and Cdc42 activated by HG.Moreover,rapamycin repaired the pattern of F-actin distribution induced by HQ reducing the formation of stress fiber,focal adhesion,lamellipodia and filopodia.Conclusions:HG activates the PI3K-Akt-mTOR pathway and induces EMT of per-itoneal mesothelium cells,and rapamycin suppresses the process by inhibiting the acti-vation of Rho GTPases(RhoA,Rac1,and Cdc42).