The Roles And Molecular Mechanisms Of Retinoic Acid Receptors In The Renal Tubular Epithelial Cell Injury Induced By Hypoxia/Reoxygenation

Part I The expression of retinoic acid receptors and its significance in the renal tubular epithelial cell injury induced by hypoxia/reoxygenationOBJECTIVE: To explore the significance of retinoic acid receptors(RARs)in the renal tubular epithelial cell(RTEC)injury induced by hypoxia/reoxygenation(HR)by detecting the expression of RARs and changes of other cell injury indexes.METHODS: Cells cultured in vitro were divided into three groups: control group(control),hypoxia/reoxygenation for 48 hour group(HR 48)and hypoxia/reoxygenation for 72 hour group(HR 72).Cell in normal group was cultured in carbon dioxide incubator in which maintained the status of 37℃,5%CO2 and 95% air.Hypoxia was induced by placing the cells into a hypoxia incubator chamber,flushing with 5% CO2 and 95% N2,and then culturing in biochemical incubator in which maintained the status of 37℃.After hypoxia for 48 hour and 72 hour,the medium was aspirated and replaced with fresh oxygenated medium(37℃),and the cells were returned to carbon dioxide incubator(37℃,5%CO2 and 95% air)for 1 hour.Finally,the correlation indexes were detected in three groups.Real time fluorescent quantitative polymerase chain reaction(real-time PCR)was performed to detect the m RNA expressions of retinoic acid receptor α(RARα),retinoic acid receptor β(RARβ),retinoic acid receptor γ(RARγ)and transforming growth factor-β1(TGF-β1).Western-blot(WB)was applied to detect the protein expressions of RARα,RARβ,RARγ,TGF-β1,collagen-IV(Col-IV)and fibronectin(FN).The red fluorescence intensity of superoxide anion(O2-)and the fluorescence intensity of mitochondrial membrane potential(MMP)were detected by using fluorescence microscope.The cell viability,enzyme activity of superoxide dismutase(SOD)and glutathione(GSH)were also detected by enzyme kinetics.Optical inverted microscope was used to observe the changes in cell morphology.RESULTS: 1.Compared to control group,the m RNA and protein expressions of RARα and RARγ in HR 48 and HR 72 group were reduced(all p<0.05).When compared with those in HR 48 group,the m RNA and protein expressions of RARα and RARγ in HR 72 group were markedly reduced(all p<0.05);However,the difference for RARβ m RNA and protein expression between the HR group(48 hour and 72 hour)and the normal group was not statistically significant(all p>0.05).2.Compared to control group,the m RNA expression of TGF-β1,and the protein expressions of TGF-β1,Col-IV and FN in HR 48 and HR 72 group were increased(all p<0.05).When compared with those in HR 48 group,the m RNA expression of TGF-β1,and the protein expressions of TGF-β1,Col-IV and FN in HR 72 group were markedly increased(all p<0.05).3.Compared to control group,the red fluorescence intensity of O2-and the fluorescence intensity of MMP in HR 48 and HR 72 group were increased,and the enzyme activity of SOD and GSH was decreased obviously(all p<0.05).When compared with those in HR 48 group,the red fluorescence intensity of O2-and the fluorescence intensity of MMP in HR 72 group were markedly increased,and the enzyme activity of SOD and GSH was decreased significantly(all p<0.05).4.Compared to control group,the cell viability in HR 48 and HR 72 group were reduced(all p<0.05);morphology of cobble-stone was disappeared,cell density was reduced,cell debris was increased.When compared with those in HR 48 group,cell viability in HR 72 group were markedly decreased(p<0.05);the change of cell morphology was more obvious,cell density was remarkablely reduced,cell debris was increased obviously.5.Correlation analysis in RTEC: ⑴ the RARα or RARγ protein expression was inversely correlated with the protein expressions of TGF-β1,Col-IV and FN,the red fluorescence intensity of O2-and the fluorescence intensity of MMP(all p<0.05);and positively correlated with cell viability and the enzyme activity of SOD and GSH(all p<0.05).⑵ there was a positive correlation between the red fluorescence intensity of O2-and the protein expressions of TGF-β1,Col-IV and FN(all p<0.05).CONCLUSION: In the RTEC damaged by hypoxia,the lower expression of RARα and RARγ was involved in hypoxia injury and fibrosis,the molecular mechanisms might be related to the O2-over-production and extracellular matrix(ECM)accumulation.Part II The role and molecular mechanisms of retinoic acid receptors in renal tubular epithelial cell injury induced by hypoxia/reoxygenationOBJECTIVE: To explore the role and molecular mechanisms of RARs in RTEC injury induced by HR through Over Expression(OE)or Knock Out(KO)to interference the expression of RARs genes.METHODS:The lentiviral vectors of RARα-OE(OEα)、RARβ-OE(OEβ)、RARγ-OE(OEγ)、RARα-KO(KOα)、RARβ-KO(KOβ)、RARγ-KO(KOγ)and negative control(neg)were synthetised by lentiviral transfection.The