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Abstract Most chronic kidney disease inevitably progress to renal fibrosis. Tubular epithelial- to-mesenchymal transition (EMT) is recognized to play major roles in renal fibrosis. Oxymatrine (OM) is a major alkaloid component found in a Chinese herb Sophora roots and has many effects. The aim is to investigate the effect of OM on renal tubular EMT and elucidate its mechanism. Mice underwent unilateral ureteral obstruction (UUO) followed by intraperitoneal injection of OM (120 mg/kg) or control vehicle. Human kidney proximal tubular cell line (HK-2) was used and EMT was induced with 5 ng/mL of transforming growth factor-β1 (TGF-β1). In vivo, renal tubulointerstitial fibrosis was induced and E-cadherin was down-regulated, while the expressions of fibronectin (FN), α-smooth muscle actin (α-SMA), TGF-β1 and its type I receptor (TGF-βRI) were up-regulated in UUO mice. In contrast, OM significantly ameliorated renal fibrotic lesions and attenuated the expressions of FN, α-SMA, TGF-β1 and TGF-βRI, but increased E-cadherin in the obstructed kidneys. In vitro, OM abolished TGF-β1-mediated E-cadherin suppression and FN, α-SMA and TGF-βRI induction in HK-2 cells in a dose-dependent manner. These observations strongly suggest that the renal protective effects of OM could be mediated by prevention of EMT and manifested as suppression of TGF-β1 and TGF-βRI expressions.
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BACKGROUND: Renal tubulointerstitial fibrosis is a common feature of the final stage of nearly all cause types of chronic kidney disease. Although classic peroxisome proliferator-activated receptor γ (PPARγ) agonists have a protective effect on diabetic nephropathy, much less is known about their direct effects in renal fibrosis. This study aimed to investigate possible beneficial effects of lobeglitazone, a novel PPARγ agonist, on renal fibrosis in mice. METHODS: We examined the effects of lobeglitazone on renal tubulointerstitial fibrosis in unilateral ureteral obstruction (UUO) induced renal fibrosis mice. We further defined the role of lobeglitazone on transforming growth factor (TGF)-signaling pathways in renal tubulointerstitial fibrosis through in vivo and in vitro study. RESULTS: Through hematoxylin/eosin and sirius red staining, we observed that lobeglitazone effectively attenuates UUO-induced renal atrophy and fibrosis. Immunohistochemical analysis in conjunction with quantitative reverse transcription polymerase chain reaction and Western blot analysis revealed that lobeglitazone treatment inhibited UUO-induced upregulation of renal Smad-3 phosphorylation, α-smooth muscle actin, plasminogen activator inhibitor 1, and type 1 collagen. In vitro experiments with rat mesangial cells and NRK-49F renal fibroblast cells suggested that the effects of lobeglitazone on UUO-induced renal fibrosis are mediated by inhibition of the TGF-β/Smad signaling pathway. CONCLUSION: The present study demonstrates that lobeglitazone has a protective effect on UUO-induced renal fibrosis, suggesting that its clinical applications could extend to the treatment of non-diabetic origin renal disease.
Subject(s)
Animals , Mice , Rats , Actins , Atrophy , Blotting, Western , Collagen Type I , Diabetic Nephropathies , Fibroblasts , Fibrosis , In Vitro Techniques , Mesangial Cells , Peroxisomes , Phosphorylation , Plasminogen Activator Inhibitor 1 , Polymerase Chain Reaction , Renal Insufficiency, Chronic , Reverse Transcription , Transforming Growth Factor beta , Transforming Growth Factors , Up-Regulation , Ureter , Ureteral ObstructionABSTRACT
Objective: To prove whether astrocyte elevated gene-1 (AEG-1) plays a role in high glucose-stimulated Rho kinase activation and epithelial-mesenchymal transition (EMT) in human renal tubular epithelial (HK-2) cells. Methods: The protein levels of AEG-1, alpha-smooth muscle actin, E-cadherin and MYPT1 were determined by Western blot. Results: AEG-1 protein level was upregulated in HK-2 cells stimulated with high glucose. AEG-1 siRNA downregulated Rho kinase protein expression and blocked high glucose-induced EMT. Conclusions: Our results show that AEG-1 acts a key role in high glucose-induced activation of Rho kinase and EMT in HK-2 cells.
