The Effect And Mechanism Study Of TFAP2A Regulating FGF7 On Renal Dysplasia Of Offsprings In Rat Intrauterine Placenta Hypoperfusion Model

Background: Intrauterine placental hypoperfusion is a common pathological basis for a variety of maternal and fetal diseases during pregnancy,and is one of the main causes of maternal pregnancy complications such as eclampsia,preterm birth,low birth weight,intrauterine growth restriction,and fetal hypoxia.Intrauterine placental hypoperfusion may increase maternal and neonatal morbidity and mortality during the perinatal and neonatal periods and has important implications for maternal and fetal outcomes.Intrauterine placental hypoperfusion results in decrease in the delivery of nutrients and oxygen to the developing fetus.In order to preferentially supply the brain,heart,adrenal glands,and liver,the fetus changes its blood flow,which affects the normal development of other organs.Studies have shown that intrauterine placental hypoperfusion has adverse effects on fetal kidney development.Kidney development is impaired,the number of nephrons is reduced,and there is a higher risk of long-term hypertension.Therefore,it is of great significance to study the effects of placental hypoperfusion during pregnancy on kidney development in offspring.Nephron development is closely regulated by a variety of growth factors and morphogenetic factors.These proteins act through a variety of signaling pathways,including the Fgf/Fgfr,Wnt/β-catenin,and Notch pathways.FGFRs and FGF ligands are highly expressed in developing kidneys and urinary organs.FGF7 is initially expressed by the cap mesenchyme and binds to Fgfr2 b to promote the development of kidney.In FGF7 deficient mice,the collecting duct and ureteral buds were reduced and the number of nephrons was reduced.Transcription factors play a central role in the network of genes that regulate nephron formation.Bioinformatics predicted that the FGF7 promoter had a conserved binding sequence of TFAP2 A.TFAP2A,a member of the AP-2 transcription factor family,regulates a variety of cellular processes,including cell growth and tissue differentiation.The TFAP2 A knockout mice died in perinatal period with severe multidirectional changes,including craniofacial changes,incomplete neural tube closure,and cardiac and renal dysplasia.At present,the protective mechanism of TFAP2 A regulating FGF7 in the kidney development of the offspring of IUPH model during the second trimester is still unclear.In this study,by constructing the IUPH model of SD rats in the second trimester,FGF7 was applied to treat the offspring of IUPH,and the potential mechanism of the protective effect of FGF7 on the offspring kidney of the model of intrauterine placenta hypoperfusion was discussed.It is of great significance for early diagnosis and treatment of renal dysplasia caused by intrauterine placenta hypoperfusion.Purpose: In this study,intrauterine placenta hypoperfusion model(IUPH)was established by bilateral uterine artery ligation in SD rats at 18.5 days of gestation.Based on proteomics data and bioinformatics analysis,the mechanism of TFAP2A/FGF7 axis in the offspring renal dysplasia of IUPH model was investigated,and its role in prenatal treatment was discussed.Methods and Materials: 1.The intrauterine placenta hypoperfusion model was established: female SD pregnant mice were randomly divided into four groups:intrauterine placenta hypoperfusion group(IUPH),sham operation group(SHAM),FGF7 placenta hypoperfusion group(IUPH+FGF7),FGF7,PI3K/AKT pathway inhibitor intrauterine placenta hypoperfusion group(IUPH+FGF7+wortmannin).On day 18.5 of gestation(E18.5),the IUPH group underwent bilateral uterine artery ligation,and the SHAM group underwent sham operation.One hour before IUPH treatment,FGF7 recombinant protein was injected into the abdominal cavity of IUPH+FGF7 group.IUPH+FGF7+wortmannin pregnant mice were injected with FGF7 recombinant protein and PI3K/AKT pathway inhibitor wortmannin.SHAM group and IUPH group were injected with equal amount of blank solvent.Subsequently,the intrauterine placenta hypoperfusion model during the second trimester was established.Median abdominal incision was performed on pregnant mice after anesthesia and disinfection,and bilateral uterine artery ligation was performed.After successful modeling,tissues of two time nodes(24h and 48 h after IUPH treatment)were harvested.Tissues treated after IUPH for24 hours were selected for TMT proteomic sequencing,and cluster analysis such as GO and KEGG were performed for differential proteins.2.q PCR was performed to detect the expression of FGF7 gene in the kidney tissues of the offspring rats,and Western blot was performed to detect the expression of FGF7,Bax,Bcl-2,PCNA,Caspase-3,PI3K/AKT pathway proteins in the kidney tissues of the offspring rats.Immunohistochemical staining and Tunel method were used to investigate the level of proliferation and apoptosis of