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Synthesis and Preliminary Evaluation of an F-18 Labeled Fluoropyridine Losartan Analog as a Novel PET Tracer for Imaging AT1 ReceptorsArksey, Natasha C. 30 April 2012 (has links)
Several cardiac diseases, including hypertrophy, cardiomyopathy, and myocardial infarction, result in the upregulation of cardiac angiotensin II type-1 receptors (AT1R). Imaging the AT1R in vivo via PET provides the potential to monitor disease progression and guide therapy accordingly. The aim of this research was to develop a novel F-18 labeled losartan analog as an AT1R PET tracer and begin evaluation in rats. Due to the longer half-life and shorter positron range of F-18, we presume that an F-18 labeled tracer will be more beneficial than current C-11 labeled tracers. Prior structure-activity relationship (SAR) studies suggested the addition of substituents to the hydroxyl group of losartan would minimally affect AT1R binding affinity. [18F]Fluoropyridine losartan ([18F]FPyrLos) was synthesized in an automated module through conjugation of [18F]fluoro-3-pent-4-yn-1-yloxypyridine ([18F]FPyKYNE) to azide-modified losartan via the Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) ‘click’ reaction. [18F]FPyrLos was produced in approximately 10% yield (decay-corrected) with > 97.5% purity and specific activities up to 4,200 mCi/µmol. MicroPET (Siemens Inveon) images of normal Sprague Dawley rats displayed high uptake in the kidneys (ratio of 8.3 compared to surrounding tissue at 10 min). Metabolite analysis in the kidneys and plasma by column-switch HPLC revealed that roughly two-thirds of the tracer was unchanged 10 min post-injection and that one labeled hydrophilic metabolite exists, accounting for roughly 6% of the total activity. Both microPET and metabolism studies displayed a dose-dependent reduction in renal uptake upon co-injection with AT1R blocker candesartan indicating specific binding. Further work in rat disease models is required to evaluate the potential of this tracer for imaging cardiac AT1R.
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Synthesis and Preliminary Evaluation of an F-18 Labeled Fluoropyridine Losartan Analog as a Novel PET Tracer for Imaging AT1 ReceptorsArksey, Natasha C. 30 April 2012 (has links)
Several cardiac diseases, including hypertrophy, cardiomyopathy, and myocardial infarction, result in the upregulation of cardiac angiotensin II type-1 receptors (AT1R). Imaging the AT1R in vivo via PET provides the potential to monitor disease progression and guide therapy accordingly. The aim of this research was to develop a novel F-18 labeled losartan analog as an AT1R PET tracer and begin evaluation in rats. Due to the longer half-life and shorter positron range of F-18, we presume that an F-18 labeled tracer will be more beneficial than current C-11 labeled tracers. Prior structure-activity relationship (SAR) studies suggested the addition of substituents to the hydroxyl group of losartan would minimally affect AT1R binding affinity. [18F]Fluoropyridine losartan ([18F]FPyrLos) was synthesized in an automated module through conjugation of [18F]fluoro-3-pent-4-yn-1-yloxypyridine ([18F]FPyKYNE) to azide-modified losartan via the Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) ‘click’ reaction. [18F]FPyrLos was produced in approximately 10% yield (decay-corrected) with > 97.5% purity and specific activities up to 4,200 mCi/µmol. MicroPET (Siemens Inveon) images of normal Sprague Dawley rats displayed high uptake in the kidneys (ratio of 8.3 compared to surrounding tissue at 10 min). Metabolite analysis in the kidneys and plasma by column-switch HPLC revealed that roughly two-thirds of the tracer was unchanged 10 min post-injection and that one labeled hydrophilic metabolite exists, accounting for roughly 6% of the total activity. Both microPET and metabolism studies displayed a dose-dependent reduction in renal uptake upon co-injection with AT1R blocker candesartan indicating specific binding. Further work in rat disease models is required to evaluate the potential of this tracer for imaging cardiac AT1R.