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OBJECTIVE@#To prove whether astrocyte elevated gene-1 (AEG-1) plays a role in high glucose-stimulated Rho kinase activation and epithelial-mesenchymal transition (EMT) in human renal tubular epithelial (HK-2) cells.@*METHODS@#The protein levels of AEG-1, alpha-smooth muscle actin, E-cadherin and MYPT1 were determined by Western blot.@*RESULTS@#AEG-1 protein level was upregulated in HK-2 cells stimulated with high glucose. AEG-1 siRNA downregulated Rho kinase protein expression and blocked high glucose-induced EMT.@*CONCLUSIONS@#Our results show that AEG-1 acts a key role in high glucose-induced activation of Rho kinase and EMT in HK-2 cells.
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[ ABSTRACT] AIM:To investigate the effect of paricalcitol ( P) on renal tubulointerstitial fibrosis and the under-lying mechanisms in diabetic nephropathy ( DN) .METHODS:DN rat model was induced by a single intraperitoneal in-jection of streptozotocin after fasting.The animals were randomly divided into 2 groups: the DN rats in paricalcitol-inter-vened group ( group P) were injected intraperitoneally with paricalcitol dissolved in propylene glycol after the day when the model was induced successfully at a dose of 0.4μg/kg (3 times a week);the DN rats in DN group ( group D) were given isopyknic propylene glycol.Normal control group ( group C) was also set up.The samples of blood, urine and renal tissue were collected after intervention of paricalcitol for 12 weeks.The biochemical indexes were measured.The renal tissues were used for pathologic observation and determining the expression of transforming growth factor-β1 (TGF-β1), Wnt-4,β-catenin and Klotho by immunohistochemistry and Western blotting.In addition, the correlation among the above indexes was analyzed.RESULTS:(1) Scr, BUN and 24 h urine protein increased significantly in group D compared with group C, while decreased in group P compared with group D ( P<0.05 ) .( 2 ) The area of renal tubulointerstitial fibrosis in-creased in group D compared with group C, while decreased in group P compared with group D (P<0.05).(3) The ex-pression of Klotho decreased, while the expression of TGF-β1, Wnt-4 and β-catenin increased in group D compared with group C (P<0.05).Compared with group D, the expression of Klotho increased, while the expression of TGF-β1, Wnt-4 andβ-catenin decreased in group P (P<0.05).(4) The expression of Klotho was negatively correlated with the fibrosis area, TGF-β1, Wnt-4 andβ-catenin (P<0.05).CONCLUSION:Paricalcitol inhibits renal tubulointerstitial fibrosis in DN by promoting the expression of renal Klotho, and inhibiting Wnt/β-catenin signaling pathway activation and TGF-β1 synthesis.
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Objective To evaluate the dynamic changes of cell mobility of renal tubular epithelial cells in the course of epithelial-mesenchymal transition(EMT) and their effect on cell cycle.Methods NRK-52E cells were cultured in vitro and treated with 5 μg/L transforming growth factor(TGF)-β1 to induce EMT.The cell mobility was assessed by using Transwell chamber assay and flow cytometry (FCW) after being treated with TGF-β1 for 4 h,8 h,12 h,24 h and 48 h.The proliferative cell cycle of NRK-52E cells were evaluated by using the FCW.Results 1.EMT was successfully induced by TGF-β1.After being treated by TGF-β1 (5 μg/L),the morphological changes of NRK-52E cells were found with loose cell arrangement and elongated fusiform change in cells body.Meanwhile,after getting treated by TGF-β1,the expressions of E-cadherin protein(epithelial marker) of NRK-52E cells were significantly decreased with time-dependent (P ＜ 0.05),while the expressions of α-smooth musle actin (α-SMA) (mesenchymal cell marker)were significantly increased with time-dependent (P ＜ 0.05).2.The Transwell chamber assay showed that compared with the control group,the cell mobility in the group treated with TGF-β1 was significantly enhanced from 12 h after getting treated with TGF-β1 (P ＜ 0.01).3.The proliferative cell cycle of NRK-52E cells showed no significant difference after being treated with TGF-β1 (P ＞ 0.05).Conclusions The migration ability of the NRK-52E cells are increased incessantly in the course of EMT,which is induced by TGF-β1 without the influence of cell proliferation in vitro.
