Ginsenoside Rh1 attenuates unilateral ureteral obstruction-induced renal interstitial fibrosis in rats

AIM:To observe the effects of ginsenoside Rh1 (G-Rh1) on unilateral ureteral obstruction (UUO)-induced renal interstitial fibrosis and to investigate the underlying mechanisms. METHODS:Male Sprague-Dawley (SD) rats (n=40) were divided into the following 4 groups:UUO-operated group (UUO group), sham-operated group (sham group), UUO-operated plus a low dose (50 mg·kg^-1·d^-1) of G-Rh1 treatment (low G-Rh1 group) and UUO-operated plus a high dose (100 mg·kg^-1·d^-1) of G-Rh1 treatment group (high G-Rh1 group). The G-Rh1 treatment was carried out by gastric gavage from the next day after the UUO operation once a day for 2 weeks (14 d). Immediately after the final dose of G-Rh1, 24 h urine was collected for the urine protein test, and then the rats were euthanized. The blood was collected for the blood urea nitrogen (BUN) and serum creatinine (SCr) assays, and the kidney was removed for pathological and biochemical evaluations. RESULTS:The levels of 24 h urine protein did not show any significant diffe-rence among the groups, while significantly increased levels of BUN and SCr in UUO group were observed (P<0.05), which was prevented by the treatment with G-Rh1 at both doses in a dose-dependent manner. Pathological evaluation showed the renal tissue damage was obvious in UUO group, which was improved by the treatment with G-Rh1 at both doses. Immunohistochemcial analysis exhibited that UUO increased renal interstitial transforming growth factor-β₁ (TGF-β₁) expression, which was also inhibited by the treatment with G-Rh1 at both doses(P<0.05). Significantly increased protein expression of renal interstitial collagen type I, α-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF) in UUO group was detected, which was suppressed by the treatment with G-Rh1 at both doses. CONCLUSION:G-Rh1 improves UUO-induced renal dysfunction and attenuates interstitial fibrosis, which is mediated via modulation of TGF-β₁-related pro-fibrogenic signaling pathway.     

Role of hypoxia stimulated expression of connective tissue growth factor in renal interstitial fibrosis

AIM: To study the expression of connective tissue growth factor (CTGF) induced by hypoxia, and the role and mechanism of hypoxia on promoting renal interstitial fibrosis. METHODS: Renal interstitial fibrosis was induced by unilateral ureteral obstruction (UUO) in rat animal model for 9 days as in vivo studies; marker of hypoxia-HIF-1α mRNA and protein, the expression of CTGF in the obstructed kidneys were assessed by RT-PCR, immunohistochemistry and Western blotting respectively. In vitro, normal rat kidney interstitial fibroblast cells (NRK-49F) were exposed to hypoxia (1%O2) for up to 6 hours, hypoxia was confirmed by detecting the expression of HIF-1α protein in cells, cellular level of CTGF mRNA and protein were assessed by RT-PCR and Western blotting respectively. RESULTS: Neither HIF-1α mRNA nor HIF-1α protein was expressed in the kidney from sham-operated group of rats. High level of HIF-1α mRNA were occurred, and strongly HIF-1α positive immunostaining were seen in the tubular and interstitial cells in kidney from UUO rats. Expression and location of CTGF protein were paralleled and relevant with the expression of HIF-1α protein in kidney of UUO rats. In cultured NRK-49F cell line, subjected to hypoxia even for 6 hours stimulated the expression of CTGF mRNA and protein. CONCLUSION: Our results indicated that hypoxia could stimulate the expression of CTGF mRNA and protein in kidney from UUO rats, which may in turn contribute to renal interstitial fibrosis.     