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Synthesis and Preliminary Evaluation of an F-18 Labeled Fluoropyridine Losartan Analog as a Novel PET Tracer for Imaging AT1 ReceptorsArksey, Natasha C. January 2012 (has links)
Several cardiac diseases, including hypertrophy, cardiomyopathy, and myocardial infarction, result in the upregulation of cardiac angiotensin II type-1 receptors (AT1R). Imaging the AT1R in vivo via PET provides the potential to monitor disease progression and guide therapy accordingly. The aim of this research was to develop a novel F-18 labeled losartan analog as an AT1R PET tracer and begin evaluation in rats. Due to the longer half-life and shorter positron range of F-18, we presume that an F-18 labeled tracer will be more beneficial than current C-11 labeled tracers. Prior structure-activity relationship (SAR) studies suggested the addition of substituents to the hydroxyl group of losartan would minimally affect AT1R binding affinity. [18F]Fluoropyridine losartan ([18F]FPyrLos) was synthesized in an automated module through conjugation of [18F]fluoro-3-pent-4-yn-1-yloxypyridine ([18F]FPyKYNE) to azide-modified losartan via the Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) ‘click’ reaction. [18F]FPyrLos was produced in approximately 10% yield (decay-corrected) with > 97.5% purity and specific activities up to 4,200 mCi/µmol. MicroPET (Siemens Inveon) images of normal Sprague Dawley rats displayed high uptake in the kidneys (ratio of 8.3 compared to surrounding tissue at 10 min). Metabolite analysis in the kidneys and plasma by column-switch HPLC revealed that roughly two-thirds of the tracer was unchanged 10 min post-injection and that one labeled hydrophilic metabolite exists, accounting for roughly 6% of the total activity. Both microPET and metabolism studies displayed a dose-dependent reduction in renal uptake upon co-injection with AT1R blocker candesartan indicating specific binding. Further work in rat disease models is required to evaluate the potential of this tracer for imaging cardiac AT1R.
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Central Mechanisms Mediating Ang II-Salt HypertensionLu, Jiao January 2016 (has links)
Abstract
Statement of problem
Plasma angiotensin II (Ang II) increases blood pressure (BP) through the activation of brain angiotensinergic pathways and the aldosterone-mineralocorticoid receptors (MR)- epithelial Na+ channel (ENaC)-endogenous ouabain (EO) pathway. The response of BP to circulating Ang II is enhanced by high salt intake, but the central mechanisms mediating this elevated response are not known.
Methods of investigation
Study 1) Male Wistar rats were divided into 4 groups and treated with regular salt diet (0.4% NaCl), high salt diet (2% NaCl), sc Ang II infusion (150 ng/kg/min), or sc Ang II infusion together with 2% salt diet for 14 days; plasma aldosterone and corticosterone levels, CYP11B2 mRNA in adrenal cortex and the mRNA levels of Ang II type 1 receptors (AT1R), CYP11B1 (11-β hydroxylase), CYP11B2 (aldosterone synthase), MR, 11βHSD2, ENaC α, ENaC β and ENaC γ in the subfornical organ (SFO), paraventricular nucleus (PVN), supraoptic nucleus (SON) and rostral ventrolateral medulla (RVLM) were measured.
Study 2) MR blockers (eplerenone, spironolactone), ENaC blocker (benzamil), AT1R blocker (losartan) or vehicles were centrally infused in rats treated with Ang II plus high salt, and BP and heart rate (HR) were recorded by telemetry; plasma aldosterone and corticosterone levels and CYP11B2 mRNA expression in adrenal cortex were measured.
