| Objective: Congenital ureter-pelvic junction obstruction(UPJO)is a common reason for hydronephrosis in children,which was commonly seen in pediatric urology.Silent mating type information regulation 2 homolog1(SIRT1)is widely expressed in kidney and plays a complex regulatory role in kidney physiology and pathology.The Ubiquitin Proteasome System(UPS)regulates various cellular functions by degrading more than 80% of cellular proteins to regulate many biological processes such as cell cycle,gene transcription and translation,cell survival and apoptosis,cell metabolism and protein quality control.SIRT1,as one of the widely expressed proteins in the body,is also regulated by UPS.This study compared the differential expression of mRNA-seq data in renal tissues of children with unilateral ureter-pelvic junction obstruction hydronephrosis with DRF<20% and DRF>40%,and analyzed RNAi SIRT1 and negative control rat renal tubular epithelial NRK52 E cells and analyzed the differential expression genes by mRNA-seq.The SIRT1 interaction protein was predicted and analyzed,and the SIRT1 related co-expression network was used to analyze the expression of SIRT1 in the kidney of children with UPJO and its regulatory target.Moreover,the establishment of Unilateral ureteral obstruction(ALP,To explore the effects of the ubiquitin proteasome system and SIRT1/FOXO1 signaling pathway on renal injury in neonatal UUO rats),and further explore the mechanism of renal injury by TGF-?1 induced rat renal tubular epithelial NRK52 E cell model.Methods: First part of this study: The renal tissue samples of children with unilateral ureter-pelvic junction obstruction hydronephrosis with DRF < 20% and DRF > 40% were collected for mRNA-seq,and the obtained differentially expressed genes were overlapped with the interacting proteins of SIRT1.Then the obtained differentially expressed genes were analyzed by GO enrichment and KEGG pathway.At the same time,the differentially expressed genes were obtained by mRNA-seq analysis of RNAi SIRT1 and negative control rat renal tubular epithelial NRK52 E cells,and the mRNA-seq analysis data of the two groups were combined.Neonatal rats were divided into Sham group and UUO group,and mRNA and protein expressions of SIRT1 and overlapping differential genes in neonatal rats were detected by qRT-PCR,immunofluorescence and Western Blot.Second part of this study: The neonatal rats were divided into Sham group,UUO group and SIRT1 inhibitor group,and the gross morphological changes of renal tissues were observed.HE staining was used to detect the pathological morphological changes of renal tissues of newborn rats,Masson staining was used to detect the renal tissue fibrosis of newborn rats,and Western Blot was used to detect the expression levels of fibrosis related proteins in renal tissues.TUNEL method was used to detect the apoptosis level of neonatal rats,Western Blot was used to detect the expression level of apoptosis-related proteins,ELISA was used to detect the content of oxidative stress factors in renal tissues,immunofluorescence was used to detect the ROS level of renal tissues,Western Blot and immunofluorescence were used to detect the expression level of markers of epithelial interstitial transformation.The protein and mRNA expression levels of SIRT1 and its downstream genes were detected by immunofluorescence,Western Blot and qRT-PCR.Rat renal tubular epithelial cells were divided into Control group,TGF-?1 group,TGF-?1+SIRT1 inhibitor + Si-NC group and TGF-?1+SIRT1 inhibitor + Si-Foxo1 group.The interaction between SIRT1 and FOXO1 protein in TGF-? group was detected by CO-IP.Western Blot was used to detect the expression level of fibrosis related proteins in rat renal tubular epithelial cells,flow cytometry was used to detect the apoptosis level of rat renal tubular epithelial cells,Western Blot was used to detect the expression level of apoptosis related proteins in rat renal tubular epithelial cells,and ELISA was used to detect the content of oxidative stress factor in rat renal tubular epithelial cells.ROS levels in rat renal tubule epithelial cells were detected by immunofluorescence,and protein expression levels of epithelial mesenchymal transformation markers in rat renal tubule epithelial cells were detected by Western Blot and immunofluorescence.Third part of this study: Neonatal rats were divided into Sham group and UUO group.Western Blot and immunohistochemistry were used to detect the expression levels of ubiquitin-proteasome system related proteins in neonatal UUO rats.Renal tubular epithelial cells were divided into Control group,TGF-?1 group and TGF-?1+ doxorubicin group.TGF-?1 induced rat renal tubular epithelial cells,and adriamycin activated the