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Renal tubulointerstitial fibrosis is the final common pathway in end-stage renal disease.Whereas molecular mechanisms underlying fibrogenesis are still not completely understood.To know the latest pathogenesis and treatment methods is of important significance to prevent disease progression and save life.
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ObjectiveTo investigate the influence of TGF-β receptor subtypes expression and their downstream signaling Smad proteins on rat renal interstitial fibrosis induced by unilateral ureteral obstruction(UUO).MethodsA total of 90 rats were randomly divided into three groups:normal control(CON),sham operation (SOR) and UUO group,and sacrificed 1,3,7,14 and 21 days after operation.Serum creatinine and urea nitrogen were detected to assess renal function.PAS and Masson staining were performed to observe histological damage in the kidneys.Quantitative RT-PCR was used to define expression of mRNA encoding TGF-β receptor subtypes and their downstream signaling Smad proteins in kidney tubular cells.Real-time PCR,Western blotting and immunofluorescence were used to monitor the time-related expression of the TGF-β receptor subtypes and their downstream signaling Smad proteins in kidney.ResultsCompared with the CON group,serum creatinine and urea nitrogen in UUO groups increased at day 3 after operation (P＜0.05) and reached their peak 21 days after operation (P＜0.01).Obvious inflammatory cell infiltration was observed in UUO group 3 days after operation,while renal tubular atrophy and renal interstitial fibrosis were observed in UUO group14 days after operation.The mRNA expressions of ALK-5,ALK-7 and TGF-βR Ⅱ increased significantly in UUO group 3 days after operation (all P＜0.05) and reached their peaks 14 days after operation (all P＜0.01).The mRNA expression of ALK-6 decreased significantly in UUO group 3 days after operation(P＜0.05) and reached its lowest level 14 days after operation (P＜0.01).The changes in the protein level of those receptors were consistent with their mRNA expressions.The protein expressions of Smad2/3 and p-Smad2/3 increased significantly in UUO group at day 3(all P＜0.05) and reached their peak at day 14 after operation(all P＜0.01).ConclusionExpressions of TGF-β receptor subtypes ALK-5,ALK-6,ALK-7,TGF-βR Ⅱ and their downstream signaling Smad2 and Smad3 proteins may influence the progress of renal interstitial fibrosis,tubular atrophy and inflammatory cell infiltration in UUO model rats.
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Objective To examine the expression of TGF-?1/Smads signaling pathway in renal tubulointerstitial fibrosis.Methods Male SD rats were divided into control,sham operation and tubulointerstitial fibrosis groups.They were sacrificed at day 3,7,14,28 after operation.Use masson coloration to examine the area of pathological changes.The level of TGF?1,p-Smad2/3 and smad7 protein was examined by immunohistochemistry staining and Western blot.The level of TGF-?1 mRNA and smad7 mRNA was analyzed by RTPCR.Results Compared with sham operation group,TGF-?1 and p-Smad2/3 were significantly increased in UUO rats,while smad7 protein reduced in it.The mRNA of TGF-?1 and smad 7 increased from day 3 to day 28.Smads protein plays an important role in the synthesis and accumulation of extracellular matrix in tubulointerstitial area.The reduction of smad 7 protein may be a major cause of the interstitial fibrosis in this model.Conclusion TGF-? /Smads protein pathway perhaps is essential in the development of tubulointerstitial fibrosis.
