Fluvastatin Inhibits The Expression Of Fibronectin In Human Peritoneal Mesothelial Cells Induced By High-glucose Peritoneal Dialysate Via SGK1Pathway

Objective: To explore the effect and mechanism of fluvastatin on theexpression of fibronectin （FN） in human peritoneal mesothelial cells （HPMC）induced by high-glucose peritoneal dialysate.Methods: After incubated in DMEM with0.01%FBS for24h, cultured HPMCwere randomly divided into groups as: control; high-glucose peritoneal dialysate（HGPDS） group; HGPDS+Flu10^-8mol/L¹0^-6mol/L group, HGPDS+GSK65039410^-5mol/L （the competitive inhibitor of SGK1） group; Flu10^-8mol/L¹0^-6mol/Lalone group; GSK65039410^-5mol/L alone group and DMSO control group. HPMCwere treated for3h,6h,12h,24h,36h,48h,72h respectively. The cellular viabilitywere detected by MTT colorimetry and the phenotype of HPMC were observed bylight microscopy. During RT-PCR, the cells were divided into groups as:①HGPDStreated for0h,1h,6h and24h respectively;②Control; HGPDS group; HGPDS+Flu10^-8mol/L¹0^-6mol/L group; HGPDS+GSK65039410^-5mol/L group; Flu10^-6mol/Lalone group and GSK65039410^-5mol/L alone group. HPMC were treated for1h or6h respectively to detect the mRNA of SGK1or FN by RT-PCR. During western blot,the cells were divided into groups as:①HGPDS treated for0h,3h,6h,12h and24hrespectively;②Control; HGPDS group; HGPDS+Flu10^-8mol/L¹0^-6mol/L group;HGPDS+GSK65039410^-5mol/L group; Flu10^-6mol/L alone group and GSK65039410^-5mol/L alone group. HPMC were treated for6h and the levels of SGK1was measured by western blot．Then the cells were divided into groups as:①HGPDStreated for0h,6h,12h,24h,48h and72h respectively;②Control; HGPDS group;HGPDS+Flu10^-8mol/L¹0^-6mol/L group, GSK65039410^-5mol/L group; Flu10^-6mol/L alone group and GSK65039410^-5mol/L alone group. HPMC were treatedfor24h and the levels of fibronectin in the culture media was measured by ELISA．Results:1. Normal HPMC monolayer showed characteristic cobblestone-likeappearance, however, loss of cell contacts with anacquisition of elongatedfibroblastoid morphology was observed after48h of HGPDS. Fluvastatin10^-6mol/Land GSK65039410^-5mol/L attenuated the influence on the cell morphology inducedby HGPDS.2. Compared with control group, the cellular viability was not changed ingroups of DMSO, fluvastatin10^-810^-6mol/L and GSK65039410^-5mol/L （P>0.05）.Lower HPMC viability was found in the groups of HGPDS from6h to72h （P<0.05）and partially restored in groups treated with different concentrations of fluvastatinand GSK65039410^-5mol/L at24h and36h. The cellular viability injured by HGPDSwas significantly improved at concentration of fluvastatin10^-6mol/L andGSK65039410^-5mol/L at the time of24h （P<0.05）.3. SGK1and FN mRNA expression measured by RT-PCR in HPMC increasedsignificantly after exposure to HGPDS, peaking at1h and6h respectively （P<0.05orP<0.01）. GSK65039410^-5mol/L significantly decreased the high mRNA expressionof SGK1and FN induced by HGPDS （P<0.01）, also the fluvastatin had the sameeffects as GSK650394in a dose-dependent manner （P<0.01）. While fluvastatin10^-6mol/L or GSK65039410^-5mol/L alone had no significant effects on SGK1andFN mRNA expression of HPMC compared with control（P>0.05）.4. SGK1and FN protein expression measured by western-blot in HPMCincreased significantly after exposure to HGPDS, peaking at6h and24h respectively （P<0.05or P<0.01）. GSK65039410^-5mol/L significantly decreased the high proteinexpression of SGK1and FN induced by HGPDS （P<0.01）, also the fluvastatin hadthe same effects as GSK650394in a dose-dependent manner （P<0.01）. Whilefluvastatin10^-6mol/L or GSK65039410^-5mol/L alone had no significant effects onSGK1and FN protein expression of HPMC compared with control（P>0.05）.Conclusion: High-glucose peritoneal dialysate can increase SGK1and FNexpression in HPMC, and the expression of SGK1peaked prior to FN. Fluvastatincould decreased the high expression of SGK1and FN induced by HGPDS, whichindicates that the protective role of fluvastatin on HPMC may be partially achievedthrough the signal pathway of SGK1. The results of this study may provide us atheoretical basis for the prevention and treatment of peritoneal fibrosis.