Antagonistic effect of thalidomide on TGF-β1-induced change of CTGF gene promoter-binding proteins in HELF cells

AIM:To investigate the antagonistic effect of thalidomide (THD) on the activation of connective tissue growth factor (CTGF) gene promoter induced by transforming growth factor-β₁ (TGF-β₁) in human embryonic lung fibroblasts (HELF). METHODS:Luciferase reporter vector driven by CTGF gene promoter was used to detect the effects of TGF-β₁ and THD on the activity of CTGF gene promoter, and DNA pull-down assay with CTGF gene promoter as a probe was used to analyze the changes of CTGF promoter-binding proteins under different conditions. RESULTS:TGF-β₁ increased the activity of reporter driven by CTGF gene promoter (P<0.01), while THD significantly inhibited TGF-β₁-induced increase in the reporter activity dose-dependently (P<0.01). At the same time, THD had inhibitory effect on the TGF-β₁-induced change of CTGF gene promoter-binding proteins (P<0.05). CONCLUSION:Regulation of CTGF gene promoter-binding proteins takes part in TGF-β₁-induced activation of CTGF gene promoter, while THD has antagonistic effect on this process.     

Effects of uremic serum of different molecular weight groups ongene and protein expression of connective tissue growth factor gene and proteinexpression in human renal tubular epithelial cells

AIM:To investigate the effects of uremic serum of different molecular weight groups on gene and protein expression of connective tissue growth factor (CTGF) in human renal tubular epithelial cells.METHODS:The serum from 40 chronic renal failure patients and 20 healthy volunteers were collected and uremic serum was segregated to three groups: >10 000 D,5 000-10 000 D,<5 000 D by 10 000 D and 5 000 D molecular weight Centricon Plus 20 Centrifugal Filter Devices.The protein expression of CTGF was examined by Western blotting.The mRNA expression of CTGF was detected by RT-PCR.RESULTS:CTGF gene expression were increased in 2.5%-20% uremic serum groups compared with that in normal control group,and it was the highest in 10% uremic serum groups.CTGF gene expression was increased significantly in molecular weight >10 000 D and 5 000-10 000 D groups,and the highest was in >10 000 D group,but it was no significant difference in <5 000 D group compared with that in normal control group.CTGF protein was increased in different molecular weight uremic serum groups compared with that in normal control group,and gradually increased following the increasing of uremic serum concentration and it was the highest in molecular weight >10 000 D group.CONCLUSION:Human renal tubulointerstitial fibrosis was accelerated significantly by uremic toxin,especially molecular weight >10 000 D uremic toxin through promoting the gene and protein expression of CTGF in renal tubular epithelial cells in patients with chronic renal failure.     

Expression of low density lipoprotein receptor-related protein in renal interstitial fibrosis

AIM: To understand the expression of low density lipoprotein receptor-related protein (LRP) in tubulointerstitial fibrosis of unilateral ureter obstruction (UUO) rats. METHODS: The localization of LRP within kidneys were assessed by immunohistochemical staining, the protein level of LRP and connective tissue growth factor (CTGF) in kidney were analysised by Western blot. RESULTS: The location of LRP positive-staining and the protein level of LRP were in the same tendency with CTGF, the level of LRP had strong correlationship with the level of CTGF (r=0.786, P＜0.01); The expression of LRP was positive correlated with tubular-interstitial injury index (r=0.800, P＜0.01). Enalapril treatment downregulated LRP level at 9 days time point. CONCLUSION: The expression of LRP was increased according to the progression of interstitial fibrosis in UUO rats, implicating that LRP may act as receptor of CTGF to mediate the profibrotic effect of CTGF.     

Up-regulation of miR-133a expression attenuates myocardial fibrosis in spontaneously hypertensive rats

AIM: To investigate the effect of up-regulated expression of microRNA-133a (miR-133a) on myocardial fibrosis in spontaneously hypertensive rats (SHR). METHODS: Wistar-Kyoto (WKY) rats with homologous normal blood pressure served as the normal control group. SHR were divided into SHR group, SHR+ adeno-associated virus (AAV) group and SHR+miR-133a-AAV group randomly. miR-133a carried by miR-133a-AAV was transfected into SHR heart by coronary perfusion. The rat tail artery pressure was monitored. The myocardial collagen deposition was observed by Masson staining. The expression of miR-133a in myocardial tissue was detected by real-time PCR. The protein levels of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) were determined by immunohistochemistry and Western blot. RESULTS: Compared with the WKY rats, the tail artery pressure of the SHR increased significantly. The expression of miR-133a in heart decreased, and the expression levels of TGF-β1 and CTGF increased (P<0.05), and myocardial fibrosis occurred. After up-regulating the expression level of miR-133a in the heart of SHR, the myocardial fibrosis was significantly reduced, and the expression levels of TGF-β1 and CTGF decreased (P<0.05). CONCLUSION: Up-regulation of the miR-133a expression improves myocardial fibrosis induced by hypertension, which may be related to inhibiting the protein expression of TGF-β1 and CTGF in myocardium.     

