In order to investigate the effects of connective tissue growth factor (CTGF) antisense oligodeoxynucleotide (ODN) on plasminogen activator inhibitor-1 (PAI-1) expression in renal tubular cells induced by transforming...In order to investigate the effects of connective tissue growth factor (CTGF) antisense oligodeoxynucleotide (ODN) on plasminogen activator inhibitor-1 (PAI-1) expression in renal tubular cells induced by transforming growth factor β1 (TGF-β1) and to explore the role of CTGF in the degradation of renal extracellular matrix (ECM), a human proximal tubular epithelial cell line (HKC) was cultured in vitro. Cationic lipid-mediated CTGF antisense ODN was transfected into HKC. After HKC were stimulated with TGF-β1 (5 μg/L), the mRNA level of PAI-1 was detected by RT-PCR. In-tracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 in the media was determined by Western blot. The results showed that TGF-β1 could induce tubular CTGF and PAI-1 mRNA expression. The PAI-1 mRNA expression induced by TGF-β1 was significantly inhib-ited by CTGF antisense ODN. CTGF antisense ODN also inhibited intracellular PAI-1 protein syn-thesis and lowered the levels of PAI-1 protein secreted into the media. It was concluded that CTGF might play a crucial role in the degradation of excessive ECM during tubulointerstitial fibrosis, and blocking the biological effect of CTGF may be a novel way in preventing renal fibrosis.展开更多
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 obstruct...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), collagenⅠ (colⅠ), 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, colⅠ 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), colⅠ (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.展开更多
In order to investigate the regulatory role of connective tissue growth factor （CTGF） on production of RANTES （regulated on activation, normal T cell expressed and secreted） in rat glomerular mesangial cells, and ...In order to investigate the regulatory role of connective tissue growth factor （CTGF） on production of RANTES （regulated on activation, normal T cell expressed and secreted） in rat glomerular mesangial cells, and the modulatory effect of lipoxin A4（LXA4） on action of CTGF, and to explore the mechanisms of action of CTGF and LXA4, cultured rat mesangial cells were treated with CTGF, with or without preincubation with LXA4. Expression of mRNA was analyzed by RT-PCR. Protein of RANTES in the supernatants was determined by ELISA. Monocyte transmigration was assessed by in vitro chemotaxis assay. Expression of p42/44 mitogen-activated protein kinase （MAPK）, phosphoinositide 3-kinase （PI3-K） and protein kinase B （PKB） was assessed by Western blotting. DNA-binding activity of nuclear factor-κB （NF-κB） was determined by electrophoretic mobility shift assay （EMSA）. To observe whether transfection of LXA4 receptor homologue gene （LRHG） into mesangial cells intensified these modulatory effects of LXA4, mesangial cells were transfected with pcDNA3.1/LRHG vector. The results showed that CTGF enhanced the mRNA expression and protein release of RANTES, and the expression of phospho （P）-p42/44 MAPK, P-PI3-K, P-PKB and NF-κB. P-p42/44 MAPK blockade inhibited the CTGF-induced expression of P-p42/44 MAPK and partially decreased the level of RANTES in supernatants. P-PI3-K blockade downregulated the CTGF-stimulated expression of P-PI3-K, P-PKB and NF-κB, and partially decreased the release of RANTES. NF-κB blockade abrogated the CTGF-activated NF-κB and partially decreased the secretion of RANTES. LXA4 dose-dependently inhibited the CTGF-stimulated above action. Transfection of LRHG into mesangial cells intensified these inhibitory effects of LXA4 on CTGF-induced release of RANTES and expression of the P-p42/44 MAPK. In conclusion, LXA4 inhibits CTGF-induced production of RANTES via PI3-K/PKB/NF-κB and p42/44 MAPK-dependent signal pathway, which is mediated by LRHG in rat mesangial cells.展开更多
The effects of mycophenolic acid (MPA) on high glucose-induced expression of transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) in mesangial cells (MC) were investigated. Rat MC were cul...The effects of mycophenolic acid (MPA) on high glucose-induced expression of transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) in mesangial cells (MC) were investigated. Rat MC were cultured in the presence of different concentrations of MPA (1.0 and 10.0 μmol/L) or MPA plus high glucose for 72 h. The expression of TGF-β and CTGF was detected by Western blot. The results showed that high glucose could induce the expression of TGF-β and CTGF in MC, but MPA could inhibit this effects. MPA did not influence the expression of TGF-β and CTGF in normal glucose. It was concluded that MPA might prevent the progression of diabetic nephropathy by inhibiting the expression of TGF-β and CTGF in MC.展开更多
