| OBJECTIVE To explore the mechanism of overexpression retinoic acid receptor alpha(RARa)in attenuating renal interstitial fibrosis(RIF)in rats,by detecting the expression of RARa,Prohibitin(PHB)and transforming growth factor-betal(TGF-β1)in unilateral ureteral obstruction(UUO)rats.METHODS Forty 6-week-old SPF male SD rats were randomly divided into four groups:sham-operation group(SHO),UUO surgery model group(UUO),UUO negative virus group(UNvg)and UUO overexpress RAR alpha group(URARa),with 10 rats in each group.Rats in UUO were double ligated and cut off the left ureter;rats in SHO were not ligated left ureter;rats in URARa and UNvg were transfected with adeno-associated virus type 9(AAV-9)that carrying RARa gene and negative control virus on the basis rats of UUO,respectively.At the end of two weeks,all rats were sacrificed.Renal was extracted for pathological examination and RIF index was calculated.The expression of RARa,PHB,TGF-β1,collagen IV(CoL-IV)and fibronectin(FN)in renal was detected by immunohistochemistry.The expression of RARa,PHB and TGF-β1 mRNA in renal were detected by real-time fluorescence PCR(RT-qPCR),the expression of RARa,PHB and TGF-β1 protein in renal were detected by immunoblotting(WB).RESULTS 1.In naked eye,there were no significant change in rats renal in SHO;However,the renal of rats were significantly swelling accompanied with thins renal cortex in UUO,UNvg and URARa;In microscope,HE staining showed that there were no significant pathological changes in rats renal in SHO.The renal tubular contraction,renal tubular epithelial cell vacuoles degeneration,interstitial inflammatory cells infiltration was appeared in rats renal in UUO and UNvg;Compared with UUO,the above pathological changes were also observed in the URARa,but the degree was significantly reduced.In fluorescent microscope,the green fluorescence was appeared in blood vessels and surrounding renal tubules of rats in the URARa and UNvg,but a little spontaneous fluorescence was appeared in SHO and UUO.2.The collagen fibers were dyed green in renal glomerular peripheral and perivascular in rats renal in SHO.In UNvg and UUO rats,not only the collagen fibers dyed green was appeared in renal glomerular peripheral and perivascula,but also a large number of collagen fibers dyed green was appeared in renal tubule and renal interstitial.Compared with UUO,the collagen fibers dyed green were significantly decreased in glomerular,blood vessels,renal tubule and renal interstitial of rat renal in the URARα;Compared with SHO,the RIF index was significantly increased in UUO(P<0.05);Compared with UUO,the RIF index was no significant change in UNvg(P>0.05),however it was significantly decreased in URARα(P<0.05).3.Compared with SHO,the mRNA and protein expression of RARa,PHB were significantly decreased in UUO,but TGF-β1 mRNA and protein,Col-IV and FN protein expression were significantly increased in UUO(all P<0.05);Compared with UUO,there was no significant change in the mRNA and protein expression of RARa,PHB,TGF-β1,Col-Ⅳ and FN protein expression in UNvg(all P>0.05);Compared with UUO,the mRNA and protein expression of RARa,PHB were significantly increased,but the TGF-β1 mRNA and protein,Col-IV and FN protein expression were significantly decreased in URARa(all P<0.05).4.Correlation analysis revealed that RARa protein expression was negatively correlation with RIF index,TGF-(31,Col-Ⅳ and FN(r=-0.75,-0.73,-0.77,-0.76,all P<0.05);PHB protein expression was negatively correlation with RIF index,TGF-β1,Col-Ⅳ and FN(r=-0.83,-0.80,-0.81,-0.79,all P<0.05);RARα protein expression was positively correlation with PHB protein expression(r=0.82,P<0.05).CONCLUSION In RIF rats induced by UUO,downregulation of RARa and PHB expression may participate in the development of RIF;upregulation of RARa expression may attenuate RIF by enhancing PHB expression. |
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