Major findings
Ang II alone caused a small increase in BP. Ang II together with 2% salt diet markedly increased the BP and plasma aldosterone level. Sc Ang II decreased 11βHSD2 and MR mRNA expression in the PVN, increased AT1R and ENaC γ expression in the PVN, and increased AT1R mRNA expression in the RVLM. Other genes tested in the four brain nuclei were not affected by sc Ang II or high salt diet. BP and plasma aldosterone increases in response to Ang II and salt, as well as CYP11B2 mRNA expression in adrenal cortex, were largely prevented by central infusion of eplerenone, spironolactone, benzamil or losartan.
Main conclusion
BP and plasma aldosterone responses to Ang II-salt are under the control of central mechanisms, and MR-AT1R activation in the brain plays a critical role in Ang II-salt induced hypertension.
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Ligand-induced conformations of extracellular loop 2 of AT1RUnal, Hamiyet 20 August 2010 (has links)
No description available.
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Busca virtual de agonistas enviesados não peptídicos do receptor de angiotensina II do tipo 1 / Angiotensin II type 1 receptor non-peptidic biased agonists\' virtual screeningMagalhães, Juliana Gallottini de 30 January 2015 (has links)
Os inibidores do receptor de angiotensina II do tipo 1 (AT1R), fármacos da classe das sartanas, são muito utilizados na terapêutica da insuficiência cardíaca. Apesar de serem eficientes por baixarem a pressão arterial, esses inibidores diminuem a contratilidade do músculo cardíaco, acentuando a patologia. Nesse sentido, os agonistas enviesados para β-arrestina do AT1R surgem como uma solução para esse problema. Estudos com o mais promissor peptídeo com ação agonista enviesada (TRV120027) mostram que ele é capaz de diminuir a pressão arterial sem causar o efeito inotrópico negativo no coração. Tendo em vista esse novo e promissor mecanismo de ação e a característica peptídica do novo agonista enviesado que restringe sua utilização, o presente trabalho visou à busca de ligantes não peptídicos com potencial ação enviesada. Foram realizados estudos de ancoramento seguidos de dinâmica molecular, no AT1R, de sete peptídeos agonistas e agonistas enviesados descritos na literatura, empregando-se os programas Surflex-Dock 2.0 e o GROMACS 4.5, além de análises de campos de interação molecular no programa GRID. Os dados das interações intermoleculares retirados da dinâmica e dos campos de interação guiaram a construção de um farmacóforo que foi utilizado posteriormente em uma busca virtual na base de dados ZINC, com o módulo UNITY 3D do pacote Sybyl-X Suite 2.0. Após ancoramento e análise visual das moléculas selecionadas na busca, foram identificadas 15 moléculas promissoras, sendo cinco delas consideradas de maior interesse. As moléculas selecionadas na busca poderão ser futuramente testadas quanto ao perfil de ação enviesada em receptores AT1R. Os resultados obtidos nesse estudo podem levar à descoberta de um novo protótipo mais eficiente e seguro para o tratamento de doenças cardiovasculares, como a insuficiência cardíaca. / Angiotensin II type 1 receptor (AT1R) inhibitors, the sartans, are widely used in the treatment of heart failure. Although they are effective for lowering blood pressure, these inhibitors decrease the contractility of the heart muscle, accentuating the pathology. Accordingly, β-arrestin biased agonists for AT1R emerge as a solution to this problem. Studies with the most promising biased agonist peptide (TRV120027) show that it is able to lower blood pressure without causing negative inotropic effect on the heart. Given this promising new mechanism of action and the peptide feature of the new agonist that restricts its use, this work aims the search for non-peptide ligands with a potential biased action. Docking studies, followed by molecular dynamics simultions, were performed for seven full and biased agonists in the AT1R, using the Surflex-Dock 2.0 and 4.5 GROMACS programs, besides molecular interaction fields analysis with GRID software. The data of intermolecular interactions from the molecular dynamic\'s analysis and the molecular interaction fields guided to the construction of a pharmacophore model which was subsequently used in a virtual screening from ZINC database, employing the 3D UNITY module from Sybyl-X Suite 2.0 package. After a docking study and visual analysis of the primary selected molecules, 15 promising molecules have been identified, five of them considered of most interest. The molecules selected in the search can be further tested for biased action on AT1R. The results of this study may lead to the discovery of a more efficient and secure lead for the treatment of cardiovascular diseases, such as heart failure.