Background Renal hypertrophy has been regarded as the early feature of diabetic nephropathy (DN), which may eventually lead to proteinuria and renal fibrosis. However, the exact mechanism of renal hypertrophy is still...Background Renal hypertrophy has been regarded as the early feature of diabetic nephropathy (DN), which may eventually lead to proteinuria and renal fibrosis. However, the exact mechanism of renal hypertrophy is still unclear. The aim of this study was to investigate the possible association of connective tissue growth factor (CTGF) with renal hypertrophy in uninephrectomized diabetic rats. Methods Seventy-two Sprague-Dawley (SD) rats were randomly divided into two groups: control group (group C, n=32) and diabetic nephropathy (group DN, n=40). Each group was re-divided into 4 subgroups according to the experimental period. The rats were sacrificed at 1, 2, 4, and 8 weeks respectively after induction of diabetes. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) after rats had received uninephrectomy. Blood glucose (BG), body weight (BW), 24-h urinary albumin excretion (24hUalb), kidney weight (KW), KW/BW, glomerular tuft area (AG), glomerular tuft volume (VG), proximal tubular area (AT) at each time point, the width of glomerular basement membrane (GBM) and tubular basement membrane (TBM) at week 8 were measured when the rats were sacrificed. Renal expression of CTGF and p27kip1 were detected by immunohistochemical staining. The relationship between CTGF expression and increasing of VG and AT was analyzed. Results There was a significant increase of 24hUalb, KW, and KW/BW from week 1 onward in diabetic rats compared to those in group C (P<0.05, respectively), diabetic rats also had a significant increase of AG, VG, and AT from week 1 onward. It was also shown that diabetic rats had a thickening of GBM [(245.7±103.0) nm vs (121.8±19.1) nm, P<0.01] and TBM [(767.7±331.1) nm vs (293.0±110.5) nm, P<0.01] at week 8. There was a weak expression for CTGF and p27kip1 in normal glomeruli and tubuli, while a significant increasing expression of CTGF and p27kip1 was found in glomeruli and tubuli in diabetic kidney from week 1 onward (P<0.05, respectively), and the extent of CTGF expression was positively correlated with AG (r=0.92, P<0.05), VG (r=0.86, P<0.05), AT (r=0.94, P<0.01) and positively correlated with the expression of p27kip1 (r=0.96, P<0.01). Conclusion The expression of CTGF increases in diabetic rat kidney at the early stage, which might be an important mediator of renal hypertrophy through arresting cell cycling.展开更多
To observe the effect of high glucose, angiotensin Ⅱ (AngⅡ) and Losartan on the expression of connective tissue growth factor (CTGF) mRNA in cultured mesangial cells (MCs) Methods MCs of SD rats were isolated and cu...To observe the effect of high glucose, angiotensin Ⅱ (AngⅡ) and Losartan on the expression of connective tissue growth factor (CTGF) mRNA in cultured mesangial cells (MCs) Methods MCs of SD rats were isolated and cultured High glucose (30 mmol/L) and AngⅡ (10 -9 , 10 - 7 , and 10 -5 mol/L) were added to the medium for 72 hours to observe the influence on CTGF mRNA expression Losartan of 10 -5 mol/L and AngⅡ of 10 -5 mol/L were added to the medium to observe the effects of Losartan on CTGF mRNA expression stimulated by AngⅡ The expressions of CTGF mRNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR) Results RT-PCR showed that high glucose and AngⅡ up-regulated the expression of CTGF mRNA, and AngⅡ stimulated the expression in a dose-dependent manner Expression of CTGF mRNA induced by AngⅡwas partially suppressed by 10 -5 mol/L Losartan (P<0 05) Conclusions High glucose and AngⅡ can enhance the expression of CTGF mRNA and thus be involved in the process of renal fibrosis Losartan can have a partial fibrogenesis-inhibiting effect, with implications for the treatment of renal展开更多
基金This project was supported by a grant from the Science & Technology Foundation of Hubei Province (No. 2003 AA 301C14).
