RESUMO
Recent studies suggested that activation of the PRR upregulates profibrotic markers through reactive oxygen species (ROS) formation; however, the exact mechanisms have not been investigated in CD cells. We hypothesized that activation of the PRR increases the expression of profibrotic markers through MAPK-dependent ROS formation in CD cells. Mouse renal CD cell line (M-1) was treated with recombinant prorenin plus ROS or MAPK inhibitors and PRR-shRNA to evaluate their effect on the expression of profibrotic markers. PRR immunostaining revealed plasma membrane and intracellular localization. Recombinant prorenin increases ROS formation (6.0 ± 0.5 vs 3.9 ± 0.1 nmol/L DCF/µg total protein, P < .05) and expression of profibrotic markers CTGF (149 ± 12%, P < .05), α-SMA (160 ± 20%, P < .05), and PAI-I (153 ± 13%, P < .05) at 10-8 mol/L. Recombinant prorenin-induced phospho ERK 1/2 (p44 and p42) at 10-8 and 10-6 mol/L after 20 minutes. Prorenin-dependent ROS formation and augmentation of profibrotic factors were blunted by ROS scavengers (trolox, p-coumaric acid, ascorbic acid), the MEK inhibitor PD98059 and PRR transfections with PRR-shRNA. No effects were observed in the presence of antioxidants alone. Prorenin-induced upregulation of collagen I and fibronectin was blunted by ROS scavenging or MEK inhibition independently. PRR-shRNA partially prevented this induction. After 24 hours prorenin treatment M-1 cells undergo to epithelial-mesenchymal transition phenotype, however MEK inhibitor PD98059 and PRR knockdown prevented this effect. These results suggest that PRR might have a significant role in tubular damage during conditions of high prorenin-renin secretion in the CD.
Assuntos
Fibroblastos/citologia , Fibroblastos/patologia , Rim/citologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular , Fibroblastos/metabolismo , Fibrose , Rim/patologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Receptor de Pró-ReninaRESUMO
[This corrects the article DOI: 10.1155/2015/726012.].
RESUMO
The mechanism of hypertension-induced renal fibrosis is not well understood, although it is established that high levels of angiotensin II contribute to the effect. Since ß-catenin signal transduction participates in fibrotic processes, we evaluated the contribution of ß-catenin-dependent signaling pathway in hypertension-induced renal fibrosis. Two-kidney one-clip (2K1C) hypertensive rats were treated with lisinopril (10 mg/kg/day for four weeks) or with pyrvinium pamoate (Wnt signaling inhibitor, single dose of 60 ug/kg, every 3 days for 2 weeks). The treatment with lisinopril reduced the systolic blood pressure from 220 ± 4 in 2K1C rats to 112 ± 5 mmHg (P < 0.05), whereas the reduction in blood pressure with pyrvinium pamoate was not significant (212 ± 6 in 2K1C rats to 170 ± 3 mmHg, P > 0.05). The levels of collagen types I and III, osteopontin, and fibronectin decreased in the unclipped kidney in both treatments compared with 2K1C rats. The expressions of ß-catenin, p-Ser9-GSK-3beta, and the ß-catenin target genes cyclin D1, c-myc, and bcl-2 significantly decreased in unclipped kidney in both treatments (P < 0.05). In this study we provided evidence that ß-catenin-dependent signaling pathway participates in the renal fibrosis induced in 2K1C rats.
Assuntos
Fibrose/genética , Hipertensão/genética , Nefropatias/genética , beta Catenina/genética , Angiotensina II/genética , Animais , Pressão Sanguínea/genética , Ciclina D1/metabolismo , Fibrose/tratamento farmacológico , Fibrose/patologia , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/patologia , Rim/efeitos dos fármacos , Rim/patologia , Nefropatias/tratamento farmacológico , Nefropatias/patologia , Lisinopril/administração & dosagem , Proteínas Proto-Oncogênicas c-myc/metabolismo , Compostos de Pirvínio/administração & dosagem , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Cyclooxygenase-2 (COX-2) is constitutively expressed and highly regulated in the thick ascending limb (TAL). As COX-2 inhibitors (Coxibs) increase COX-2 expression, we tested the hypothesis that a negative feedback mechanism involving PGE(2) EP3 receptors regulates COX-2 expression in the TAL. Sprague-Dawley rats were treated with a Coxib [celecoxib (20 mg·kg(-1)·day(-1)) or rofecoxib (10 mg·kg(-1)·day(-1))], with or without sulprostone (20 µg·kg(-1)·day(-1)). Sulprostone was given using two protocols, namely, previous to Coxib treatment (prevention effect; Sulp7-Coxib5 group) and 5 days after initiation of Coxib treatment (regression effect; Coxib10-Sulp5 group). Immunohistochemical and morphometric analysis revealed that the stained area for COX-2-positive TAL cells (µm(2)/field) increased in Coxib-treated rats (Sham: 412 ± 56.3, Coxib: 794 ± 153.3). The Coxib effect was inhibited when sulprostone was used in either the prevention (285 ± 56.9) or regression (345 ± 51.1) protocols. Western blot analysis revealed a 2.1 ± 0.3-fold increase in COX-2 protein expression in the Coxib-treated group, an effect abolished by sulprostone using either the prevention (1.2 ± 0.3-fold) or regression (0.6 ± 0.4-fold vs. control, P < 0.05) protocols. Similarly, the 6.4 ± 0.6-fold increase in COX-2 mRNA abundance induced by Coxibs (P < 0.05) was inhibited by sulprostone; prevention: 0.9 ± 0.3-fold (P < 0.05) and regression: 0.6 ± 0.1 (P < 0.05). Administration of a selective EP3 receptor antagonist, L-798106, also increased the area for COX-2-stained cells, COX-2 mRNA accumulation, and protein expression in the TAL. Collectively, the data suggest that COX-2 levels are regulated by a novel negative feedback loop mediated by PGE(2) acting on its EP3 receptor in the TAL.