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Busca virtual de agonistas enviesados não peptídicos do receptor de angiotensina II do tipo 1 / Angiotensin II type 1 receptor non-peptidic biased agonists\' virtual screeningJuliana Gallottini de Magalhães 30 January 2015 (has links)
Os inibidores do receptor de angiotensina II do tipo 1 (AT1R), fármacos da classe das sartanas, são muito utilizados na terapêutica da insuficiência cardíaca. Apesar de serem eficientes por baixarem a pressão arterial, esses inibidores diminuem a contratilidade do músculo cardíaco, acentuando a patologia. Nesse sentido, os agonistas enviesados para β-arrestina do AT1R surgem como uma solução para esse problema. Estudos com o mais promissor peptídeo com ação agonista enviesada (TRV120027) mostram que ele é capaz de diminuir a pressão arterial sem causar o efeito inotrópico negativo no coração. Tendo em vista esse novo e promissor mecanismo de ação e a característica peptídica do novo agonista enviesado que restringe sua utilização, o presente trabalho visou à busca de ligantes não peptídicos com potencial ação enviesada. Foram realizados estudos de ancoramento seguidos de dinâmica molecular, no AT1R, de sete peptídeos agonistas e agonistas enviesados descritos na literatura, empregando-se os programas Surflex-Dock 2.0 e o GROMACS 4.5, além de análises de campos de interação molecular no programa GRID. Os dados das interações intermoleculares retirados da dinâmica e dos campos de interação guiaram a construção de um farmacóforo que foi utilizado posteriormente em uma busca virtual na base de dados ZINC, com o módulo UNITY 3D do pacote Sybyl-X Suite 2.0. Após ancoramento e análise visual das moléculas selecionadas na busca, foram identificadas 15 moléculas promissoras, sendo cinco delas consideradas de maior interesse. As moléculas selecionadas na busca poderão ser futuramente testadas quanto ao perfil de ação enviesada em receptores AT1R. Os resultados obtidos nesse estudo podem levar à descoberta de um novo protótipo mais eficiente e seguro para o tratamento de doenças cardiovasculares, como a insuficiência cardíaca. / Angiotensin II type 1 receptor (AT1R) inhibitors, the sartans, are widely used in the treatment of heart failure. Although they are effective for lowering blood pressure, these inhibitors decrease the contractility of the heart muscle, accentuating the pathology. Accordingly, β-arrestin biased agonists for AT1R emerge as a solution to this problem. Studies with the most promising biased agonist peptide (TRV120027) show that it is able to lower blood pressure without causing negative inotropic effect on the heart. Given this promising new mechanism of action and the peptide feature of the new agonist that restricts its use, this work aims the search for non-peptide ligands with a potential biased action. Docking studies, followed by molecular dynamics simultions, were performed for seven full and biased agonists in the AT1R, using the Surflex-Dock 2.0 and 4.5 GROMACS programs, besides molecular interaction fields analysis with GRID software. The data of intermolecular interactions from the molecular dynamic\'s analysis and the molecular interaction fields guided to the construction of a pharmacophore model which was subsequently used in a virtual screening from ZINC database, employing the 3D UNITY module from Sybyl-X Suite 2.0 package. After a docking study and visual analysis of the primary selected molecules, 15 promising molecules have been identified, five of them considered of most interest. The molecules selected in the search can be further tested for biased action on AT1R. The results of this study may lead to the discovery of a more efficient and secure lead for the treatment of cardiovascular diseases, such as heart failure.
