| Objective:Congenital obstructive kidney disease is the main cause of chronic kidney disease in children.More than 50%of children with chronic kidney disease are congenital kidney and urinary tract malformations,and most of them have urinary tract obstruction.Obstructive nephropathy is complicated by kidney damage,and urinary tract obstruction is often associated with renal dysplasia.Although surgery is the main treatment for obstructive nephropathy.some patients still have renal damage that does not recover or even progresses,and long-term renal function deteriorates.Hemodialysis and kidney transplantation are eventually needed,which seriously affects children and There is no good treatment for the quality of life of the family.Therefore,it is of great social significance to study in-depth the mechanism of kidney injury in the early stage of obstructive kidney disease,to intervene in the disease progression in the early stage,and to preserve renal function.Our previous studies have shown that the key sensor of energy metabolism,oxidative stress sensor,histone deacetylase(Sirtuin type 1,SIRT1)is a key protein for epithelial-mesenchymal transition and apoptosis in the kidney.We further confirmed the transformation of SIRT1 in renal tubular epithelial cells with EMT in partial unilateral ureteral obstruction(PUUO)and complete unilateral ureteral obstruction(CUUO)animal models.SIRT1 played a role in promoting fibrosis during the early stage of fibrosis in unilateral ureteral obstruction animal models.SIRT1 interference vector delayed the progression of renal tubular epithelial cell fibrosis by regulating the Smad3 pathway.However,it is still unclear how SIRT1 is regulated.Further study of the upstream regulatory mechanism of SIRT1 is helpful to find new targets for early treatment of renal injury in obstructive kidney disease.We performed a whole-genome sequencing of renal parenchymal specimens of children with hydronephrosis at the junction of the renal pelvis and ureter at the early stage of surgery.We compared the two groups with renal function less than 30%and renal function greater than 40%.Mi R-181d-5p and circ-The expression of 0045861 is significantly different.Targetscan and microRNA.org were used to predict the presence of binding sites between SIRT1 and miR-181d-5p,and circnet was used to predict the presence of binding sites between circ-0045861 and miR-181d-5p at TNRC6C.We further performed q RT-PCR to verify the expression of miR-181d-5p and SIRT1 in the control group,2 and 5 days after UUO in rats with partially ureteral obstructed kidneys,and miR-181d-5p in renal tissues in UUO group.The expression was down-regulated and gradually decreased with the increase of renal fibrosis,while the expression of SIRT1gradually increased with the increase of renal fibrosis,suggesting a correlation between the expression of miR-181d-5p and SIRT1.Therefore,we propose a new hypothesis that circ-0045861 may inhibit the action of miR-181d-5p by competitively binding miRNAs combined with 3'UTR,thereby upregulating the transcription of SIRT1 mRNA,eventually leading to abnormal expression of SIRT1 protein and strengthening SIRT1.Involved in the regulation of PI3K/AKT signaling pathway by FOXO1,which eventually led to the development of renal injury in obstructive kidney disease.By constructing a circRNA-miRNA-mRNA multi-dimensional regulatory network,it is expected to clearly understand the relationship between circRNA,microRNA and SIRT1 signal pathway expression,and the relationship between the development and development of renal fibrosis in obstructive nephropathy,and from the perspective ofRNA methylation modification A deep understanding of the pathogenesis of the disease from the level of transcription provides new ideas for early diagnosis and treatment of obstructive kidney disease,and provides new clues for the development and development mechanism of other chronic kidney diseases,and provides new targets for early diagnosis and treatment of these diseases.Methods:1.Verification of cytokine indicators such as circ-0045861,mir-181d-5p,SIRT1 in vitro and in vivo.Renal tubular epithelial cells were cultured in vitro,and the expression levels of mir-181d-5p,circ-0045861 and SIRT1 in renal tubular epithelial cells were detected by TGF-?1 induced fibrosis and H202 induced cell fibrosis.Newborn rats were selected to make a unilateral ureteral obstruction model,and the expression levels of miR-181d-5p,circ-0045861 and SIRT1 after UUO were detected in vivo.2.Through in vivo and in vitro experiments,interfere with or overexpress circ-0045861 and miR-181d-5p,and explore the regulatory mechanism of circ-0045861/miR-181d-5p on SIRT1's involvement in early renal injury.Cell-level regulation of circ-0045861/miR-181d-5p was used to detect renal tubular epithelial cells undergoing EMT,fibrosis,apoptosis,and changes in SIRT1/PI3K/AKT/FOXO1 oxidative stress pathways.Renal tubular epithelial cells are cultured in vitro,which promotes epithelial-mesenchymal transition(EMT)through mechanical stress or TGF-?1,and induces oxidative stress in cells through H2O2.The cells were divided into three groups.normal group,fibrosis group,and oxidative stress.To these three groups,circ-0045861 or mir-181d-5p inhibitors or mimics were added,and renal tubular epithelial cells were cultured in vitro to construct mir-181d-5p reporter gene plasmid for SIRT1 3'-UTR binding site,circ-0045861 reporter gene plasmid for SIRT13'-UTR binding site,circ-0045861 reporter gene binding site for miR-181d-5p Plasmid,the binding site was constructed into the pmiR-RB-REPORT luciferase reporter gene vector,and a mutant binding site plasmid was constructed to