Expression of Sonic hedgehog pathway-related genes in kidney tissues of rats with unilateral ureteral obstruction

AIM: To investigate the role of Sonic hedgehog (Shh) signaling pathway in renal interstitial fibrosis in the rats with unilateral ureteral obstruction (UUO). METHODS: Forty-eight male Sprague-Dawley rats were divided randomly into sham operation group and UUO model group with 24 rats each. The kidneys were excised on day 3, 7, and 14, and the deposition of collagen fiber in the kidneys was detected with HE and Masson staining. Immunohistochemical analysis was performed to evaluate the expression of Shh signaling pathway-related proteins, including Shh, Smo,Ptch1 and Gli1. The contents of TGF-β₁ and Shh in the kidney tissues were determined by ELISA. Real-time RT-PCR was used to detect the mRNA expression of TGF-β₁, Col I, Col III and Shh signaling-related genes.RESULTS: Fibrosis observed with HE and Masson staining was obviously increased in UUO kidneys, and aggravated as time prolonged. The contents of TGF-β₁, Col I and Col III were also increased. In addition, the expression of Shh, Smo and Gli1 was markedly increased in obstructive kidneys, and the expression of Ptch1 was decreased (P<0.01), suggesting that Shh signaling was activated. The level of Shh in UUO rats was associated with the content of TGF-β₁. CONCLUSION: Shh signaling is activated in the progress of renal interstitial fibrosis in UUO rats, and the possible mechanism triggering the fibrogenic response is that Shh signaling promotes the expression of TGF-β₁.     

Expression of CTGF and its role in the streptozotocin-induced diabetic rat kidney

AIM:To investigate the change of connective tissue growth factor(CTGF) expression and its role in streptozotocin-induced diabetic rat kidney. METHODS:Male SD rats were randomly divided into two groups: control(sham operated rats, group C，n=32) and diabetic rats (group DN，n=35). Rats in each group were sacrificed at 1, 2, 4, and 8 weeks respectively after induction of diabetes. Body weight（BW）, blood glucose（BG）, 24-hour urine volumn（UV）, kidney weight, KW/BW,24-hour urinary albumin excretion (24Ualb), creatinine clearance (Ccr), kidney weight (KW), KW/BW, glomerular area (A_G), proximal tubular area (A_T) and the width of GBM、TBM at each time point were measured. Expression of CTGF and α-SMA were detected by immunostaining. RESULTS:There was a significant increase of 24 h Ualb, Ccr, KW/BW, A_G, V_G and the expression of CTGF in glomeruli and tubuli from week 1 onward in diabetic rats compared with those in group C (P<0.05, P<0.01, respectively), and an increasing expression of α-SMA mainly located in dilated tubuli from week 4 was found in group DN, which was more evident at week 8 accompanied by the decrease in A_T. Diabetic rats also had a significant increase in A_T from week 1 onward, which peaked at week 4. CONCLUSION:In the early stage of DN, the time-dependently upregulated CTGF might mediate the renal hypertrophy, which might be associated with the subsequent tubular epithelial-mesenchymal transdifferentiation (EMT) and renal fibrosis.     

Effect of drug-containing serum of Fushen Jiangzhuo formula on anti-fibrosis factors HGF and BMP-7 in rat renal interstitial fibroblasts with renal tubulointerstitial lesion

AIM: To study the protective effects and mechanism of Fushen Jiangzhuo formula (FSJZ) on the rat renal interstitial fibroblasts with renal tubulointerstitial lesion. METHODS: The serum containing FSJZ and blank control serum were prepared. The rat model of mesangial proliferative glomerulonephritis (MsPGN) was established and extended the time of modeling to 20th weeks for developing tubulointerstitial lesion naturally. The rat renal interstitial fibroblasts at the end of 12, 16, 20 week of modeling were isolated and cultured. The effects of FSJZ on the expression of hepatocyte growth factor (HGF) and bone morphogenetic protein-7 (BMP-7) at mRNA and protein levels in the pathological renal interstitial fibroblasts were determined. RESULTS: The anti-fibrosis factors HGF and BMP-7 were changed in pathological renal interstitial fibroblasts. Renal tubulointerstitial lesion significantly down-regulated the expression of HGF and BMP-7, while the drug containing serum of FSJZ significantly up-regulated the expression of HGF and BMP-7. CONCLUSION: Drug containing serum of FSJZ has protective effect on pathological renal interstitial fibroblasts by regulating the mRNA and protein expression of HGF and BMP-7.     