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Curta administração de GW501516 melhora o estado inflamatório do tecido adiposo branco, o dano hepático e a inflamação renal de camundongos alimentados com dieta rica em frutose / Short administration of GW501516 improves inflammatory state of white adipose tissue, liver damage and renal inflammation in mice fed a high-fructose dietD'Angelo Carlo Magliano 05 August 2015 (has links)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A superativação do eixo ECA/AT1r está intimamente relacionada à síndrome metabólica e no organismo tem grande relação com o quadro de inflamação. A administração de frutose, seja por dieta ou pela água, tem sido usada como um modelo para a indução da superatividade desse eixo e para o estudo das vias inflamatórias relacionadas ao AT1r. Com isso, o objetivo deste trabalho foi avaliar se a administração de GW510156 poderia diminuir a superativação do eixo ECA/AT1r e consequentemente diminuir os danos causados pela dieta rica em frutose. Para isso foram utilizados camundongos machos C57Bl/6 que receberam uma dieta contendo 47% de frutose durante oito semanas ou uma dieta controle. Após oito semanas, os grupos foram redivididos aleatoriamente para o início da administração do GW501516 durante três semanas, totalizando quatro grupos experimentais. Os animais tratados apresentaram uma melhora da pressão arterial sistólica e também dos parâmetros urinários como proteinúria e ácido úrico. Houve ainda uma melhora dos triglicerídeo e ácido úrico plasmáticos. No tecido adiposo branco, o GW501516 foi capaz de diminuir a expressão dos componentes do eixo ECA/AT1r e também amenizou a inflamação causada pela dieta rica em frutose. No fígado, não houve alterações significativa do eixo, porém a fosforilação de JAK2 dependente de AT1r foi diminuída e consequentemente houve uma menor ativação das células estreladas no grupo que recebeu o GW501516. Além disso, as proteínas e genes relacionados à β-oxidação foram aumentados com o tratamento e aqueles relacionados à lipogênese de novo, diminuídos o que resultou em menor esteatose no parênquima hepático. Os rins apresentaram uma melhora da inflamação induzida pelo eixo, apesar de o eixo também não ter apresentado diferenças significativas com o tratamento. Também não foram encontradas diferenças significativas na expressão proteica e gênica das proteínas antioxidantes. Com esses resultados podemos concluir que a curta administração do GW501516 pôde aliviar os efeitos inflamatórios e a esteatose hepática causada pela dieta rica em frutose, podendo ser pensado como uma nova ferramenta terapêutica no tratamento da superativação do eixo ECA/AT1r. / High-activation of ACE/AT1r axis is closely linked to metabolix syndrome and low-grade inflammation state. Fructose administration in water or in diet has been proposed as a model to study the high-activity of this axis and AT1r-related inflammatory pathways. In this view, we aimed to evaluate if GW501516 administration could decrease the high-activation of ACE/AT1r axis and consequently fructose damage. To this males mice C57Bl/6 were fed a high-fructose diet (47%) during eight weeks or standard-chow diet. After eight weeks, the groups were randomly divided to start treatment with GW501516, totaling four experimental groups. Animals treated with GW501516 presented an improvement of systolic blood pressure and in urinary parameters, as proteinuria and uric acid. Also was verified an improvement in plasmatic triglycerides and uric acid. In white adipose tissue, GW501516 was able to decrease the components of this axis and improved inflammation as well. In liver, it was not found differences in axis, but JAK2 phosphorylation AT1r-dependent was decreased and consequently it was found a diminished activations of hepatic stellate cells. In addition, proteins and genes related to β-oxidation were increased with GW501516 and those related to lipogenesis de novo, were diminished, improving hepatic parenchyma. Kidneys presented an improvement of inflammation state, although it was not found differences in axis with treatment. Also, it was not found differences in gene and protein expression in relation to anti-oxidants proteins. These results show that short-administration of GW501516 could alleviate inflammatory effects and hepatic steatosis caused by high fructose diet, suggesting that GW501516 could be a new therapeutic option to treat the high activation of ACE/AT1r axis.