文摘In order to investigate the effects of connective tissue growth factor (CTGF) antisense oligodeoxynucleotide (ODN) on plasminogen activator inhibitor-1 (PAI-1) expression in renal tubular cells induced by transforming growth factor β1 (TGF-β1) and to explore the role of CTGF in the degradation of renal extracellular matrix (ECM), a human proximal tubular epithelial cell line (HKC) was cultured in vitro. Cationic lipid-mediated CTGF antisense ODN was transfected into HKC. After HKC were stimulated with TGF-β1 (5 μg/L), the mRNA level of PAI-1 was detected by RT-PCR. In-tracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 in the media was determined by Western blot. The results showed that TGF-β1 could induce tubular CTGF and PAI-1 mRNA expression. The PAI-1 mRNA expression induced by TGF-β1 was significantly inhib-ited by CTGF antisense ODN. CTGF antisense ODN also inhibited intracellular PAI-1 protein syn-thesis and lowered the levels of PAI-1 protein secreted into the media. It was concluded that CTGF might play a crucial role in the degradation of excessive ECM during tubulointerstitial fibrosis, and blocking the biological effect of CTGF may be a novel way in preventing renal fibrosis.
文摘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), collagenⅠ (colⅠ), 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, colⅠ 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), colⅠ (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.
文摘In order to investigate the regulatory role of connective tissue growth factor （CTGF） on production of RANTES （regulated on activation, normal T cell expressed and secreted） in rat glomerular mesangial cells, and the modulatory effect of lipoxin A4（LXA4） on action of CTGF, and to explore the mechanisms of action of CTGF and LXA4, cultured rat mesangial cells were treated with CTGF, with or without preincubation with LXA4. Expression of mRNA was analyzed by RT-PCR. Protein of RANTES in the supernatants was determined by ELISA. Monocyte transmigration was assessed by in vitro chemotaxis assay. Expression of p42/44 mitogen-activated protein kinase （MAPK）, phosphoinositide 3-kinase （PI3-K） and protein kinase B （PKB） was assessed by Western blotting. DNA-binding activity of nuclear factor-κB （NF-κB） was determined by electrophoretic mobility shift assay （EMSA）. To observe whether transfection of LXA4 receptor homologue gene （LRHG） into mesangial cells intensified these modulatory effects of LXA4, mesangial cells were transfected with pcDNA3.1/LRHG vector. The results showed that CTGF enhanced the mRNA expression and protein release of RANTES, and the expression of phospho （P）-p42/44 MAPK, P-PI3-K, P-PKB and NF-κB. P-p42/44 MAPK blockade inhibited the CTGF-induced expression of P-p42/44 MAPK and partially decreased the level of RANTES in supernatants. P-PI3-K blockade downregulated the CTGF-stimulated expression of P-PI3-K, P-PKB and NF-κB, and partially decreased the release of RANTES. NF-κB blockade abrogated the CTGF-activated NF-κB and partially decreased the secretion of RANTES. LXA4 dose-dependently inhibited the CTGF-stimulated above action. Transfection of LRHG into mesangial cells intensified these inhibitory effects of LXA4 on CTGF-induced release of RANTES and expression of the P-p42/44 MAPK. In conclusion, LXA4 inhibits CTGF-induced production of RANTES via PI3-K/PKB/NF-κB and p42/44 MAPK-dependent signal pathway, which is mediated by LRHG in rat mesangial cells.
