| Renal fibrosis is often considered to be a potential pathological process of chronic kidney disease(CKD),which is the final pathological change of chronic kidney injury or adaptive repair of injury.The factors that cause renal fibrosis are also diverse.In addition to common chronic diseases such as diabetes and high blood pressure,kidney damage caused by various causes is an important cause of long-term renal fibrosis.Epithelial mesenchymal transition(EMT)plays an important role in renal fibrosis.In this process,the gradual loss of epithelial markers such as E-cadherin and the increase in mesenchymal markers such as α-smooth muscle actin(α-SMA)are characteristic features.In the long-term repeated or severe stimulation,renal tubular epithelial cells(TECs)damage and dedifferentiation,interstitial inflammatory cell infiltration,and then secrete various pro-inflammatory and pro-fibrotic cytokines.Under the combination of the above factors,the number of renal interstitial myofibroblasts increased significantly,leading to excessive deposition of extracellular matrix(ECM)-such as fibronectin(FN)and collagen I(Collagen I,COL-I),normal kidney tissue is gradually replaced,and then fibrosis is formed.In many studies of fibrosis mechanisms,TGF-β/Smad signaling pathway plays a key role,and transforming growth factor β(TGF-β)is a key cytokine.Cathepsin S(CTSS)is derived from the lysosomal cysteine protease family and is involved in the degradation of various ECM components such as collagen,laminin,fibronectin and elastin.In our previous study of the effects of pressure perfusion on different degrees of hydronephrosis,CTSS was found to be significantly elevated.Therefore,we speculate that CTSS may be involved in the occurrence of renal fibrosis and further participate in its regulation.In this study,we explored the role of CTSS in the fibrosis caused by different degrees of hydronephrosis caused by pressure perfusion and performed a specific mechanism analysis.The experimental results showed that the expression of CTSS increased significantly in the renal fibrosis model and the TGFβ-induced renal tubular epithelial fibrosis model.CTSS can regulate the deposition of ECM,and can also affect the fibrosis by interfering with the EMT process through the TGF-β/Smad signaling pathway.In summary: Our study shows that CTSS can affect ECM deposition and EMT,and can regulate renal fibrosis through TGF-β/Smad signaling pathway.Part 1: Fibrosis changes in kidneys with varying degrees of HydronephrosisObjective: To investigate the changes of fibrosis in different degrees of hydronephrosis,and further give gradient pressure perfusion to observe the effect of perfusion pressure on fibrosis in kidneys with hydronephrosis.Method: Different degrees of hydronephrosis models were constructed by unilateral ureteral ligation(UUO).Ultrasound examination confirmed the formation of mild(M group)and severe(S group)hydronephrosis after 3d and 7d,respectively.Each group was given gradient pressure perfusion(0,20,60,100 mm Hg)and divided into four subgroups(M0-M3,S0-S3)according to the perfusion pressure.The degree of fibrosis was assessed by Masson staining,and detection the expression of of alpha-smooth muscle actin(α-SMA),fibronectin(FN),and E-cadherin(E-Ca)and collagen I(Collagen I,COL-I)by immunohistochemistry,western blot,and q PCR.Result: Compared with the control group(N group),the expression levels of COL-I,FN and α-SMA in the M0 group and the S0 group were significantly increased,and E-Cadherin was significantly decreased.The M group was given gradient perfusion pressure,and there was no significant change in the expression of COL-I and FN in each subgroup.When the S group was exposed to gradient perfusion pressure,the expression levels of COL-I and FN increased significantly when the perfusion pressure reached 60 mm Hg.In the M group and the S group,when the pressure reached 20 mm Hg,a significant increase in the expression level of α-SMA was observed.In the S group,when the perfusion pressure reached 20 mm Hg,the expression of E-cadherin was remarkably lowered,and