| Objective:To study the effects of endothelin receptor A (ETRA) targeted plasmid vector-mediated small interfering RNA(siRNA)on renal ischemia-reperfusion injury in rats.Methods:(1)Two different siRNA sequences(siRNA#1,siRNA#2,)targeting ETRA Gene were designed and synthysized, their plasmid vectors including cGFP gene were constructed. Plasmid vectors were transfected into A-10 cell line with liposome.The efficiency of plasmid transfection was tested by density of GFP with fluorescence microscopy.The efficiency of gene silencing was determined by quantification of ETRA mRNA with real-time PCR. (2) 28 male SD rats were randomized into four groups:Sham group, siRNA group, Vector group, PBS group. Renal ischemia-reperfusion injury was induced by clamping the left renal pedicle for 1 hour, and then reperfused for 24 hours.siRNA were administrated 48 hours before clamping. 100 ug of ETRA siRNA plasmid DNA were diluted in 1 ml of PBS and injected into left renal vein by a hydrodynamic injection. Blood was collected and the left kidney tissue was harvested for analysis 24 hours after reperfusion.Serum creatinine and blood urea nitrogen were examined.ETRA mRNA,ET-1 mRNA,TNF-αmRNA,IL-6 mRNA,MIP-2 mRNA,MCP-1 mRNA were quantificated by real-time PCR.ETRA protein level was detected by western blotting. Snap frozen sections of kidney tissue were made for the detection of GFP expression in order to verify the transfection efficiency. Renal histopathology was analysed for tissue damage.Results:(1)Plasmid treated group showed obvious GFP expression.SiRNA#1 had higher transfection efficiency than siRNA#2 (P<0.05).(2)Plasmid treated group(siRNA and Vector group) had significant GFP expression. SiRNA group showed lower expression levels of both ETRA mRNA and protein than the Vector and PBS groups (P<0.05).Serum creatinine and BUN were significantly decreased in siRNA group when compared to Vector and PBS groups (P<0.05).Pathological changes,including renal tubular epithelium necrosis, vacuolization, tubular casts,were more severe in Vector and PBS groups,and semi-quantitative score of renal structural damage showed siRNA group had significant lower score than that of Vector and PBS groups (P<0.05).The expresson levels of ET-1 mRNA, TNF-αmRNA, IL-6 mRNA, MIP-2 mRNA and MCP-1 mRNA in kidney tissue were lower in siRNA group than Vector and PBS groups (P<0.05).Conclusion:(1)ETRA targeted siRNA plasmid vector can be successfully transfected into A-10 cells using liposome, ETRA siRNA#1 had a higher silencing efficiency than the other one.(2) The hydrodynamic retrograde renal vein injection could successfully transfect plasmid into kidneys. (3) ETRA siRNA can partially inhibit the expression of ETRA in kidney, which may improve the renal function and renal structure damage during renal I/R injury.(4)ETRA silencing resulted in reduced levels of ET-1 mRNA, inflammatory cytokines TNF-αmRNA, IL-6 mRNA and chemokines MIP-2 mRNA, MCP-1 mRNA during renal ischemia-reperfusion injury.
|
PDF Full Text Request