文摘The effects of mycophenolic acid (MPA) on high glucose-induced expression of transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) in mesangial cells (MC) were investigated. Rat MC were cultured in the presence of different concentrations of MPA (1.0 and 10.0 μmol/L) or MPA plus high glucose for 72 h. The expression of TGF-β and CTGF was detected by Western blot. The results showed that high glucose could induce the expression of TGF-β and CTGF in MC, but MPA could inhibit this effects. MPA did not influence the expression of TGF-β and CTGF in normal glucose. It was concluded that MPA might prevent the progression of diabetic nephropathy by inhibiting the expression of TGF-β and CTGF in MC.
基金This research is supported by the National Natural Science Fundation (No. 30471732), Jiangsu Provincial Natural Science Fundation (No. 2002052), and the Jiangsu Key Medical Talent Project (No. 2002072).
文摘Background Renal hypertrophy has been regarded as the early feature of diabetic nephropathy (DN), which may eventually lead to proteinuria and renal fibrosis. However, the exact mechanism of renal hypertrophy is still unclear. The aim of this study was to investigate the possible association of connective tissue growth factor (CTGF) with renal hypertrophy in uninephrectomized diabetic rats. Methods Seventy-two Sprague-Dawley (SD) rats were randomly divided into two groups: control group (group C, n=32) and diabetic nephropathy (group DN, n=40). Each group was re-divided into 4 subgroups according to the experimental period. The rats were sacrificed at 1, 2, 4, and 8 weeks respectively after induction of diabetes. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) after rats had received uninephrectomy. Blood glucose (BG), body weight (BW), 24-h urinary albumin excretion (24hUalb), kidney weight (KW), KW/BW, glomerular tuft area (AG), glomerular tuft volume (VG), proximal tubular area (AT) at each time point, the width of glomerular basement membrane (GBM) and tubular basement membrane (TBM) at week 8 were measured when the rats were sacrificed. Renal expression of CTGF and p27kip1 were detected by immunohistochemical staining. The relationship between CTGF expression and increasing of VG and AT was analyzed. Results There was a significant increase of 24hUalb, KW, and KW/BW from week 1 onward in diabetic rats compared to those in group C (P<0.05, respectively), diabetic rats also had a significant increase of AG, VG, and AT from week 1 onward. It was also shown that diabetic rats had a thickening of GBM [(245.7±103.0) nm vs (121.8±19.1) nm, P<0.01] and TBM [(767.7±331.1) nm vs (293.0±110.5) nm, P<0.01] at week 8. There was a weak expression for CTGF and p27kip1 in normal glomeruli and tubuli, while a significant increasing expression of CTGF and p27kip1 was found in glomeruli and tubuli in diabetic kidney from week 1 onward (P<0.05, respectively), and the extent of CTGF expression was positively correlated with AG (r=0.92, P<0.05), VG (r=0.86, P<0.05), AT (r=0.94, P<0.01) and positively correlated with the expression of p27kip1 (r=0.96, P<0.01). Conclusion The expression of CTGF increases in diabetic rat kidney at the early stage, which might be an important mediator of renal hypertrophy through arresting cell cycling.
基金ThestudywassupportedbythegrantsfromtheNationalNatureScienceFoundationofChina (No 39870 2 97)
文摘To observe the effect of high glucose, angiotensin Ⅱ (AngⅡ) and Losartan on the expression of connective tissue growth factor (CTGF) mRNA in cultured mesangial cells (MCs) Methods MCs of SD rats were isolated and cultured High glucose (30 mmol/L) and AngⅡ (10 -9 , 10 - 7 , and 10 -5 mol/L) were added to the medium for 72 hours to observe the influence on CTGF mRNA expression Losartan of 10 -5 mol/L and AngⅡ of 10 -5 mol/L were added to the medium to observe the effects of Losartan on CTGF mRNA expression stimulated by AngⅡ The expressions of CTGF mRNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR) Results RT-PCR showed that high glucose and AngⅡ up-regulated the expression of CTGF mRNA, and AngⅡ stimulated the expression in a dose-dependent manner Expression of CTGF mRNA induced by AngⅡwas partially suppressed by 10 -5 mol/L Losartan (P<0 05) Conclusions High glucose and AngⅡ can enhance the expression of CTGF mRNA and thus be involved in the process of renal fibrosis Losartan can have a partial fibrogenesis-inhibiting effect, with implications for the treatment of renal