Assuntos
Ciclo-Oxigenase 2/biossíntese , Rim/enzimologia , Receptores de Prostaglandina E Subtipo EP3/fisiologia , Animais , Western Blotting , Inibidores de Ciclo-Oxigenase 2/farmacologia , Dinoprostona/análogos & derivados , Dinoprostona/farmacologia , Dinoprostona/fisiologia , Retroalimentação Fisiológica/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Imuno-Histoquímica , Rim/efeitos dos fármacos , Rim/metabolismo , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Masculino , Néfrons/metabolismo , RNA/biossíntese , RNA/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Prostaglandina E Subtipo EP1/metabolismo , Receptores de Prostaglandina E Subtipo EP3/efeitos dos fármacosRESUMO
Estradiol (E(2)) accelerates oviductal egg transport through intraoviductal non-genomic pathways in unmated rats and through genomic pathways in mated rats. This shift in pathways has been designated as intracellular path shifting (IPS), and represents a novel and hitherto unrecognized effect of mating on the female reproductive tract. We had reported previously that IPS involves shutting down the E(2) non-genomic pathway up- and downstream of 2-methoxyestradiol. Here, we evaluated whether IPS involves changes in the genomic pathway too. Using microarray analysis, we found that a common group of genes changed its expression in response to E(2) in unmated and mated rats, indicating that an E(2) genomic signaling pathway is present before and after mating; however, a group of genes decreased its expression only in mated rats and another group of genes increased its expression only in unmated rats. We evaluated the possibility that this difference is a consequence of an E(2) non-genomic signaling pathway present in unmated rats, but not in mated rats. Mating shuts down this E(2) non-genomic signaling pathway up- and downstream of cAMP production. The Star level is increased by E(2) in unmated rats, but not in mated rats. This is blocked by the antagonist of estrogen receptor ICI 182 780, the adenylyl cyclase inhibitor SQ 22536, and the catechol-O-methyltransferase inhibitor, OR 486. These results indicate that the E(2)-induced gene expression profile in the rat oviduct differs before and after mating, and this difference is probably mediated by an E(2) non-genomic signaling pathway operating on gene expression only in unmated rats.
Assuntos
Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Oviductos/efeitos dos fármacos , Comportamento Sexual Animal/fisiologia , Transdução de Sinais/fisiologia , Animais , Análise por Conglomerados , AMP Cíclico/metabolismo , Regulação para Baixo/fisiologia , Feminino , Perfilação da Expressão Gênica , Genoma/efeitos dos fármacos , Genoma/fisiologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Oviductos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genéticaRESUMO
Estradiol (E(2)) accelerates oviductal egg transport through intraoviductal nongenomic pathways in cyclic rats and through genomic pathways in pregnant rats. This shift in pathways, which we have provisionally designated as intracellular path shifting (IPS), is caused by mating-associated signals and represents a novel and hitherto unrecognized phenomenon. The mechanism underlying IPS is currently under investigation. Using microarray analysis, we identified several genes the expression levels of which changed in the rat oviduct within 6 hours of mating. Among these genes, the mRNA level for the enzyme catechol-O-methyltransferase (COMT), which produces methoxyestradiols from hydroxyestradiols, decreased 6-fold, as confirmed by real-time PCR. O-methylation of 2-hydroxyestradiol was up to 4-fold higher in oviductal protein extracts from cyclic rats than from pregnant rats and was blocked by OR486, which is a selective inhibitor of COMT. The levels in the rat oviduct of mRNA and protein for cytochrome P450 isoforms 1A1 and 1B1, which form hydroxyestradiols, were detected by RT-PCR and Western blotting. We explored whether methoxyestradiols participate in the pathways involved in E(2)-accelerated egg transport. Intrabursal application of OR486 prevented E(2) from accelerating egg transport in cyclic rats but not in pregnant rats, whereas 2-methoxyestradiol (2ME) and 4-methoxyestradiol mimicked the effect of E(2) on egg transport in cyclic rats but not in pregnant rats. The effect of 2ME on egg transport was blocked by intrabursal administration of the protein kinase inhibitor H-89 or the antiestrogen ICI 182780, but not by actinomycin D or OR486. We conclude that in the absence of mating, COMT-mediated formation of methoxyestradiols in the oviduct is essential for the nongenomic pathway through which E(2) accelerates egg transport in the rat oviduct. Yet unidentified mating-associated signals, which act directly on oviductal cells, shut down the E(2) nongenomic signaling pathway upstream and downstream of methoxyestradiols. These findings highlight a physiological role for methoxyestradiols in the female genital tract, thereby confirming the occurrence of and providing a partial explanation for the mechanism underlying IPS.