when the pressure reached 60 mm Hg,the same change was observed in the M group.Conclusion: Compared with mild hydronephrosis,the fibrosis of severe hydronephrosis is more severe;and severe hydronephrosis is less tolerant to perfusion pressure.Part 2 Relationship between CTSS and renal fibrosis with different degrees of hydronephrosisObjective: To investigate the role of CTSS in both fibrosis of renal with hydronephrosis and tubular epithelial fibrosis induced by TGF-β.Method: Different degrees of hydronephrosis models were constructed by unilateral ureteral ligation(UUO).Ultrasound examination confirmed the formation of mild(M group)and severe(S group)hydronephrosis after 3d and 7d,respectively.The degree of fibrosis was assessed by Masson staining.Detect the expression of alpha-smooth muscle actin(α-SMA),fibronectin(FN),collagen I(Collagen I,COL-I)and E-cadherin(E-Ca)by immunohistochemistry,western blot,and q PCR.The inhibitor group(Mi,Si)was given the same treatment as the CTSS-specific inhibitor LY3000328 was given.The degree of fibrosis and changes in fibrosis-related indicators were also examined.In vitro,immunohistochemistry,western blot and q PCR were used to detect The expression of α-SMA,E-Ca,FN and COL-I before and after si RNA-CTSS were used in the fibrosis model of mouse renal tubular epithelial cells(TCMK-1)induced by TGF-β.Result: The expression levels of CTSS were significantly increased in the M group and the S group compared with the N group,but a significant increase in the expression levels of COL-I and FN was observed only in the S group.Compared with the N group,the expression levels of TGF-β1 and α-SMA in the M group and the S group were significantly increased,while the expression of E-cadherin was significantly decreased.When CTSS expression was inhibited,the expression levels of TGF-β1,α-SMA,fibronectin and COL-I increased to different degrees,while the expression of E-cadherin decreased.A significant increase in the amount of CTSS expression was observed in the TGF-β1-stimulated TCMK-1 cell line.The expression levels of α-SMA,COL-I and FN were significantly increased while the expression of E-cadherin was significantly decreased.The opposite change occurred after si RNA-CTSS intervention.Conclusion: In the model of hydronephrosis and TGF-β-induced fibrosis,the expression of CTSS was significantly increased;inhibition of CTSS can aggravate fibrosis.It is suggested that there is a close relationship between CTSS and renal fibrosis.Part 3 Molecular mechanism of CTSS regulating renal fibrosisObjective: Investigate the regulatory role of CTSS in TGF-β/Smad signaling pathway,and to determine the specific molecular mechanism of CTSS in regulating of UUO-induced renal fibrosis and TGF-β-induced fibrosis.Method: UUO method was used to construct different degrees of hydronephrosis models.The expressions of TGF-β,Smad2,Smad3,p Smad2 and p Smad3 were detected by western blot and q PCR.Mean to assess the activation of the TGF-β/Smad signaling pathway.In the inhibitor group(Mi group,Si group),CTSS-specific inhibitors were given after ureteral ligation,and the expressions of TGF-β,Smad2,Smad3,p Smad2 and p Smad3 were detected by western blot and q PCR.TGF-β induced fibrosis model was established.The expressions of Smad2,Smad3,p Smad2 and p Smad3 were detected by western blot and q PCR before and after si RNA-CTSS intervention.Result: In the UUO-induced renal fibrosis model,the expression levels of TGF-β1,Smad2,Smad3 and p Smad2/3 were significantly increased in the M group and the S group compared with the N group.When CTSS expression was inhibited,the expression levels of TGF-β1,Smad2,Smad3 and p Smad2/3 increased to varying degrees.A significant increase in CTSS was observed in the TGF-β1-stimulated TCMK-1 cell line.ECM deposition and EMT are also enhanced.The opposite result occurred after si RNA-CTSS intervention.Conclusion: Hydronephrosis,perfusion pressure,and CTSS can all affect the TGF-β signaling pathway;thus affecting the progression of renal fibrosis... |
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