co-transfect the inhibitor of miR-181d-5p or circ-0045861 in renal tubular epithelial cells/Amplifier and reporter gene,to detect the activity of the luciferase reporter gene,and verify the presence of binding sites for circ-0141134/miR-181d-5p/SIRT1 mRNA.Fluorescence in situ hybridization maps the expression of circ-0045861 and miR-181d-5p,and detects up-or down-regulation of circ-0045861 or miR-181d-5p on renal tubular epithelial cells EMT,apoptosis,fibrosis,oxidative stress,SIRT1/p53signaling pathway-related genes and protein expression.3.Make animal models of partial ureteral obstruction in newborn rats,and test the effects of up-or down-regulation of circ-0045861 or miR-181d-5p on renal EMT,fibrosis,apoptosis and SIRT1 oxidative stress pathway after partial ureteral obstruction.In previous studies,podocyte transformation was observed 2 days after partial ureteral obstruction in neonatal rats,and renal tubular epithelial cells were transformed 5 days after partial ureteral obstruction.Therefore,renal parenchymal injection or mice were injected 1day,2 days,3 days,and 5 days after partial ureteral obstruction.Tail vein injection of circ-0045861 or miR-181d-5p mimics or inhibitors to detect the expression of circ-0045861/miR-181d-5p before and at different time points after ureteral obstruction,and related to apoptosis,EMT,and fibrosis Gene and protein,SIRT1 and PI3K/AKT/FOXO1 signal pathway related genes and protein expression dynamic changes.4.Statistical analysis.Real-time PCR data is expressed as 2-??Ctvalue,and Western blot analysis is expressed as relative density.All experimental data are expressed as meanąstandard deviation.SPSS19.0 statistical software is used for statistical analysis.For the data that obey the normal distribution,the two-sample t test is used for comparison between the experimental group and the control group.For the data that does not obey the normal distribution,the Mann-Whitney U test is used for the comparison between the experimental groups,and p<0.05 is considered statistically significant.difference.Result:We performed whole-genome sequencing on children with UPJO who had a renal function greater than 40%and children who had a renal function less than 30%and found the expressions of circRNA-0045861 and miR-181d-5p were different.It was found that the expression of circ-0045861 and SIRT1 was up-regulated and the expression of miR-181d-5p decreased in fibrotic HK-2 cells.We designed circ-0045861 siRNA and miR-181d-5p mimics.Adding si-circ-0045861 to HK-2 cells can inhibit the apoptosis and fibrosis of fibrotic HK-2 cells by over-expressing miR-181d-5p.Circ-0045861 can be combined with miR-181d-5p by double luciferase experiments,and fish experiments have demonstrated that circ-0045861 and mir-181d-5p can be co-localized in HK-2 cytoplasm.In addition,our further double luciferase experiments demonstrated that miR-181d-5p and SIRT1 mRNA have binding sites.In UUO animal models and fibrotic HK-2 cell models,miR-181d-5p expression decreases and SIRT1 expression increases.We constructed miR-181d-5p mimics and transfected them into UUO models and fibrotic HK-2 cells.We found that miR-181d-5p mimics can inhibit UUO models and fibrotic HK-2 cells.Expression of death-related proteins(p53,JNK,bax,caspase-9)and fibrosis-related proteins(?-sma,vim),and inhibited the expression of oxidative stress-related proteins(3-NT,Trx,p53).The decline of renal function in obstructive nephropathy is regulated by the m6A methylation modification ofRNA.The progression of obstructive nephropathy is related to metabolic processes,regulation of cellular processes,and tightly related cellular processes.The axon receiving signal pathway,Gn RH signal pathway signal pathway and the decline of renal function in obstructive nephropathy.Among them,lncRNA ENSMUST00000149811.2,ENSMUST00000209614.Are regulated by m6A methylation modification in UUO model.Conclusion:In this section,we perform whole-genome sequencing of the kidney tissue of children with UPJO who have greater than 40%renal function and less than30%who have renal function,and find that the course of obstructive kidney disease progresses with cell balance metabolism,single-cell biological processes,Extracellular matrix accumulation and other biological metabolic processes are closely related,while the decline of renal function in obstructive nephropathy is mainly regulated by the cell adhesion signal pathway and the endoplasmic reticulum protein formation signal pathway,among which hsa?circ?0006577,hsa?circ?0045861 and the kidneys of children with UPJO Tissue renal fibrosis is closely related and deserves further study.Circ-0045861 plays an important role in the regulation of renal tubular epithelial cells.Inhibiting circ-0045861 can inhibit the fibrosis and apoptosis of renal tubular epithelial cells.The expression of miR-181d-5p is decreased in renal tubular epithelial mesenchymal transition.Overexpression of miR-181d-5p can inhibit fibrosis and apoptosis of renal tubular epithelial cells.While circ-0045861 and miR-181d-5p do have binding sites,miR-181d-5p and SIRT1 mRNA also have binding sites.In the pathogenesis of obstructive nephropathy,circ-0045861 as ceRNA combines with miR-181d-5p to regulate the expression of SIRT1 and further regulate oxidative stress-induced apoptosis and fibrosis in obstructive nephropathy.Through this study,it was found that the decline of renal function in obstructive kidney disease is regulated by the m6A methylation modification ofRNA.Among them,m6A is involved in the progression of multiple obstructive kidney disease and is the key to the development of the disease. |
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