ubiquitin-proteasome system.Western Blot was used to detect the expression levels of ubiquitin-proteasome system related proteins,SIRT1 and FOXO1 proteins and fibrosis related proteins,and flow cytometry was used to detect cell apoptosis.Western Blot was used to detect apoptosis-related proteins,immunofluorescence was used to detect ROS levels,ELISA was used to detect oxidative stress factors,and Western Blot and immunofluorescence were used to detect EMT-related proteins.Results: First part of this study: 1427 genes including SIRT1,ADIPOQ and GSTT1 were upregulated in renal tissues of children with unilateral pelvic junction obstruction,compared with those with DRF > 40%.1099 genes including FOXO1,PPARGC1 A and HLA-DRB5 were down-regulated.Overlapping analysis was performed on the differentially expressed genes of mRNA-seq and SIRT1 interaction protein.FOXO1 and PPARGC1 A could be used as the target genes of SIRT1 in UPJO.FOXO1 co-expressed genes are mainly enriched in carboxylic acid metabolism,organic acid metabolism,monocarboxylic acid metabolism,carboxylic acid decomposition and other biological processes,and are mainly involved in metabolic pathways,fatty acid degradation and degradation of valine,leucine and isoleucine.The co-expressed genes of PPARGC1 A are mainly enriched in biological processes such as protein localization in nucleoli,p53-like mediators regulating signal transduction,monocarboxylic acid metabolism and carboxylic acid decomposition.Compared with the negative control cells,2382 genes were up-regulated and 2138 genes were down-regulated in NRK52 E cells of rat renal tubular epithelium.The up-regulated genes were mainly concentrated in biological processes such as tissue development,protein metabolism regulation and transcriptional activity of ubiquitinrelated proteins.SIRT1 mRNA and protein expression were up-regulated,while FOXO1 and PPARGC1 A mRNA and protein expression were down-regulated.Second part of this study: SIRT1 inhibitor reversed hydronephrosis in renal tissues of UUO neonatal rats,improved renal histopathological injury,decreased renal fibrosis and the expression levels of fibrosis related proteins ?-SMA,Fibronectin and Collgen I,and inhibited renal cell apoptosis.Decreased the expression levels of apoptosis related proteins Bax and Cleaved caspase3,increased the expression level of Bcl-2 protein,decreased the level of reactive oxygen species,increased the content of GSH,decreased the content of MDA,and increased the expression level of EMT marker e-cadherin protein in kidney tissue.Decreased the protein expression levels of N-cadherin and Vimentin,decreased the mRNA and protein expression levels of SIRT1,and increased the mRNA and protein expression levels of FOXO1 and PPARGC1 A.SIRT1 interacts with FOXO1 protein,and SIRT1 inhibitors reduce TGF-?1-induced rat renal tubular epithelial cells and the expression levels of fibrosis related proteins ?-SMA,Fibronectin and Collgen I,and reduce TGF-?1-induced apoptosis of rat renal tubular epithelial cells.Decreased the expression levels of apoptosis-related proteins Bax and Cleaved caspase3,increased the expression levels of Bcl-2 protein,decreased the level of reactive oxygen species(ROS),increased the content of GSH and decreased the content of MDA in rat renal tubular epithelial cells induced by TGF-?1.TGF-?1 induced the expression of EMT marker E-cadherin protein in rat renal tubular epithelial cells,decreased the expression of N-cadherin and Vimentin protein,and silencing FOXO1 reversed the effect of SIRT1 inhibitor.Third part of this study: The expression levels of ubiquitin-proteasome system-related proteins Nedd4,Smulf1,MURF and atrogin-1 in newborn UUO rats were decreased.The ubiquitin-proteasome system activator reversed the inhibitory effect of TGF-?1 on the expression of Nedd4,Smulf1,MURF and atrogin-1 proteins in rat renal tubular epithelial cells,decreased SIRT1 mRNA and protein expression,and increased FOXO1 mRNA and protein expression.Decreased the expression levels of fibrosis related proteins ?-SMA,Fibronectin and Collgen I,inhibited apoptosis,decreased the expression levels of apoptosis related proteins Bax and cleaved caspase3,increased the expression level of Bcl-2 protein,decreased the level of reactive oxygen species,and increased the content of GSH.Decreased MDA content,increased EMT marker e-cadherin protein expression level,decreased n-cadherin and Vimentin protein expression level.Conclusion: The ubiquitin-proteasome system mediates SIRT1/FOXO1 signaling pathway to promote pathological damage of renal tissue in UUO neonatal rats,and increase renal fibrosis,renal tubular epithelial cell apoptosis,renal oxidative stress and EMT. |
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