RESUMO
BACKGROUND/AIMS: Fluorofenidone [1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone, AKF-PD], a novel pyridone agent, showed potent antifibrotic properties. The aim of the present study was to investigate the effects of AKF-PD on diabetic nephropathy and kidney fibrosis, and to obtain an insight into its mechanisms of action. METHODS: We administered AKF-PD to diabetic db/db mice for 12 weeks. Moreover, we performed in vitro cultures using murine mesangial cells exposed to high ambient glucose concentrations. RESULTS: AKF-PD reduced renal hypertrophy, mesangial matrix expansion and albuminuria in the db/db mice. The upregulated expression of α1(I)- and α1(IV)-collagen and fibronectin mRNAs, transforming growth factor-ß1 (TGF-ß1), α-smooth muscle actin (α-SMA), and tissue inhibitors of metalloproteinase 1 (TIMP-1) mRNAs and proteins was inhibited by AKF-PD treatment in the renal cortex of db/db mice. The maximal effective dose of AKF-PD was about 500 mg/kg body weight. AKF-PD inhibited the upregulated expression of α1(I)- and α1(IV)-collagens, TGF-ß1, TIMP-1 and α-SMA induced by high glucose concentrations in cultured mesangial cells. CONCLUSIONS: Our data indicate that AKF-PD diminishes the abnormal accumulation of mesangial matrix through the inhibition of upregulated expression of TGF-ß target genes in kidneys of db/db mice, resulting in attenuation of renal fibrosis and amelioration of renal dysfunction despite persistent hyperglycemia. Therefore, AKF-PD, a potent antifibrotic agent, holds great promise in the treatment of diabetic nephropathy.
Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Hipoglicemiantes/farmacologia , Nefropatias/tratamento farmacológico , Rim/fisiopatologia , Piridonas/farmacologia , Albuminas/análise , Animais , Glicemia , Técnicas de Cultura de Células , Colágeno/fisiologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Matriz Extracelular/metabolismo , Fibronectinas/fisiologia , Fibrose/patologia , Fibrose/fisiopatologia , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Hiperglicemia/fisiopatologia , Hipoglicemiantes/uso terapêutico , Rim/patologia , Córtex Renal/fisiopatologia , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos , Camundongos Knockout , Piridonas/uso terapêutico , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/fisiologiaRESUMO
AIM: Fluorofenidone (1-(3-fluorophenyl)-5-methyl-2-(1H)-pyridone) is a novel pyridone agent. The aim of the present study is to investigate the effects of fluorofenidone on angiotensin (Ang)II-induced fibrosis and the involved molecular mechanism in rat proximal tubular epithelial cells. METHODS: NRK-52E cells, a rat proximal tubular epithelial cell line, were incubated with medium containing AngII, with or without nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI), losartan, fluorofenidone (2, 4 and 8 mmol/L) and pirfenidone (8 mmol/L) for 24 h. Cells in the serum-free medium were controls. The expression of three subunits of NADPH oxidase, including p47phox, Nox-4 and p22phox, were determined by real-time reverse transcription polymerase chain reaction (RT-PCR) and western blot. NADPH oxidase activity was measured directly by superoxide dismutase (SOD) inhibitable cytochrome C reduction assay. The generation of reactive oxygen species (ROS) was measured by dichlorofluorescein fluorescence analysis. The mRNA and protein expression of collagen I and transforming growth factor (TGF)-beta1 were determined by real-time RT-PCR and enzyme-linked immunosorbent assay. RESULTS: Fluorofenidone significantly inhibited TGF-beta1 and collagen I expression upregulation induced by AngII or TGF-beta1 respectively. Moreover, fluorofenidone greatly reduced the elevation of expression and activity of NADPH oxidase and inhibited ROS generation induced by AngII in rat proximal tubular epithelial cells. These responses were also attenuated by DPI, losartan, and pirfenidone. CONCLUSION: Fluorofenidone acted as an anti-oxidative and anti-fibrotic agent through the mechanisms of blocking NADPH oxidase-dependent oxidative stress and inhibiting TGF-beta1 expression in rat proximal tubular epithelial cells.
