The Study On Establishment Of Horseshoe Kidney Animal Model Induced By Retinoic Acid And The Molecular Mechanism

Background and Objective:Horseshoe kidney is common in renal fusion malformation. And it is usually accompanies complications. It is unclear about the formation of horseshoe kidney. And it may be related to the decreased migration ability and enhanced adhering capacity. It is clear that the canonical Wnt signaling pathway which is related with kidney development as well as migration and adhesion. However, Retinoic acid could inhibit the nuclear reaction of the canonical Wnt signaling pathway and decreased the migration ability at the same time enhanced adhering capacity. The possible signaling pathway was inferred as retinoic acid/retinoic acid receptor(RA/RAR)—Wnt signaling pathway (Wnt5a/CaMK ?/TAK1/NLK) and the nuclear components (?-catenin/Lef/Tcf) — adhering Slug (Snai2) and migration molecule Rho (Rhob) --horseshoe kidney. The aim of this study:?To Establish a mouse model of horseshoe kidney using RA, and to lay a foundation for the study of the pathogenesis of horseshoe kidney;?To compare the renal tissue of mice with horseshoe kidney and their littermates with normal renal morphology through transcriptome sequencing and find out the related differential genes;?To screen and verify the possible genes which contribute to the formation of horseshoe kidney.Methods:?Six-eight-week-old mature female ICR mice were mated with male ones, and the identification of a vaginal semen plug was defined as embryonic Day 1 (Eld). At E8d, pregnant mice were injected with RA intraperitoneally. Fetal mice were taken out at E13d, E15d and E17d, respectively. The morphology of fetal mice and their kidneys were observed and recorded with stereomicroscope. ?Qualified kidneys of 2 fetal mice at E17d with horseshoe kidney (numbered E17RA8, E17RA9) and 2 of their littermates mice with normal renal morphology (numbered E17RA2, E17RA4) were analyzed by transcriptome sequencing, such as the analysis of differential genes, the analysis of cluster genes, GO function and Pathway. Genes both related with RA and involved in the development of the kidney were selected as candidates. ?erifying differentially expressed genes and key factors of possible signaling pathway using horseshoe kidney and pig embryo kidney cell with q-PCR in order to explore the molecular mechanism of horseshoe kidney caused by RA.Results: ?The tail length of fetal mice with horseshoe kidney is a quarter or a half of that of fetal mice with normal renal morphology. Among all the 11 fetal mice acquired at E13d,3 of them had horseshoe kidney,2 of them had "U" shaped kidneys without fusion, and 6 of them had normal renal morphology. As for the 4 fetal mice acquired at E15d, all of them had horseshoe kidney. Two pregnant mice were killed at E17d, one had 13 fetal mice (3 with horseshoe kidney,9 with normal morphology,1 was unclear) and the other had 12 fetal mice (8 with horseshoe kidney,4 with normal morphology). ?Qualified kidneys of littermate mice with horseshoe kidney (numbered E17RA8, E17RA9) and normal renal morphology (numbered E17RA2, E17RA4) were compared through transcriptome sequencing. Genes whose expression difference were more than 1 times were screened out. There were 328 differential genes between E17RA8 and E17RA2, and 802 differential genes between E17RA8 and E17RA4,562 differential genes between E17RA9 and E17RA2 and 1061 differential genes between E17RA9 and E17RA4. Among all the differential genes mentioned above,63 of them were chosen after clustering analysis. However, none of them were related with RA or kidney development and a possible pathway was inferred by literature review. ? q-PCR was used to verify key genes involved in the possible pathway we inferred and found that the expression of Rarb, Wnt5a, Tcf3 and Snai2 were up-regulated and the migration molecule Rhob gene was down-regulated. The results of the key factors above in pig embryo kidney cell under stimulating of RA was as same as horseshoe kidney.Conclusions:?Horseshoe kidney malformations of mouse could be induced by RA and this model could be used to investigate its molecular mechanism; ?The formation of horseshoe kidney induced by RA may be related to the Wnt signaling pathway which could up-regulate adhering molecule and down-regulate migration molecule.