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Curta administração de GW501516 melhora o estado inflamatório do tecido adiposo branco, o dano hepático e a inflamação renal de camundongos alimentados com dieta rica em frutose / Short administration of GW501516 improves inflammatory state of white adipose tissue, liver damage and renal inflammation in mice fed a high-fructose dietD'Angelo Carlo Magliano 05 August 2015 (has links)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A superativação do eixo ECA/AT1r está intimamente relacionada à síndrome metabólica e no organismo tem grande relação com o quadro de inflamação. A administração de frutose, seja por dieta ou pela água, tem sido usada como um modelo para a indução da superatividade desse eixo e para o estudo das vias inflamatórias relacionadas ao AT1r. Com isso, o objetivo deste trabalho foi avaliar se a administração de GW510156 poderia diminuir a superativação do eixo ECA/AT1r e consequentemente diminuir os danos causados pela dieta rica em frutose. Para isso foram utilizados camundongos machos C57Bl/6 que receberam uma dieta contendo 47% de frutose durante oito semanas ou uma dieta controle. Após oito semanas, os grupos foram redivididos aleatoriamente para o início da administração do GW501516 durante três semanas, totalizando quatro grupos experimentais. Os animais tratados apresentaram uma melhora da pressão arterial sistólica e também dos parâmetros urinários como proteinúria e ácido úrico. Houve ainda uma melhora dos triglicerídeo e ácido úrico plasmáticos. No tecido adiposo branco, o GW501516 foi capaz de diminuir a expressão dos componentes do eixo ECA/AT1r e também amenizou a inflamação causada pela dieta rica em frutose. No fígado, não houve alterações significativa do eixo, porém a fosforilação de JAK2 dependente de AT1r foi diminuída e consequentemente houve uma menor ativação das células estreladas no grupo que recebeu o GW501516. Além disso, as proteínas e genes relacionados à β-oxidação foram aumentados com o tratamento e aqueles relacionados à lipogênese de novo, diminuídos o que resultou em menor esteatose no parênquima hepático. Os rins apresentaram uma melhora da inflamação induzida pelo eixo, apesar de o eixo também não ter apresentado diferenças significativas com o tratamento. Também não foram encontradas diferenças significativas na expressão proteica e gênica das proteínas antioxidantes. Com esses resultados podemos concluir que a curta administração do GW501516 pôde aliviar os efeitos inflamatórios e a esteatose hepática causada pela dieta rica em frutose, podendo ser pensado como uma nova ferramenta terapêutica no tratamento da superativação do eixo ECA/AT1r. / High-activation of ACE/AT1r axis is closely linked to metabolix syndrome and low-grade inflammation state. Fructose administration in water or in diet has been proposed as a model to study the high-activity of this axis and AT1r-related inflammatory pathways. In this view, we aimed to evaluate if GW501516 administration could decrease the high-activation of ACE/AT1r axis and consequently fructose damage. To this males mice C57Bl/6 were fed a high-fructose diet (47%) during eight weeks or standard-chow diet. After eight weeks, the groups were randomly divided to start treatment with GW501516, totaling four experimental groups. Animals treated with GW501516 presented an improvement of systolic blood pressure and in urinary parameters, as proteinuria and uric acid. Also was verified an improvement in plasmatic triglycerides and uric acid. In white adipose tissue, GW501516 was able to decrease the components of this axis and improved inflammation as well. In liver, it was not found differences in axis, but JAK2 phosphorylation AT1r-dependent was decreased and consequently it was found a diminished activations of hepatic stellate cells. In addition, proteins and genes related to β-oxidation were increased with GW501516 and those related to lipogenesis de novo, were diminished, improving hepatic parenchyma. Kidneys presented an improvement of inflammation state, although it was not found differences in axis with treatment. Also, it was not found differences in gene and protein expression in relation to anti-oxidants proteins. These results show that short-administration of GW501516 could alleviate inflammatory effects and hepatic steatosis caused by high fructose diet, suggesting that GW501516 could be a new therapeutic option to treat the high activation of ACE/AT1r axis.