TAK1 regulates fibrosis of renal tubular epithelial cells by regulating p38 MAPK signaling pathway

AIM:To investigate the effect of transforming growth factor-β (TGF-β) activated kinase 1(TAK1) on renal tubular epithelial fibrosis. METHODS:The renal tubular epithelial cell line HK-2 was used as the research object. After induced by TGF-β1, real-time PCR and Western blot were used to detect the expression of TAK1 in the HK-2 cells. TAK1 shRNA lentivirus was used to infect HK-2 cells, real-time PCR and Western blot were used to determine the interference effect on TAK1 expression in the HK-2 cells with TGF-β1 stimulation. Under the condition of treating with p38 MAPK activator anisomycin, the levels of type I collagen and type Ⅲ collagen in the supernatant, and the protein levels of α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF) and p-p38MAPK^{Thr 180/Tyr 182} in the HK-2 cells with TAK1 knock-down were determined by ELISA and Western blot, respectively. RESULTS:TGF-β1 significantly increased the expression of TAK1 in the HK-2 cells(P<0.05). TAK1 shRNA significantly decreased the expression of TAK1 in the HK-2 cells with TGF-β1 stimulation. Type I collagen and type Ⅲ collagen secreted by the HK-2 cells after treatment with TGF-β1 were increased, the protein levels of α-SMA, CTGF and p-p38MAPK^{Thr 180/Tyr 182} were also increased(P<0.05). Knock-down of TAK1 expression significantly inhibited the secretion of type I and type Ⅲ collagen, reduced the protein levels of α-SMA, CTGF and p-p38MAPK^{Thr 180/Tyr 182} in the TGF-β1-induced HK-2 cells(P<0.05). Treatment with p38 MAPK activator reversed the inhibitory effect of TAK1 knock-down on the secretion of type I and type Ⅲ collagens, and the protein levels of α-SMA, CTGF and p-p38 MAPK^{Thr 180/Tyr 182} in the HK-2 cells(P<0.05). CONCLUSION:Knock-down of TAK1 expression attenuates the TGF-β1 induced fibrosis of renal tubular epithelial cells by inhibiting p38 MAPK signaling pathway.     

Role of connective tissue growth factor in high glucose-induced epithelial-mesenchymal transition in podocytes

AIM: To investigate the role of connective tissue growth factor (CTGF) in high glucose-induced epithelial-mesenchymal transition (EMT) in podocytes. METHODS: The differentiated podocytes cultured under different conditions for 24 h at 37 ℃ were divided into 4 groups: control (5 mmol/L glucose solution), 5 mmol/L glucose solution supplemented with 50 μg/L CTGF, high glucose (30 mmol/L glucose solution) and high glucose supplemented with CTGF (50 μg/L). The morphology of cultured podocytes was observed under phase-contrast microscope. To study the relevant markers of EMT, the mRNA and protein expression was analyzed by real-time RT-PCR and Western blotting, respectively. In addition, the effect of CTGF inhibition with anti-CTGF antibody on high glucose-induced EMT in podocytes was investigated. RESULTS: High glucose induced phenotypic transition in the podocytes from an arborization morphology to a cobblestone morphology. After addition of CTGF to high glucose culture, the podocytes developed a feature of spindle. Under high glucose condition, podocytes underwent EMT, as the epithelial marker (nephrin) was decreased and the mesenchymal marker (desmin) was increased. Exogenous CTGF showed the effect of synergy on high glucose-induced EMT. Moreover, high glucose-induced EMT in podocytes was prevented by CTGF inhibition with anti-CTGF antibody. CONCLUSION: CTGF is involved in high glucose-induced EMT in podocytes. Inhibition of CTGF might prevent podocytes from injury in diabetes.     