Assuntos
Colágeno Tipo I/antagonistas & inibidores , Fibrose/tratamento farmacológico , NADPH Oxidases/fisiologia , Piridonas/farmacologia , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Animais , Células Cultivadas , Colágeno Tipo I/genética , Rim/patologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , NADPH Oxidases/genética , Ratos , Superóxidos/metabolismo , Fator de Crescimento Transformador beta1/genéticaRESUMO
OBJECTIVES: The development of novel antifibrotic agent candidates for the treatment of diabetic nephropathy. The present study was designed to investigate the potential mechanism of fluorofenidone involving the downregulation of CTGF expression induced by TGF-beta1 and the related signaling pathway in mouse mesangial cells (MMCs). METHODS: Mouse mesangial cells were applied to explore the involvement of MAPK in TGF-beta1 signal pathway to CTGF, and the regulation of fluorofenidone. The activation of three major members of MAPK, including ERK1/2, P38 and JNK was detected by Western blot; the expression of CTGF was investigated by real time PCR and Western blot. RESULTS: Fluorofenidone significantly reduced the phosphorylation of ERK1/2, P38 and JNK induced by TGF-beta1. Fluorofenidone, PD98059 and SB203580 could partially inhibit TGF-beta1-induced expression of CTGF in mouse mesangial cells, however, JNK inhibitor II had no effect. CONCLUSIONS: The antifibrotic effects of fluorofenidone are suggested to be mediated byits actions through inhibition of MAPK activation and consequent reduction of CTGF expression.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/biossíntese , Fator de Crescimento do Tecido Conjuntivo/genética , Fibrinolíticos/farmacologia , Células Mesangiais/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Piridonas/farmacologia , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/farmacologia , Animais , Western Blotting , Regulação para Baixo/efeitos dos fármacos , Células Mesangiais/efeitos dos fármacos , Camundongos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: To investigate the effect of enalapril on renal interstitial fibrosis in rats with unilateral ureteral obstruction(UUO). METHODS: UUO model was induced by ligating the left ureter in rats. Male Sprague-Dawley(SD) rats were randomly divided into a sham-operated group(n=16), a UUO model group(n=24), and an enalapril treated group(n=24). The rats were treated with 10 mg/kg.d by gastric gavage in the enalapril treated group from 24 h before the operation, and the rats were treated with the identical dose of normal saline in the other 2 groups. The rats were sacrificed at 3,7,14, and 21 days after UUO. Pathological changes of the renal tissue were observed by HE and Masson staining, the mRNA expression of collagen I (Col I) was detected by real-time PCR, and the protein expression of connective tissue growth factor (CTGF) was detected by Western blot. RESULTS: The renal interstitial damage index, relative collagen area and the expression of Col I mRNA and CTGF in the renal tissues in the model group increased with the prolongation of obstruction. Enalapril significantly reduced the renal interstitial damage index and relative collagen area, and inhibted the expression of Col I mRNA and CTGF. There was significant difference on day 3,7,and 14 (P<0.05), but not on day 21 (P>0.05). CONCLUSION: Enalapril significantly attenuates renal interstitial fibrosis by supressing the expression of Col I mRNA and CTGF.
Assuntos
Colágeno Tipo I/biossíntese , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Enalapril/uso terapêutico , Nefrite Intersticial/prevenção & controle , Nefroesclerose/prevenção & controle , Animais , Colágeno Tipo I/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Masculino , Nefrite Intersticial/etiologia , Nefroesclerose/etiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Obstrução Ureteral/complicaçõesRESUMO
OBJECTIVE: To explore the degradation mechanism of losartan on extracellular matrix in rats with diabetic nephropathy. METHODS: The rat model of diabetic nephropathy was established by streptozotozin(STZ) injection, and the rats were randomly divided into 3 groups: (a normal group, a model group and a losartan group). For 16 weeks, the serum creatinine and urea nitrogen were measured, and glomerular sclerosis index(GSI) were caculated. The expression of collagen Type IV,connective tissue growth factor and transforming growth factor-beta1 were examined by Western blot and real time-PCR respectively. RESULTS: Blood urea nitrogen, GSI and the expressions of collagen Type IV and CTGF protein in the losartan group were lower than those in the model group(all P<0.05), and the expressions of collagen Type IV mRNA,TGF-beta1 mRNA and CTGF mRNA were lower than those in the model group (all P<0.05). CONCLUSION: Losartan modulates glomerular sclerosis and decreases the accumulation of collagen Type IV by inhibiting TGF-beta1 and CTGF.
