The Mechanism Study Of P66SHC In Renal Fibrosis Of Obstructive Nephropathy

Objective:The purpose of this study was to verify the expression of P66SHC in congenital hydronephrosis and to investigate its role in epithelial cell injury,EMT,and renal fibrosis.We performed RNA-seq on mouse renal tubular epithelial cells overexpressing P66SHC,and analyzed related differential expression,in order to find its downstream mechanism,which may provide future treatment for congenital hydronephrosis.Methods:1.Animal model:Newborn male and female C57/BL mice were randomly divided into complete unilateral ureteral obstruction（CUUO）group and SHAM group within 48h after delivery.In CUUO group they were fixed on the operating table in supine position under isoflurane and oxygen general anesthesia,laparotomy was performed through the left abdominal incision under the microscope.Two 8-0 sutures were used to ligate the ureter（the two operative nodes were 0.3-0.5cm apart）,and the ureter between the two operative nodes was severed.SHAM group only free ureter.The mice were returned to their mothers after they recovered from the anesthesia.Samples were harvested at 7 and 14 days after surgery.2.Cell culture and establishment of EMT model:TCMK-1were maintained in MEM at 37℃with 5%CO₂.The following experiments were performed:when the cells grew to 30%confluent,they were cultured with TGF-β1（final concentration of 10ng/m L）.After 48 hours of culture,the cells were harvested for different experiments.3.The expression of P66SHC in patients was detected by qRT-PCR and Western blot.4.The m RNA expression of P66SHC in animal model and EMT model was detected by qRT-PCR.5.The proteins expression of P66SHC,α-SMA and E-cadherin in animal model and EMT model was detected by Western blot.6.Kidney tissue sections were collected and fixed in 4%paraformaldehyde at room temperature at least overnight,and were applied to Hematoxylin-eosin（H&E）and Masson staining.Immunohistochemistry（IHC）of p66Shc was performed on paraffin sections（4μm）.7.TCMK-1 were transfected with RNAi for 48h.The interference efficiency was verified by qRT-PCR and Western blot.RNAi and TGF-β1（10 ng/ml）were transfected simultaneously in TCMK-1 for 48h.The proteins expression of P66SHC,α-SMA and E-cadherin was detected by Western blot.8.TCMK-1 were transfected with ad V for 48h.The overexpression efficiency was verified by qRT-PCR and Western blot.ad V and TGF-β1（10ng/ml）were transfected simultaneously in TCMK-1 for 48h.The proteins expression of P66SHC,α-SMA and E-cadherin was detected by Western blot.9.TCMK-1 were transfected with RNAi for 48h.Then cells were harvested for Apoptotic flow、ROS flow、JC-1.10.TCMK-1 were transfected with ad V for 48h.Then cells were harvested for Apoptotic flow、ROS flow、JC-1.11.TCMK-1 were transfected with RNAi and ad V.Cell proliferation was measured by CCK8 assay at 12h,24h,48h,and 72h.12.Mice in CUUO group were intraperitoneally injected with in vivo si RNA,and samples were taken 14 days later.MASSON staining was performed and protein expressions of P66SHC,α-SMA,E-cadherin,Bax and Bcl2 were detected by Western blot.13.Statistical analysis was performed using SPSS.Two-way ANOVA was used for cell proliferation curves.Student’s t-test and one-way ANOVA test were used to determine statistical differences between the two groups.All values are expressed as mean±standard deviation.Experiments were performed with at least three replicates,and significance was set at P<0.05.Results:1.The results of qRT-PCR and Western blot showed that the expression of P66SHC was increased in the kidney tissues of congenital hydronephrosis patients 2.qRT-PCR results showed that the expression of P66SHC in EMT cell model was higher than that in control group after TGF-β1 induction for 48h（P<0.05）.In animal model,the expression of P66SHC in CUUO group was up-regulated compared with that in SHAM group on the same day,and the expression of P66SHC gradually increased with the increase of obstruction time（P<0.05）.3.Western blot results showed that the EMT cell model was successfully constructed,which showed increasedα-SMA expression and decreased E-cadherin expression.And P66SHC was up-regulated（P<0.05）.In animal model,α-SMA and P66SHC expressions were increased in CUUO group,while E-cadherin expression was decreased（P<0.05）.With the increase of obstruction days,the expression changes of related molecules were more obvious.4.HE staining showed that the boundary between the renal cortex and medulla was blurred,atrophic and deformed.The renal tubule lumen was dilated and the structure was destroyed.The nephron was reduced in CUUO group.MASSON staining showed that collagen fiber deposition was increasing with the increasing of obstruction days.IHC staining showed that the P66SHC positive signal was located in the cytoplasm of renal tubule cells,and brownish yellow or brown particles could be seen in the cytoplasm.5.After knockdown the expression of P66SHC in cells,the results of CCK8 showed that cell proliferation was promoted（P<0.05）.Flow cytometry showed that apoptosis decreased（P<0.05）and ROS production decreased（P<0.05）.The results of JC-1 staining showed that the mitochondrial damage was alleviated（P<0.05）.6.After overexpressing P66SHC,the cell proliferation was inhibited by CCK8（P<0.05）.Flow cytometry showed that apoptosis increased（P<0.05）and ROS production increased（P<0.05）.The results of JC-1 staining showed that P66SHC increased mitochondrial damage（P<0.05）.7.After transfecting RNAi and ad V of P66SHC in TCMK-1 EMT models,Western blot results showed that the increase of P66SHC expression would aggravate the EMT process,which was manifested as the increase ofα-SMA and the decrease of E-cadherin（P<0.05）.When P66SHC expression was inhibited,the increase ofα-SMA was inhibited,and the downregulation of E-cadherin was decreased（P<0.05）.8.After inhibiting P66SHC expression in CUUO group mice,MASSON staining showed that renal fiber deposition was reduced.Western blot results showed that after inhibiting the expression of P66SHC in kidney,α-SMA expression was decreased,E-cadherin expression was increased（P<0.05）,EMT process was alleviated,the ratio of Bax to Bcl2 was decreased（P<0.05）,and apoptosis level was decreased.9.After overexpression of P66SHC,RNA-seq was performed,and GO analysis,KEGG analysis and GSEA analysis were performed.Conclusion:The results showed that the expression of P66SHC was increased in patients,CUUO mice and EMT-producing cells.In renal tubular epithelial cells,P66SHC acts as a damage molecule to promote ROS generation、mitochondrial damage and apoptosis,and inhibit cell proliferation.Knockdown of P66SHC in vitro inhibited EMT levels,while overexpression of P66SHC promoted EMT progression.After inhibiting P66SHC in vivo,the level of renal fibrosis and apoptosis was reduced.By analyzing the results of RNA-seq,we found that P53 signaling pathway,Calcium signaling pathway and m TOR signaling pathway had a significant difference.