Apoptosis Induced By SFRP4 In Autosomal Dominant Polycystic Kidney Disease Through Inhibition Of The Wnt/?-catenin And PI3K/AKT Signaling Pathway

Objective: To explore the expression of s FRP4 in autosomal dominant polycystic kidney disease(ADPKD) and the underlying mechanism of s FRP4 induced apoptosis in ADPKD.Methods: 1.Experiment in vivo:(1)Serological experiment: 12 samples of healthy people and 20 samples of ADPKD patients' serum were collected and the level of s FRP4 in the serum was evaluated by ELSIA method.(2)Histology experiment: 3 normal renal tissue samples and 3 polycystic renal tissues samples were taken for histology experiment. Immunohistochemistry was used to detect the expression of s FRP4 in the renal tissues; Apoptosis cells existing in the samples were evaluated by TUNEL assay. RT-PCR was applied to analysis the level of s FRP4 m RNA?caspase-3 m RNA?GSK3? m RNA??-catenin m RNA and cyclind1 m RNA in the samples; The protein level of the s FRP4 ?cleaved-caspase-3?pro-caspase-3?p-GSK3??GSK3???-catenin?p-?-catenin?cyclind1?AKT?p-AKT in the renal tissues was explored by Western blot assay.(3) Zebrafish experiment: Microinjection of 200 pg hs FRP4 m RNA in zebrafish embryos, pronephros cysts were observed after 55 hours. 2. Experimrnt in vitro:(1) HEK-293 T cells were divided into 3 groups: Normal group?Pc DNA6 group and s FRP4 group. Flow cytometry assay was performd to estimate apoptosis rate of cells. The levels of cleaved-caspase-3 and pro-caspase-3 were observed by Western blot assay.(2) HEK-293 T cells were divided into 3 groups: control group, Wnt3? group and Wnt3?+ s FRP4 cotransfection group. Western blot method was utilized to measure the protein expressions of ?-catenin, p-AKT, AKT, NF-?B, Bax, cleaved-caspase9 and pro-caspase9.Results: 1. The serum concentrations of s FRP4 were notably higher in ADPKD than normal(P<0.001). 2. Compared with normal control, the markedly elevated s FRP4 mainly appeared in cytoplasm of renal tubular epithelial cells and the apoptosis of renal tubular epithelial cells increased in ADPKD. 3. Except GSK3?, the levels of s FRP4, caspase-3, ?-catenin and cyclind1 m RNA in the ADPKD was higher than the normal control. Western blot assay demonstrated the levels of s FRP4, cleaved-caspase-3, p-GSK3?, ?-catenin and cyclind1 protein are higher than the control group, but GSK3?, p-?-catenin and p-AKT/AKT are less than the control. 4. Overexpression s FRP4 in zebrafish could induce pronephric cysts formation. 5. Both of cells apoptosis rate and cleaved-caspase-3/cleaved-caspase-9 protein level were increased after s FRP4 overexpressed in HEK-293 T cells, but the levels of ?-catenin?p-AKT and NF-?B were decreased.Conclusions: s FRP4 is strikingly up in ADPKD. It could promote cystogenesis of the zebrafish pronephros. It could result in the abnormal apoptosis in the renal tissues of ADPKD through the inhibition of the classic Wnt/?-catenin and PI3K/AKT signaling pathway.