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Objective To observe the effect of PI3K/AKT signal passageway on renal tubulointerstitial fibrogenesis of UUO rats.Methods Thirty Sprague Dawley rats were divided into two groups,the sham-operated group and the uuo model group.The model group's left ureter was dissociated to be obstructed.The sham-operated group was dissociated left ureter only without obstruction.Five rats of each group were killed respectively at 5,10,15(days after) surgery.Histological changes of renal tubular interstitium was observed by HE and Masson staining.Immunohistochemistry was performed on left renal tubular interstitium for AKT1,phospho-AKT1,TGF-?1 in renal(tubulointerstitum) at each time point.Results AKT1,phospho-AKT1and TGF-?1 were faintly stained in the sham-operated kidneys.Immunostaining of AKT1,phospho-AKT1 and TGF-?1 in renal tubulointerstitium increased(progressively) starting from 5 to 15 days after UUO(vs sham-operated group,P
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Objective: To observe the protective effect of BushenHuoxueFang on renal interstitial fibrosis in rats and try to explore the mechanism. Methods: The renal obstruction model of rats was established by unilateralureteral obstruction ,and then they were divided into four groups randomly, including sham-opration group,model group,Lotensin group, BushenHuoxueFang high-dose and low dose group.The morphological changes of kidney tissue were observed,the level of PDGF-BB was detected by Western-blot on the third,seventh,fourteenth and twenty first,twenty eighth day Results: Compared with the sham-operation group, there were different degrees of kidney lesions in UUO group and the treatment groups. The level of PDGF-BB in UUO group was significantly higher than that in sham-operation group from the third day(P
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Objective: To observe the protective effect of WenyangHuoxueFang on renal interstitial fibrosis in rats and try to explore the mechanism.Methods: The renal obstruction model of rats was established by unilateral ureteral obstruction(UUO) ,and then they were divided into four groups randomly,including UUOW group(treated with WenyangHuoxueFang),UUOF group(treated with Fosinopril),UUO group and the sham-operation group.The morphological changes of kidney tissue were observed,the level of RBP was detected by ELISA,the expression of MMP-3 and TIMP-1 was detected by immuno-histochemistry.Results: Compared with the sham-operation group,there were different degrees of kidney lesions in UUO group and the treatment groups.The level of RBP in UUO group was significantly higher than that in sham-operation group.Compared with UUO group,the RBP expression in UUOF group and UUOW group was significantly lower(P0.05).The expressions of MMP-3 and TIMP-1 in UUO group were increased,and the amplitude of increasing expression of TIMP-1 was higher than that of MMP-3.The treatment groups had higher expression of MMP-3(P
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In order to explore the role of connective tissue growth factor (CTGF) in the pathogenesis of renal tubulointerstitial fibrosis, 48 Wistar rats were randomly divided into sham-operated and unilateral ureteral obstruction (UUO) group. On the postoperative day 1, 3, 7 and 14, the rats were killed and the kidneys were removed. The renal tubulointerstitial injury index was evaluated according to the MASSON staining. The mRNA levels of CTGF, transforming growth factor-β1(TGF-β1), collagen Ⅰ (col Ⅰ ), and plasminogen activator inhibitor-1 (PAI-1) were detected using reverse transcriptional-polymerase chain reaction (RT-PCR). Immunohistochemistry was performed to evaluate the protein expression of the above factors, and the relations among them were analyzed. Quantitative expression of CTGF protein in the kidneys was also assessed using Western blot. The results showed that TGF-β1 mRNA level was increased at first day after UUO, followed by a marked elevation of CTGF mRNA level, which began to increase 3 days after UUO (P＜0.01). With the progression of the disease, the mRNA expression of CTGF, col Ⅰ and PAI-1 was increased progressively. Immunohistochemistry revealed that the CTGF protein expression was significantly increased in fibrotic areas and tubular epithelial cells 3 days after UUO. On the post-UUO day 7, the protein level of CTGF was positively related to the renal tubulointerstitial injury index (r =0.62, P＜0.01), the expression of TGF-β1 (r=0.85, P＜0.01), col Ⅰ (r=0.78, P＜0.01),and PAI-1(r=0.76, P＜0.01). Upon Western blot analysis, CTGF protein expression began to increase 3 days after UUO, and appeared progressively throughout the time course (P＜0. 01, as compared with sham-operated group). It is concluded that CTGF can be induced by TGF-β and mediate various profibrotic actions of this cytokine, such as increasing extracellular matrix (ECM)synthesis and decreasing ECM degradation. The increased expression of CTGF may play a crucial role in the development and progression of tubulointerstitial fibrosis.
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10 000 u,5 000~10 000u and 10 000 u group,and the gene expression was slightly increased in molecular weight 5 000～10 000 u group,but no significant difference of gene expression and protein secretion in 10 000 u uremic toxin,through promoting the gene expression and protein excretion of TGF-?_1 of renal tubular epithelial cells in patients with chronic renal failure.