The Effect Of Wnt4 Gene Silencing On Renal Interstitial Fibrosis Of UUO Rat

[Objection] Silencing the expression of Wnt4 gene, then observe the effection in the blockback of Wnt/?-catenin signaling pathway, to explore the feasibility of Wnt4 si RNA to delay the process of renal interstitial fibrosis. Aim to offer a new theoretical support to the treatment of chronic kidney disease.[Methods]1?According to the design principles of sh RNA, 4 couples target for Wnt4 gene were designed, we selected the best effective one to synthesize Wnt4 si RNA lentivirus vector,then packaged the lentivirus and detected the titer;2?12 healthy male SD mice were divided into 4 groups randomly(N=3),different doses of negative lentivirus were injected into Renal capsule respectively: 1×105ifu?1×106 ifu?1×107 ifu?2×107 ifu. We collected the renal tissue after 3 days to make frozen section, to test the efficiency of infection under the microscope, and determined the optimal dosage of the lentivirus.3?128 healthy male SD mice(7 weeks old, weight about 180-220g)were divided into 4 groups randomly :(1)Unilateral ureteral separation Group(CON,N =32)(2)Unilateral ureteral obstruction group(UUO,N = 32)(3)Negative silence group(negative group, N = 32);(4)the Wnt4 gene silence group(silence group, N= 32).after the operation of 3th, 7th, 10 th, 14 thdays we killed 8 rats of each group,extracted kidney tissues, observed the changes of renal structure by electron microscope, HE stain and Masson stain; detected the expression position of Wnt4,?-catenin, ?-SMA and vimentin protein by immunohistochemical stain;detected the m RNA relative expression of Wnt4, ?-catenin, ?-SMA and vimentin by RT-PCR.[Results](1)Wnt4 si RNA lentivirus vector was constructed successfully,the final ti ter was 4 ×108TU/ml;(2)Frozen section: the titer of 1×107ifu had a high efficiency of infection;(3)HE staining, Masson staining and Electron microscopy: Compared with the UUO and negative groups, the damage of renal tubular and interstitial in silence group was reduced;(4) Immuno- histochemistry: wnt4, ?-catenin, ?-SMA and vimentin proteins had a large expression in the collecting duct epithelial cells, some vimentin proteins expressed in mesangial region;(5) RT-PCR : Compared with the same time in UUO and negative group, The expression wnt4 m RNA decreased in silence group of the 10 th, 14th(P<0.05);There is no obvious difference in the expression of ?-catenin in the UUO, negative and silence group, Compared with the same time in UUO and negative group, the expression of ?-SMA and vimentin m RNA decreased in silence group of the 14th(P<0.05);(6)Correlation analysis of m RNA relative expression:the m RNA relative expression of wnt4 in UUO group is positive correlated to ?-catenin and ?-SMA,The m RNA relative expression of wnt4 in silence group have almost no relation to?-catenin, but it is positive related to ?-SMA;[Conclusion](1)In UUO model, Wnt4 gene was activated, join Epithelial-Mesenchymal Transition by Wnt4/?-catenin signaling pathway, to promote the renal interstitial fibrosis.(2) Wnt4 si RNA can reduce the protein and m RNA expression of wnt4 in the renal of UUO rat specifically and effectively;(3)Silencing Wnt4 gene can decrease the Epithelial-Mesenchymal Transition rate,thus alleviated the process of renal interstitial fibrosis of UUO rats. But have no much difference to ?-catenin,suggested that maybe Wnt4/?-catenin signaling pathway was not the single one contributed to the Epithelial-Mesenchymal Transition induced by non-injured factors.