| Part?The expression of RAR/MMP signal pathway and their roles in renaltissue of glomerulosclerosis rats treated by all-trans retinoic acidOBJECTIVE To investigate the expression of retinoic acid receptors/matrix metalloproteinases(RAR/MMP)signal pathway and their roles in renal tissue of glomerulosclerosis(GS)rats treated with all-trans retinoic acid(ATRA).METHODS Healthy male Wistar rats(n=30,6-weeks-old)were randomly divided into three groups: sham operation group(SHO group),model group(GS group),model group treated with ATRA(ATRA group)(Each group n=10).Rats in SHO and ATRA group were subjected to uninephrectomy,and after 1 week,a single tail vein injection of Adriamycin(ADR)at a dose of 5 mg/kg,then ATRAgroup treated with ATRA(20 mg/kg · d)while GS group treated with equivalent normal saline(NS).Rats in SHO group subjected to a sham operation and treated with NS.All rats from the aforementioned three groups were sacrificed at the end of the 12 th week post-operative period and the kidneys were removed.Change of glomerular podocyte foot process were observed by transmission electron microscopy(TEM),histological changes of glomerulus were observed by HE staining and glomerular sclerosis index(GSI),cell apoptosis of glomerular were detected by terminal-deoxynucleotidyl transferse-mediated nick end labeling(TUNEL),glomerular protein expression of collagen-IV(Col-IV)and fibronectin(FN)was detected by immunohistochemistry,m RNA and protein expression of RAR?,RAR?,RAR?,MMP2,MMP9,transforming growth factor-?1(TGF-?1)and nephrin was detected by real-time fluorescent quantitative polymerase chainreaction(RT-PCR),immunohistochemistry and WB.RESULTS 1.Compared with SHO group,visible foot process fusion or disappear,and glomerular basement membrane(GBM)was cover by a layer of the cytoplasm in GS group;Compared with GS group,foot process fusion was remit in ATRA group.2.Compared with SHO group,glomeruli balloon adhesions and incrassation,hyperplasia of endotheliocyte and mesangial cell,accompanied with extracellular matrix(ECM)accumulation and focal segmental sclerosis,and GSI was significantly increased in GS group(P<0.05);Compared with GS group,GSI was significantly reduced in ATRA group(P<0.05).3.Compared with SHO group,cell apoptosis rate of glomerular was increased in GS group(P<0.05);Compared with GS group,apoptosis rate was significantly reduced in ATRA group(P<0.05).4.Compared with group SHO,expression of Col-IV,FN,TGF-?1 protein,and expression of TGF-?1 m RNA were increased in GS group(all P<0.05);Compared with GS group,expression of Col-IV,FN,TGF-?1 significantly reduced in ATRA group(all P<0.05).5.Compared with group SHO,m RNA and protein expression of RAR?,RAR?,MMP2,MMP9,nephrin were significantly reduced in GS group(all P<0.05);m RNA and protein expression of RAR?,RAR?,MMP2,MMP9,nephrin in ATRA group significantly upregulation when compared with GS group(all P<0.05);while there was no significant difference in m RNA and protein expression of RAR? in those three groups(P>0.05).6.Correlation analysis: RAR? and RAR? protein expression were negatively correlated with GSI,apoptosis rate,Col-IV,FN,TGF-?1,and positively correlation with MMP2,MMP9,nephrin(all P<0.01);RAR? protein expression had no significantly correlation with GSI,apoptosis rate,Col-IV,FN,TGF-?1,MMP2,MMP9,nephrin(all P>0.05).CONCLUSION Low expression of RAR?/RAR? might involve in the progression of GS,and ATRA exerted protective role in GS might through RAR(?,?)/MMP(2,9)signal pathway to downregulating TGF-?1 and ECM expression,and to maintain nephrin expression.Part?All-trans retinoic acid alleviate ADR-induced podocytes lesion throughRAR/MMP signal pathway in vitroOBJECTIVE To explore the association of RAR/MMP signal pathway in podocyte lesion induced by ADR in vitro,and their potential molecular mechanisms in ATRA treatment ininjured podocytes.METHODS The lentiviral vectors of Rara-RNAi,Rarb-RNAi,Rarg-RNAi,negative control vector of CON077 were synthesized.The cells were divided into seven groups: normal control group(NC group),podocyte injury induced by ADR group(ADR group),ATRA group,RAR?(-)group(transfected with vectors of Rara-RNAi),RAR?(-)group(transfected with vectors of Rarb-RNAi),RAR?