Role of connective tissue growth factor in airway remodeled in rats induced by repeated bacterial infection

AIM: To investigate the role of connective tissue growth factor (CTGF) in airway remodeling in rats induced by repeating Pseudomonas aeruginosa (PA) infection. METHODS: The rats were intratracheally injected with PA for 12 times to induce chronic lung inflammation. The pathological changes of the trachea and lungs were observed, and the thickness of the trachea wall and vessel wall was measured. At the same time, the methods of immunochemistry and real-time quantitative RT-PCR were used to determine the protein and mRNA expression of CTGF in the lung tissues. The relevance between pathological changes and CTGF expression was also analyzed. RESULTS: From the 4th week, the thickness of the trachea wall and vessel wall in infectious group was larger than that in NS group (P<0.05). At the 16th week, obvious chronic inflammation in all grade bronchi appeared, the trachea walls were thickened and the lumens were narrowed in the infected animals. The expression of CTGF was significantly up-regulated (P<0.01) with positive correlations to the thickness of the trachea wall and vessel wall (r=0.880, r=0.829,P<0.01). CONCLUSION: Airway remodeling in rats is induced by repeated injection of PA. CTGF may play some roles in the pathogenesis of airway remodeling.     

Relationship between peritubular capillary injury and renal interstitial fibrosis in mice with unilateral ureteral obstruction

AIM: To observe the injury of peritubular capillary (PTC), hypoxia and interstitial fibrosis after unilateral ureteral obstruction (UUO), and to explore the effects of PTC injury and hypoxia on interstitial fibrosis in mouse model of UUO. METHODS: Forty-eight male KM mice were randomly divided into control group and UUO group. On the 1st, 3rd, 7th and 14th days, 6 mice in each group were sacrificed. The changes of pathomorphism in the kidney were observed by HE and Masson staining. The expression levels of thrombospondin-1 (TSP-1), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α), and PTC density were detected by immunohistochemistry. The protein expression of VEGF was also determined by Western blotting. RESULTS: No histological abnormalities of the kidneys were observed in sham-operated mice. The expression of TSP-1 was increased 1 day after UUO, and significantly increased on the 3rd, 7th and 14th days(P<0.05). The expression of VEGF was obviously decreased(P<0.05). PTC density was gradually decreased. The expression of HIF-1α was gradually increased, and renal interstitial area was gradually expanded. PTC density was negatively correlated with the expression of TSP-1 and HIF-1α (r=-0.874 and r=-0.930, respectively). VEGF expression was positively correlated with PTC density (r=0.745). PTC density was negatively correlated with the area of renal fibrosis (r=-0.787). HIF-1α expression was positively correlated with the area of renal fibrosis (r=0.835, P<0.05). CONCLUSION: In mouse UUO model, the expression of TSP-1 is increased. The expression of VEGF is reduced. The peritubular capillary injury and tissue hypoxia are aggravated, and renal interstitial fibrosis area is expanded. Ischemia and hypoxia may play an important role in the progression of UUO.     

Role of miR-219 and TGFBR2 in pathogenesis of renal fibrosis

AIM:To study the role of microRNA-219 (miR-219) in regulation of transforming growth factor-β receptor type 2 (TGFBR2) in renal fibrosis. METHODS:The renal fibrosis patients (n=70) were selected in this stu-dy, and 20 cases of healthy people were selected as control group. RT-qPCR was used to detect the expression of miR-219 in the serum of the patients with renal fibrosis and control group, and the expression of miR-219 in NRK49F cells after stimulation with angiotensin Ⅱ(AngⅡ) was detected. The protein expression of α-smooth muscle actin (α-SMA) in the NRK49F cells transfected with miR-219 mimics after stimulation with AngⅡ was determined by Western blot. The potential target gene TGFBR2 of miR-219 was screened and verified by the method of luciferase reporter gene. RT-qPCR and Western blot were used to detected the effect of miR-219 mimics on the expression of TGFBR2 at mRNA and protein levels, and the mRNA expression of α-SMA, connective tissue growth factor (CTGF), type I collagen α1 (COL1A1) and COL3A1 in the NRK49F cells was also detected, respectively. The unilateral ureteral occlusion (UUO) mouse model was established and the expression of miR-219 in the renal tissue was monitored. The morphological change of renal fibrosis was observed in the UUO mice after injection of miR-219, and the mRNA expression levels of COL1A1 and COL3A1 were detected. RESULTS:The expression level of miR-219 in the patients with renal fibrosis was significantly lower than that in control group, and the expression of miR-219 in the UUO mice was decreased significantly (P<0.01). The expression level of miR-219 was significantly decreased in the NRK49F cells after AngⅡ stimulation, and miR-219 mimics inhibited the protein expression of α-SMA(P<0.01). miR-219 mimics had a targeted regulatory effect on TGFBR2 gene, which inhibited the mRNA and protein expression of TGFBR2. miR-219 mimics inhibited the mRNA expression of α-SMA, CTGF, COL1A1 and COL3A1. miR-219 also down-regulated the mRNA expression of COL1A1 and COL3A1 in the UUO mice and inhibited the process of renal fibrosis. CONCLUSION:miR-219 inhibits the development of renal fibrosis by inhibiting the expression of TGFBR2, which may become a new target for the diagnosis and treatment of renal fibrosis.     