Assuntos
Colágeno Tipo IV/biossíntese , Fator de Crescimento do Tecido Conjuntivo/biossíntese , Nefropatias Diabéticas/patologia , Glomerulosclerose Segmentar e Focal/prevenção & controle , Losartan/farmacologia , Animais , Colágeno Tipo IV/genética , Fator de Crescimento do Tecido Conjuntivo/genética , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/metabolismo , Glomerulosclerose Segmentar e Focal/etiologia , Losartan/uso terapêutico , Masculino , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Distribuição Aleatória , Ratos , Ratos Wistar , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genéticaRESUMO
OBJECTIVE: To investigate the mechanism of losartan treating glomerulosclerosis and to observe the effect of losartan on the expressions of TGF-beta1, p-Smad2/3, and Smad7 in the renal tissues of 5/6 nephrectomized rats. METHODS: Male Wistar rats were randomly divided into a sham-operated group, a 5/6 nephrectomized model group, and a losartan treated group. The rats in the model group and the losartan treated group were performed 5/6 nephrectomy by the method with 2 procedures. Twelve weeks after of the operation, all rats were killed. The 24-hour urinary protein, serum creatinine, and urea nitrogen were detected. Pathological changes of the renal tissues were observed by HE and Masson staining, and the expressions of TGF-beta1, p-Smad2/3, and Smad7 were detected by immunohistochemical staining. RESULTS: The 24-hour urinary protein, serum creatinine, urea nitrogen, and the relative area of collagen in the renal tissues of the rats in the model group significantly increased (P<0.01), and losartan could reduce these indexes. The expressions of TGF-beta1 and p-Smad2/3 were just at a low level in the renal tissues of the rats in the sham-operated group, and were strongly positive in the model group; but losartan could decrease the expressions of TGF-beta1 and p-Smad2/3 (P<0.01). The expression of Smad7 in the model group was fewer than that in the sham-operated group (P<0.01), but losartan could improve the expression of Smad7 (P<0.01). CONCLUSION: Losartan may implement its anti-glomerulosclerosis by affecting TGF-beta1, p-Smad2/3, and Smad7 of TGF-beta/Smads pathway of the renal tissues of 5/6 nephrectomized rats.
Assuntos
Rim/metabolismo , Losartan/farmacologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Proteína Smad7/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Rim/efeitos dos fármacos , Masculino , Nefrectomia/métodos , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: To explore the effect of p27 in the renal tubule on the process of renal interstitial fibrosis caused by unilateral ureteral obstruction (UUO) in rats, and to examine the expression changes of p27 after enalapril intervention and to interpret the anti-fibrotic mechanism. METHODS: Ninety rats were randomly divided into the sham-operated group (SOR), UUO group,and UUO+enalapril treatment group [enalapril: 10 mg/(kg.d)]. The rats of each group were respectively sacrificed on 7, 14, 21 days post-operatively. The renal pathological changes were dynamically observed by HE. The expression and dynamic changes of p27 were detected by immunohistochemistry. The level of p27 mRNA were detected by RT-PCR. RESULTS: The expression of p27 in renal tubular epithelial cells and p27 mRNA were strongly positive in the SOR group. With degree of interstitial fibrosis aggravating, the expression of p27 mRNA was gradually reducing. Enalapril could improve the expression of p27 on the 14th and 21st days after the UUO. CONCLUSION: (1) This study supports a causative role of p27 in the formation of fibrosis of renal mesenchyme in rats with UUO. (2) The anti-fibrotic mechanism of enalapril is partly the improvement of p27 expression.
Assuntos
Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Enalapril/uso terapêutico , Nefroesclerose/tratamento farmacológico , Nefroesclerose/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Inibidor de Quinase Dependente de Ciclina p27/genética , Enalapril/farmacologia , Feminino , Túbulos Renais/metabolismo , Nefroesclerose/etiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Obstrução Ureteral/complicaçõesRESUMO
OBJECTIVE: To investigate the expression of P21 in renal interstitial fibrosis rats and the effect of enalapril on it. METHODS: Sprague Dawley rats were randomly divided into 3 groups: a sham operation group,a unilateral urethral obstruction group, and an enalapril treatment group. The expression of P21 in renal tubular epithelial cells on the process was detected by immunohistochemistry at different time spots (7, 14, 21 d after UUO, sham-surgery or enalapril treatment). The expression of p21 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Seven days after the surgery, significant differences were found in P21 expression between UUO and SOR renal tubular cells. With degree of interstitial fibrosis aggravating, P21 expression increased. Enalapril can inhibit its expression. CONCLUSION: In the kidney of UUO rats, P21 expression increased and enalapril possessed significant inhibitory effects on the procedure. P21 may participate in the pathogenesis of renal tubule-interstitial fibrosis.