offspring kidneys.3.Serum creatinine,urinary protein,urinary creatinine and MDA concentrations were detected at 12 weeks after birth(PD12W)to investigate renal function of the offspring rats.4.Renal tubular epithelial cell line HK2 cells were cultured in vitro under hypoxia conditions to simulate the hypoxia stimulation of intrauterine fetal rat renal tissue by IUPH,and FGF7 or/and PI3K/AKT pathway inhibitors were added into the medium to detect the changes in protein expression levels of FGF7 and PI3K/AKT pathways by WB.WB,Edu,Tunel,cck8 and flow cytometry were performed simultaneously to evaluate cell proliferation and apoptosis.5.Tfap2 a transcription factor was overexpressed or inhibited in tool cells,and FGF7 protein levels were detected by western blot,and double luciferase and Chip experiments were conducted to clarify the regulatory relationship.Results: 1.A total of 51 differentially expressed proteins(DEPs)were identified by TMT proteomic sequencing,among which 29 DEPs were up-regulated and 22 DEPs were down-regulated in the intrauterine placental hypoperfusion group.GO enrichment analysis showed that DEPs were enriched in important biological processes such as iron binding,oxygen binding and heme binding.KEGG enrichment analysis showed that the enrichment pathway was African trypanosomiasis,cholesterol metabolism and ferroptosis pathway.Among them,S100a8 and S100a9 proteins were highly expressed in IUPH group,and it was reported that FGF7 inhibited the expression of S100a8 and S100a9 proteins.2.q PCR and Western blot experiments showed that the m RNA and protein levels of FGF7 in fetal renal tissue of E19.5 and E20.5 in IUPH group were decreased.With FGF7 treatment,FGF7 m RNA and protein levels increased.3.WB results showed that the protein levels of Bax and Caspase-3 in IUPH group were increased,while the protein levels of Bcl-2 and PCNA were decreased.After FGF7 treatment,the protein levels of Bax and Caspase-3 decreased,while the protein levels of Bcl-2 and PCNA increased.4.Immunohistochemistry and Tunel showed that the level of Bax protein in IUPH group was increased,while the level of PCNA protein was decreased.After FGF7 treatment,the level of Bax protein decreased and the level of PCNA protein increased.In IUPH group,cell proliferation decreased and apoptosis increased.After FGF7 treatment,cell proliferation increased and apoptosis decreased.FGF7 has therapeutic effect.When PI3K/AKT pathway inhibitors were administered,the therapeutic effect of FGF7 was inhibited.5.WB experiment showed that the expression levels of P-PI3K/PI3 K and P-AKT/AKT were decreased in IUPH group,while the expression levels of P-PI3K/PI3 K and P-AKT/AKT were increased after FGF7 treatment.6.In the detection of renal function,serum creatinine,the level of urinary protein/urinary creatinine and MDA concentrations in IUPH group were higher than those in SHAM group,and renal function was improved after FGF7 treatment.The effect of FGF7 on renal function was attenuated when PI3K/AKT pathway inhibitors were administered simultaneously.7.In the experiment of the effect of FGF7 on HK2 cells under hypoxia condition,WB experiment showed that the protein levels of FGF7,Bcl-2,PCNA,P-PI3K/PI3 K and P-AKT/AKT were decreased in IUPH group,while the protein levels of Bax and Caspase-3 were increased.After FGF7 treatment,protein levels of FGF7,Bcl-2,PCNA,P-PI3K/PI3 K and P-AKT/AKT were increased.The therapeutic effect of FGF7 was attenuated when PI3K/AKT pathway inhibitors were administered simultaneously.8.cck8,flow cytometry,Edu and TUNEL experiments all showed that cell proliferation decreased and apoptosis increased in IUPH group,and cell proliferation increased and apoptosis decreased after FGF7 treatment.When PI3K/AKT pathway inhibitors were administered,the therapeutic effect of FGF7 was inhibited.9.In tool cells,Tfap2 a transcription factor was overexpressed,and FGF7 expression was increased,while Tfap2 a transcription factor expression was inhibited,and FGF7 expression was decreased.Meanwhile,both dual luciferase and Chip experiments showed that TFAP2 A promoted FGF7 transcription.Conclusion: 1.During the development of the offspring kidney of the uteroplacental hypoperfusion model,FGF7 is under expressed in the kidney tissue.2.IUPH can cause increased apoptosis and decreased proliferation of kidney cells,and decrease the activation of PI3K/Akt pathway.After treatment with FGF7,it can reduce the occurrence of increased apoptosis,and increase the activation of PI3K/Akt pathway and cell proliferation.The therapeutic effect of FGF7 was attenuated when PI3K/Akt pathway inhibitors were also administered.3.The renal function of the offspring rats of IUPH was decreased,and the renal function was improved to a certain extent after FGF7 treatment.4.The TFAP2A/FGF7 axis is involved in the development of the kidney of intrauterine placenta hypoperfusion by regulating the PI3K/Akt signaling pathway.