Expression of TGF-β1 and Smad2/3 in kidney of STZ-induced diabetic nephropathy rats

AIM: To study the role of TGF-β/Smad pathway in the development of renal fibrosis in diabetic nephropathy.METHODS: Rats were induced to diabetic nephropathy by using tail intravenous injection of STZ.The expression of TGF-β₁, Smad2/3 protein and mRNA in kidney were examined at 2, 4, 8 and 16 weeks after STZ induction.CTGF, collagen-Ⅲ, PAI-1 mRNA expression in kidney at 16 weeks of STZ-induced diabetic nephropathy and normal rats were studied by RT-PCR.RESULTS: Weak TGF-β₁, Smad2/3 protein were detected in normal renal tissues while strong TGF-β₁, Smad2/3 staining were observed in renal tissues of diabetic nephropathy (0.057±0.030/0.223±0.040;0.017±0.010/0.153±0.010, respectively, P<0.05).The TGF-β₁, Smad2/3 protein expression were constantly high with the development of diabetic nephropathy and fibrosis (0.153±0.010, 0.122±0.050, 0.141±0.070 and 0.216±0.030 for 2, 4, 8 and 16 weeks, respectively).The TGF-β₁, Smad2 mRNA expression also increased with the development of diabetic nephropathy (2.86, 3.25 fold compared to control, respectively).The expression of TGF-β₁, Smad2, CTGF, collagen-Ⅲ and PAI-1 mRNA were significantly higher in kidney of 16 week diabetic nephropathy rats than that in normal ones (3.92, 2.95, 1.57, 1.95 and 1.97 folds compare to control, respectively, P<0.05).CONCLUSION: The results indicate that TGF-β₁/ Smad2 pathway activity might play an important role in pathophysiological process of diabetic nephropathy.It may be involved in diabetic renal fibrosis through up-regulation of CTGF and PAI-1 to promote extracellular matrix deposition.     

Effect of Ginkgo biloba extract on the expression of connective tissue growth factor in the initiating stage of fibrosis in lungs of rats

AIM: To study the effect of Ginkgo biloba extract (GbE) on the expression of connective tissue growth factor (CTGF) in the initiating stage of pulmonary fibrosis of rats after administration of bleomycin (BLM).METHODS: The expression of CTGF in lungs was detected by Western blotting. The content of hydroxyproline was assayed by the method of chloramines T. The content of malondialdehyde (MDA) in plasma was investigated by colorimetry.RESULTS: On day 14 after administration of BLM, the contents of CTGF in lungs and MDA in plasma in BLM+NS group were higher than those in NS group, respectively (P<0.05; P<0.01). On day 30 after BLM, the contents of hydroxyproline in lungs and MDA in plasma in BLM+NS group were higher than those in NS group, respectively (both P<0.01). Treatment with GbE ameliorated the above changes induced by BLM. CONCLUSION: GbE ameliorates the up-regulation of CTGF in the initial stage of fibrosis in lungs of rats after administration of BLM. GbE prevents the hyperoxidative injury in lungs of rats after BLM, which might be one of mechanisms underling the effect of GbE on CTGF.