Assuntos
Enalapril/uso terapêutico , Nefroesclerose/tratamento farmacológico , Nefroesclerose/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Enalapril/farmacologia , Túbulos Renais/metabolismo , Masculino , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Obstrução Ureteral/complicaçõesRESUMO
OBJECTIVES: The present study was designed to investigate the inhibitory effects of fluorofenidone on Ang II-induced apoptosis in renal tubular cells and the related signaling pathway. METHODS: Rat proximal tubular epithelial cells (NRK-52E) were used to examine the anti-apoptosis effects of fluorofenidone. Cell proliferation was assessed by methyl thiazolyl tetrazolium assay. Apoptosis was examined by AO/EB staining and TUNEL assay. The expression of Fas/FasL pathway members, including Fas, FasL, Bax, Bcl-2, Caspase-8, and Caspase-3 was detected by real-time RT-PCR and/or Western blot, respectively. The activity of Caspase-8 and Caspase-3 was detected by spectrophotometry. RESULTS: Fluorofenidone didn't affect the proliferation of NRK-52E cells, but significantly inhibited the apoptosis of NRK-52E cells induced by Ang II. Fluorofenidone significantly reduced Ang II-induced increases in Fas, FasL, Bax, Caspase-8 and Caspase-3 at the mRNA level. Consistent with these observations, fluorofenidone also prevented Ang II-mediated up-regulation of FasL and Bax at the protein level. Additionally, Ang II-induced activation of Caspase-8 and Caspase-3 as well as Ang II-initiated downregulation of Bcl-2 at both mRNA and protein levels was all prevented by fluorofenidone. CONCLUSIONS: Fluorofenidone can inhibit Ang II-induced apoptosis of renal tubular cells through blockage of the Fas/FasL pathway.
Assuntos
Angiotensina II/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Proteína Ligante Fas/antagonistas & inibidores , Túbulos Renais Proximais/efeitos dos fármacos , Piridonas/farmacologia , Receptor fas/antagonistas & inibidores , Angiotensina II/metabolismo , Animais , Caspase 3/genética , Caspase 3/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Inibidores de Caspase , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Receptor fas/genética , Receptor fas/metabolismoRESUMO
AIM: To investigate the effects and mechanism of losartan on expression of CTGF induced by high glucose. METHODS: Mouse mesangial cells (MMCs) were cultured in vitro, initially, MMCs were stimulated by high glucose(25 mmol/L glucose) for 24 h, 48 h, 72 h, the phosphorylation of ERK1/2 was assessed by Western blot. Then MMCs were randomly divided into 5 groups: (1) Low glucose group (5.6 mmol/L glucose); (2)Sorbitol group (5.6 mmol/L glucose + 19.4 mmol/L sorbitol); (3) High glucose group (25 mmol/L glucose); (4) Losartan group (25 mmol/L glucose + 10(5) mol/L losartan); (5) ERK inhibitors group (25 mmol/L glucose + 25 micromol/L PD98059). After 48 hours, the phosphorylation of ERK1/2 were detected by Western blot. After 72 hours, the protein and mRNA expression level of CTGF were assessed by Western blot and real-time PCR. RESULTS: High glucose induced the phosphorylation of ERK1/2 in a time-dependent manner. The protein expression of phosphor-ERK1/2 and CTGF were increased in high glucose group comparing with low glucose group(P<0.01), and reduced in losartan group and ERK inhibitors group comparing with high glucose group(P<0.05). The mRNA expression of CTGF was increased in high glucose group comparing with low glucose group(P<0.01) , and reduced in losartan group and ERK inhibitors group comparing with high glucose group(P<0.01). CONCLUSION: Losartan can inhibit high glucose-induced CTGF expression in mouse mesangial cells, and the mechanisms maybe involve the interruption of ERK1/2 MAPK pathway.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Glucose/farmacologia , Losartan/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Western Blotting , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/genética , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
AIM: To explore the expression of p27 and the changes of TGF-beta in the renal tubule on the process of renal interstitial fibrosis caused by unilateral ureteral obstruction (UUO) in rats and to investigate the relationship between TGF-beta and p27 in renal interstitial fibrosis rats. METHODS: Sixty rats were randomly divided into sham-operated group (SOR) and UUO group. Ten rats from each group were sacrificed on days 7, 14 and 21 after operation respectively. The renal pathological changes were dynamically observed by HE . The expression and dynamic changes of p27 were detected by immunohistochemistry. The levels of p27 mRNA and TGF-beta mRNA were detected by RT-PCR. RESULTS: The expression of p27 in renal tubular epithelial cells and p27mRNA was strongly positive in SOR group. With the aggravation of interstitial fibrosis, the expression of p27 gradually decreased in UUO group while the expression of TGF-beta increased. There was a negative correlation between TGF-beta and p27. CONCLUSION: P27 is involved in the fibrosis of renal mesenchyme in rats with UUO. The mechanism of TGF-beta accelerating fibrosis may relate to the reduction of p27 expression in UUO rats.