| AIM: Detecting the expression of the apoptosis index of podocyte, nephrin,podocin, TGF-β1andα-SMA, to study the postponed effects of all-transretinoic acid(ATRA) on injury podocyte which induced by adriamycin(ADR).METHODS: Cells were divided into4groups: negative control group (NC),group of injury podocyte induced by adriamycin(ADR)(AI), group of negativecontrol group treated with ATRA(AC) and group of podocyte injury modeltreated with ATRA(AA). After been filled with the bottom of plate, thepodocytes of group AI and AA were first subjected to DMED medium whichcontaining0.15ug/ml ADR for24hours to construct podocyte injury model.After ADR intervention24hours, podocyte of group NC and AI was grown innormal DMED medium until the end, meanwhile group AC and AA wassubjected to standard DMED medium which containing0.1μM ATRA. AfterATRA intervention24hours, podocytes were collected for histological andmolecular biology examination(n=12). Light microscope and scanning electronmicroscopy were used to observe cells morphology and ultra-structure changes;flow cytometry was used to detect apoptotic index of podocyte；MTT was used to draw cell growth curve; Western-blot and real-time RT-PCR were used for thedetection of transforming growth factor-β1（TGF-β1）, α-smooth muscle actin（α-SMA）,nephrin,podocin protein and mRNAexpressions.RESULTS:(1)Compared with that in group NC, podocytes in group AIarrangement disorder and sparse, cell body of some podocytes shrinkage, footprocess shrink and even disappear; most of the podocytes after ATRAintervention were in order arrangement, and unconspicuous in foot processshrink.(2) Cell growth curves of foot cells in group NC and AC were showed theapproximate "S" type. podocyte cell growth inhibition in group AI wassignificantly more than that in group AA. Cell growth inhibition in podocytes ofgroup AA was significantly abatement when compared with that in podocytes ofgroup AI.(3) Compared with that in group NC, glomeruli podocytes apoptosisrate in group AI was significantly increased (P<0.05). Compared with that ingroup AI, glomeruli podocytes apoptosis rate in group AA was significantlydecreased (P<0.05).(4) Both protein and mRNA expressions of TGF-β1,α-SMA in podocytes of group AI were significantly increased than that in groupNC(P<0.05); and both protein and mRNA expressions of TGF-β1,α-SMA inpodocytes of group AC and group AA were significantly decreased than that ingroup AI(P<0.05).(5)Both protein and mRNA expressions of nephrin, podocinin podocytes of group AI were significantly decreased than that in groupNC(P<0.05); and both protein and mRNA expressions of nehprin, podocin inpodocytes of group AC and group AA were significantly increased than that ingroup AI(P<0.05).(6)The protein expression of nephrin and podocin werenegative correlated with protein level of α-SMA and TGF-β1in group AI(allP＜0.05). The protein expression ofα-SMA was positively correlated with protein level of TGF-β1in group AI(P＜0.05).Cell apoptosis index waspositively corretated with protein level of TGF-β1in group AA(P＜0.05).CONCLUSION: ATRA may inhibit podocyte apoptosis and transdifferentiationin vitro，promote podocyte differentiation, thereby reducing podocyte injurywhich were caused by adriamycin. AIM: Detecting the expression of matrix metalloproteinase-2(MMP-2), matrixmetalloproteinase-9(MMP-9), retinoic acid receptor-α(RAR-α), retinoic acidreceptor-α(RAR-β), and retinoic acid receptor-γ(RAR-γ), to study molecularmechanisms of all-trans retinoic acid mitigate glome-rular podocyte injury which were induced by adriamycin in ivtro.METHODS: Cells were randomly divided into3groups: negative control group(NC), group of injury podocyte induced by adriamycin(ADR)(AI) and group ofpodocyte injury model treated with ATRA(AA). The construction of podocyteinjury model and the method of the intervention in every group are the same asthe partⅠ. Light microscope and scanning electron microscopy were used to observe cells morphology changes; MTT assay was used to detect podocyteviability of each group;Gelatin zymography was used for MMP-2and MMP-9enzymic activity detection; Western-blot was used for the detection of MMP-2,MMP-9, RAR-α, RAR-β, RAR-γ,α-SMA and TGF-β1protein expressions.Real-time RT-PCR were used for the detection of MMP-2, MMP-9, RAR-α,RAR-β and RAR-γ mRNA expressions.RESULTS:(1) Compared with that in group NC, foot process atrophy or vanishin group AI was observed under electron microscopy; but foot process fusion,retraction is not obvious in group AA.(2) Compared with that in group NC,podocyte viability of group AI was significantly reduced(P<0.05). Comparedwith that in group AI, podocyte viability of group AA was significantlyinhanced(P<0.05).(3)Compared with that in group NC, both protein and mRNAexpressions of MMP-2,MMP-9in podocytes of group AI were significantlydecreased (P<0.05); but protein expression of TGF-β1was significantlyincreased (P<0.05). Compared with that in group AI, both protein and mRNAexpressions of MMP-2,MMP-9in podocytes of group AA were significantlyincreased (P<0.05), but protein expression of TGF-β1was significantlydecreased (P<0.05).(4) Both protein and mRNA expressions of RAR-α,RAR-γ in podocytes of group AI were significantly decreased than that in groupNC(P<0.05); and both protein and mRNA expressions of RAR-α,RAR-γ inpodocytes of group AA were significantly increased than that in groupAI(P<0.05).(5) Both protein and mRNA expressions of RAR-βin podocytes ofgroup AI were decreased than that in group NC, but there was no significantlydifference between them(P>0.05); and both protein and mRNA expressions ofRAR-βin podocytes of group AA were significantly increased than that in group AI, but there was no significantly difference between them(P>0.05).(6)Compared to group NC, MMP-2and MMP-9enzymatic activities of glomerulifoot cells in group AI were significantly attenuated(P<0.05).Compared to groupAI, MMP-2and MMP-9enzymatic activities of glomeruli foot cells in group AIwere significantly inhanced(P<0.05).(7) The protein expression of RAR-αandRAR-γ were positively correlated with protein level of MMP-2and MMP-9ingroup AI(all P＜0.05), and negative correlated with protein level of TGF-β1andα-SMA(all P＜0.05).There was no correlation between protein expressionsof RAR-β and MMP-2/MMP-9(P>0.05).The protein expression of MMP-2and MMP-9was positively correlated with podocyte viability in group AI(P<0.05), and negative correlated with protein level of TGF-β1andα-SMA(all P＜0.05).CONCLUSION:In process of podocyte injury which induced by adriamycin,ATRA may up-regulate expressions of MMP-2and MMP-9by binding toRAR-α and/or RAR-γ, so as to mitigate glomeruli podocyte injury. |
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