cells were divided into nine groups: control group,HR group,HR OEα group,HR OEβ group,HR OEγ group,HR KOα group,HR KOβ group,HR KOγ group and HR neg.RTEC in control group was cultured routinely,RTEC in HR group was subjected to hypoxia for 48 hour,and RTEC in gene intervention group was suffered from gene intervention agents for 96 hour in preference to hypoxia for 48 hour.The method of hypoxic treatment was the same as the part I.real-time PCR was performed to detect the m RNA expressions of RARα,RARβ,RARγ and TGF-β1.WB was used to measure the protein expressions of RARα,RARβ,RARγ,TGF-β1,Col-IV and FN.The red fluorescence intensity of O2-and the fluorescence intensity of MMP were detected by fluorescence microscope.The cell viability,enzyme activity of SOD and GSH were also detected by enzyme kinetics.Transmission electron microscopy was applied to observe the changes in cell morphology ulteriorly.RESULTS: 1.Compared to control group,the m RNA and protein expressions of RARα and RARγ in HR group were reduced(all p<0.05).When compared with those in HR group,the m RNA and protein expressions of RARα,RARβ and RARγ in HR OEα group,HR OEβ group and HR OEγ group were markedly increased,respectively;and the m RNA and protein expressions of RARα,RARβ and RARγ in HR KOα group,HR KOβ group and HR KOγ group were markedly decreased,respectively(all p<0.05).However,there was no statistical differences for the m RNA and the protein expressions of RARα,RARβ and RARγ between HR neg group and HR group(all p>0.05).2.Compared to control group,the m RNA expression of TGF-β1,and the protein expressions of TGF-β1,Col-IV and FN in HR group were increased(all p<0.05).When compared with those in HR group,the m RNA expression of TGF-β1,and the protein expressions of TGF-β1,Col-IV and FN in HR OEα group and HR OEγ group were reduced markedly;the m RNA expression of TGF-β1 and the protein expressions of TGF-β1,Col-IV and FN in HR KOα group and HR KOγ group were increased markedly(all p<0.05).However,the difference for the m RNA expression of TGF-β1 and the protein expressions of TGF-β1,Col-IV and FN between HR group,HR neg group,HR OEβ group and HR KOβ group was not statistically significant(all p>0.05).3.Compared to control group,the red fluorescence intensity of O2-and the fluorescence intensity of MMP in HR group were increased,and the enzyme activity of SOD and GSH was decreased obviously(all p<0.05).When compared with those in HR group,the red fluorescence intensity of O2-and the fluorescence intensity of MMP in HR OEα group and HR OEγ group were markedly reduced,and the enzyme activity of SOD and GSH was elevated significantly;the red fluorescence intensity of O2-and the fluorescence intensity of MMP in HR KOα group and HR KOγ group were markedly increased,and the enzyme activity of SOD and GSH was decreased significantly(all p<0.05).However,there was no statistical differences for the red fluorescence intensity of O2-,the fluorescence intensity of MMP and the enzyme activity of SOD and GSH between HR group,HR neg group,HR OEβ group and HR KOβ group(all p>0.05).4.Compared to control group,the cell viability in HR group was reduced markedly(p<0.05);the microvilli on RTEC surface was reduced,and heterochromatin agglutination was displayed.When compared with those in HR group,cell viability in HR OEα group and HR OEγ group was markedly increased(all p<0.05),the cell injury was ameliorative;cell viability in HR KOα group and HR KOγ group was obviously decreased(all p<0.05),and the cell damage was aggravating,even RTEC rupture could be found.However,the difference for cell viability between HR group,HR neg group,HR OEβ group and HR KOβ group was not statistically significant(all p>0.05),and the change of cell morphology was unconspicuous.5.Correlation analysis in RTEC: ⑴ the RARα protein expression was inversely correlated with the protein expressions of TGF-β1,Col-IV and FN,the fluorescence intensity of O2-and MMP(all p<0.05),and positively correlated with cell viability,the enzyme activity of SOD and GSH(all p<0.05);⑵ the RARγ protein expression was inversely correlated with protein expressions of TGF-β1,Col-IV and FN,the fluorescence intensity of O2-and MMP(all p<0.05),and positively correlated with cell viability,the enzyme activity of SOD and GSH(all p<0.05).⑶ there was a positive correlation between the red fluorescence intensity of O2-and the protein expression of TGF-β1,Col-IV and FN(all p<0.05).CONCLUSION:In the hypoxia damaged RTEC,the increased expression of RARα or RARγ plays a protective role against oxidative damage and fibrosis,and the decreased expression of RARα or RARγ can aggravated the oxidative damage and fibrosis.The possible molecular mechanism is that RARα or RARγ can reduce the O2-production,increase the enzyme activity of SOD and GSH,down-regulate TGF-β1 expression and then reduce ECM accumulation.