(-)group(transfected with vectors of Rarg-RNAi),and virus negative control group(VNC group)(transfected with vectors of CON077).Podocytes were subjected to complete medium which containing 0.15?g/ml ADR for 24 h to construct podocytes injury model.Podocytes in ATRA group,RAR?(-)group,RAR?(-)group,RAR?(-)group,and VNC group were subjected ADR injury then treated with ATRA for 24 h.Podocytes in normal group cultured in normal medium till the end of experiment.Podocytes in all groups were collected for related detection after ATRA incubating for 24 h.Cells microscope changes were detected by light and scanning electron microscopy(SEM),cell viability was detected by MTT assay,cell apoptosis were detected by flow cytometry,m RNA expression of RAR?,RAR?,RAR?,TGF-?1,nephrin,podocin,MMP2,MMP9 in each group were detected by RT-PCR,protein expression of RAR?,RAR?,RAR?,MMP2,MMP9 in each group were detected by WB.RESULTS 1.The high transfection efficiency lentiviral vectors of Rara-RNAi,Rarb-RNAi,Rarg-RNAi were identified and transfected to podocytes successfully.2.Under light microscopy and SEM,podocytes in NC group display with polygon,clear outline,arrangement order,and visible foot process;while increased atrophy and cell debris,foot process effacement in ADR group;Compared with ADR group,podocytes in ATRA,RAR?(-),VNC group showed less atrophy and cell debris,however,podocytes in RAR?(-)group and RAR?(-)group showed less change.3.Compared with NC group,cell viability was significantly inhibited in ADR group(P<0.05);Compared with ADR group,cell viability were significantly increased in ATRA group and RAR?(-)group(all P<0.05),while cell viability were showed no significantly change(all P>0.05)in RAR?(-)group and RAR?(-)group;There were no significantly change between ATRA group and VNC group(P>0.05).4.Compared with NC group,cell apoptosis was significantly increased in ADR group(P<0.05);Compared with ADR group,cell apoptosis were significantly reduced in ATRA group and RAR?(-)group(all P<0.05),while cell apoptosis were showed no significantly change in RAR?(-)group and RAR?(-)group(all P>0.05);there were no significantly change between ATRA group and VNC group(P>0.05).5.Compared with NC group,m RNA and protein expression of RAR? and RAR? were significantly reduced(all P<0.05)in ADR group,while m RNA and protein expression of RAR? showed no significantly change(P>0.05);Compared with ADR group,m RNA and protein expression of RAR? were significantly increased in ATRA group,RAR?(-)group,RAR?(-)group(allP<0.05)but significantly reduced in RAR?(-)group(P>0.05),m RNA and protein expression of RAR? were no significantly change in ATRA group,RAR?(-)group,RAR?(-)group(all P>0.05)but significantly reduced in RAR?(-)group(P<0.05),m RNA and protein expression of RAR? were significantly increased in ATRA group,RAR?(-)group,RAR?(-)group(all P<0.05)but significantly reduced in RAR?(-)group(P>0.05);m RNA and protein expression of RAR?,RAR?,RAR? between ATRA group and VNC group was no significantly change(all P>0.05).6.Compared with NC group,m RNA and protein expression of MMP2 and MMP9,m RNA expression of nephrin,podocin were significantly reduced(all P<0.05),while m RNA expression of TGF-?1 significantly increased(P<0.05)in ADR group;compared with ADR group,m RNA and protein expression of MMP2 and MMP9,m RNA expression of nephrin,podocin were significantly increased(all P<0.05),while TGF-?1 significantly reduced(all P<0.05)in ATRA group and RAR?(-)group;Compared with ADR group,there was no significantly change of MMP2,MMP9,nephrin,podocin,TGF-?1 expression in RAR?(-)group and RAR?(-)group(all P>0.05).7.RAR? and RAR? protein expression were positively correlated with MMP2 and MMP9 protein expression(all P<0.05),while negatively correlated with cell apoptosis(all P<0.05),RAR? protein expression was not correlated with MMP2,MMP9,cell apoptosis(all P>0.05);MMP2 and MMP9 protein expression were negatively correlated with cell apoptosis(all P<0.05).CONCLUSION 1.ATRA reduced podocytes damage associated with inducing MMP2 and MMP9 expression.2.ATRA might through RAR?/? to upregulate MMP2/MMP9 expression,to prompt injured podocytes differentiation,increase cell viability and reduced apoptosis. |
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