The Protective Effect Of Dexamethasone On Rat Antiserum-induced Human Podocyte Injury

Objective:To explore the effect of dexamethasone on human podocytes treated by rat anti-serum.Methods:1.Cell culture:Cells were grown on dishes at 33 ? in the presence of lU/ml ITS in RPMI 1640 medium supplemented with 10%FBS,100 U/ml penicilin and 100 U/ml strieptomycin.To induce differentiation,podocytes were maintained at 37?under 5%CO2 to allow differentiation for at least 14 days.Differentiated podocytes were cultured for 12h in RPMI 1640 mediun(1%FBS)before exposed to various experimental conditions.2.Cultured human podocytes were randomly divided into 4 groups:? Normal control group;? Rat anti-serum(RA)(20?l/ml)group:The podocytes were treated with RA or RA plus myosin9 polypeptide(P)for 24h,48h,72h respectively;?Inactivated RA(20?l/ml)group:The podocytes were treated with Inactivated RA or inactivated RA plus normal rat serum respectively for 72h;? DEX(10-5mol/L)group:The podocytes were treated with RA or RA+P plus DEX for 72h.The morphology of the podocytes were observed by inverted microscope and fluorescence microscope.The expressions of nephrin,myosin9 and a-tubulin were detected by Western blot and RT-PCR respectively.3.MTT group:Podocytes were divided into 7 groups:? normal control group;? RA group;? Inactivated RA group;? Inactivated RA+ normal rat serum group;? RA+P group;? RA+DEX group;? RA+P+DEX group.The viability of cells was determined by MTT colorimetry at 24h,48h,and 72h respectively.Results:1.The morphology changes of the podocytesCompared with normal control group,the inactivated RA had no significant effect on cell morphology of podocytes;The morphology of podocytes were changed in inactivated RA plus normal rat serum group,RA group and RA+P group.The morphology of podocytes were reserved after treated with dexamethasone.2.Effect of RA on the expression of nephrinCompared with the normal control group,the expressions of nephrin protain and mRNA in inactivated RA group showed no significant differcence(P>0.05).After treated with RA for 24h,48h,72h,the expressions of nephrin and mRNA was gradually decreased in a time-dependent manner(P<0.05).3.Effect of inactivated RA on the expression of nephrinAfter treated for 72h,the expression of nephrin was found significant decreased in RA group and inactivated RA plus normal rat serum group(P<0.05).4.Effect of RA plus myosin9 polypeptide on expressions of nephrin,myosin9 and a-tubulinCompared with the normal control group,the expressions of nephrin,myosin9,a-tubulin protains and mRNAs were found no significant differcence in inactivated RA group(P>0.05),but they were found markedly decreased in RA group and RA+P group(P<0.05).5.Effect of dexamethasone on the expressions of nephrin,myosin9 and?-tubulin of RA/RA+P-treated podoctyesDexamethasone could upregulated expressions of nephrin,myosin9,?-tubulin protains and mRNAs in either RA group or RA+P group compared with the normal control group(P<0.05 or P<0.01)6.Changes of podocytes viabilityThe results of MTT revealed that inactivated RA had no significant effect on cell viability of podocytes(P>0.05).While lower cell viability was found in inactivated RA plus normal rat serum group,RA group,RA+P group at 72h(P<0.05 or P<0.01).Compared with the RA group or RA+P group,the viability of podocytes improved significantly in DEX group(P<0.05).Conclusions:1.The morphology of podocytes were markedly changed by RA/RA+P.So as the cell viability and the expressions of nephrin,myosin9 and ?-tubulin.2.Dexamethasone could not only upregulated expressions of nephrin,myosin9,a-tubulin protains and mRNAs treated with RA/RA+P,but also reverse the cell morphology and viability.Objective:To investigate the effect of fluvastatin(Flu)on the expression of nephrin and angiotensin ?(Ang ?)in high glucose-treated human podocytes.Methods:Cultured human podocytes were randomly divided into several groups as follows:(1)normal glucose(NG,5.5mmol/L D-glucose)group;(2)DMSO(lmg/ml);(3)high glucose(HG,30mmol/L D-glucose)group:the podocytes were treated with high glucose for 0h,6h,12h and 24h respectively;(4)Flu(10-7?10-5mol/L)group,HG+Flu(10-7,10-6mol/L)group:The podocytes were treated with various concentration of Flu or HG plus different concentrations of Flu for 24h.The cellular viability were detected by MTT colorimetry.The expressions of Ang? and nephrin were detected by radioimmunoassay and Western Blot respectively.Results:The results of MTT revealed that the viability of podocytes was found no significant difference in DMSO,Flu10-7M and 10-6M group compared with control(P>0.05),but lower in Flu10-5M group and HG group at the time of 24h(P<0.05).Compared with the HG group,the viability of podocytes was improved markedly in HG+Flu group when the concentration of Flu was 10-7M and 10-6M at the time of 24h(P<0.05).Compared with the normal control group,the expression of nephrin was found no significant difference in DMSO group,Flu10-7M and10-6M group(P>0.05),while it decreased in Flu10-5M group and HG group at the time of 24h(P<0.05).The induction of nephrin progressively decreased from Oh to 24h in a time-dependence manner(P<0.05),so as the upregulation of the synthesis of Ang ?(P<0.05).The expression of nephrin was improved and the elevated Ang ? synthesis was reversed in HG+Flu10-7M and 10-6M group in a does-dependent manner(P<0.05).Conclusion:Fluvastatin could protect the podocytes via ameliorating ectopic expressions of nephrin and Ang ? induced by high glucose.