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Efeito da variação do conteúdo de K+ na dieta sobre a expressão renal de AT1R, ATRAP e WNKs. / Effect of varying the K+ content of the diet on renal expression of AT1R, ATRAP and WNKs.Neri, Elida Adalgisa 11 August 2014 (has links)
O mecanismo mais importante para a homeostase do K+ é o controle da secreção de K+ no néfron distal. O objetivo deste trabalho foi avaliar em animais submetidos à depleção de K+ por sete dias, a expressão de AT1R da ATRAP e algumas vias de sinalização como as WNK1, KS-WNK1 e WNK4. Estes animais apresentaram menor ganho de peso corporal, hipertrofia renal, isostenúria, e redução da FE de Na+ e K+, com aumento de Ang II, sem alterar a aldosterona. Verificamos aumento da expressão de AT1R mais acentuado em lisado celular e o aumento de ATRAP foram iguais nas frações de lisado total, membranas total e apical. Não detectamos variação nos níveis de RNAm dessas proteínas. A depleção de K+ induziu a fosforilação de c-Src, ERK1/2 e p38, bem como aumento dos RNAm de WNK1 e WNK4, e redução do RNAm de KS-WNK1. Considerando nossos resultados, a depleção aumenta a ação da Ang II, provavelmente devido à hiperexpressão de AT1R, sem diminuir a expressão de ATRAP. A hiperexpressão de WNK1 e WNK4, associada à redução da KS-WNK1. / The most important mechanism for the K+ homeostasis on varying the content of this ion in the diet is the control of K+ secretion in the distal nephron. Since angiotensin II (Ang II) is an important modulator of K+ secretion, the aim of this study was to evaluate, in animals subjected to K+ depletion for seven days, the expression level of angiotensin type 1 receptor (AT1R) and the AT1R-associated protein (ATRAP). Moreover, it was intended to evaluate the possible activation of some signaling pathways triggered by Ang II via AT1R. We also looked for evaluate the expression of ion transporters and \'\'with no lysine kinases\'\' (WNKs) WNK1, KS-WNK1 and WNK4 in these animals, since some of the effects of angiotensin II in the distal tubular segments are mediated by these kinases. The animals subjected to K+ depletion have showed lower body weight gain, renal hypertrophy, marked polyuria, isosthenuria, and significant reduction in FE Na+ and K+, and increased plasmatic Ang II levels, without changing the aldosterone levels. We found that the expression of ATRAP and AT1R is increased in all cell fractions analyzed, with the highest rise in the AT1R in total cell lysate and ATRAP increase was not significant in the apical membrane. We did not detect changes in mRNA levels of these proteins, suggesting no changes in the transcription rate. The mRNA levels of Na+/H+ exchanger isoform 3 (NHE3) and Cl-/Formate (CFEX), abundant in proximal tubuleswere not altered as well. Regarding signaling pathways, K+ depletion induced c-Src, ERK1/2 and p38 phosphorylation, as well as a significant increase in WNK1 and WNK4 mRNA , and reduced KS- WNK1 mRNA. Considering our results, K+ depletion increases Ang II action in renal tissue, probably due to the overexpression of AT1R, and that effect is not associated to the decreased expression of ATRAP. However, the total cell lysate AT1R increasing, was greater than that of ATRAP. The overexpression of WNK1 and WNK4 associated with (to) the reduction of KS - WNK1 appears to be important for K+ secretion inhibition in K+-depleted animals. The inhibitory activity of WNK4 on ROMK channels depends on its dephosphorylation, which depends on the activation of c-Src. The activation of c-Src was evidenced by the increase in K+ -depleted animals phosphorylation.
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