O papel dos receptores AT1 na hipertensão induzida por Angiotensina II: análise do perfil de ativação das vias de sinalização e dos complexos mitocondriais em células mesangiais; e avaliação da resposta pressórica e natriurética - de camundongos knockout para receptores AT1 no túbulo proximal
Data
2020-02-19
Autores
Leite, Ana Paula de Oliveira [UNIFESP]
Orientadores
Casarini, Dulce Elena [UNIFESP]
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A desregulação do Sistema renina angiotensina aldosterona é um dos fatores chaves na hipertensão arterial sistêmica (HAS). Os tratamentos com losartan, bloqueador do receptor AT1 além de melhorar diminuir a pressão arterial (PA) melhora as lesões de órgão alvo, entretanto em alguns pacientes, os efeitos do bloqueador não são tão eficientes. As células mesangiais glomerulares (CM) e o túbulo proximal são alvo da ação da angiotensina II (ANG II) e estão relacionados com lesão de órgão alvo em patologias como a HAS. O aumento da formação de ANGII nas CM pode causar lesão glomerular, promovendo o crescimento celular, hipertrofia e fibrose. Investigamos a sinalização de proteínas fosforiladas e as respostas mitocondriais à ANG II parácrina ou intracelular em CM de murinos (in vitro) e avaliamos a função renal do receptor AT1 em camundongos nocaute para este receptor no túbulo proximal (in vivo). Métodos: As CM foram cultivadas em meio DMEM e Ham's F-12, com 80% de confluência, carenciadas de soro fetal bovino. As células foram tratadas com ANG II na ausência ou presença de inibidores dos receptores AT1 (losartan) e/ou AT2 (PD 123319). As proteínas p-PKCα, p-ERK1/2, p38, p65 e p50 de NF-кB foram analisadas por Western Blotting (WB). As CM, foram submetidas ao processo de transfecção com vetores adenovirais que codificam uma proteína de fusão intracelular de ANG II, a mito-ECFP-ANG II, tratadas com Losartan ou com dupla transfecção com mito-GFP-AT2, para análise complexos mitocondriais proteicos I a V por WB. Os animais nocaute para o receptor AT1 no túbulo proximal e os animais selvagem, de ambos os sexos foram divididos em doze grupos (n=8/grupo), C57BL/6J (C57, selvagem) e mutantes (PT-AT1 -/-, PT). Esses aninais foram infundidos com ANG II por meio de bomba osmótica por 2 semanas (40 ng / min, i.p.), tratados simultaneamente com losartan (20 mg/kg/dia, v.o.) para estudar os papéis do AT1 extrarrenal e no túbulo proximal. Esses animais tiveram a PA verificada via caudal e submetidos a gaiola metabólica para a analise de Na+, Cl- e K+ na urina. Foi realizada a análise histológica do rim esquerdo de machos para observação da deposição de colágeno. A ANG II aumentou significativamente a expressão de p-ERK1/2, p-PKCα, p38 e NF-kb e essas respostas foram atenuadas pelo losartan e/ou PD123319. O mito-ECFP/ANG II aumentou o complexo proteico mitocondrial I a V, o qual foi atenuado pelo losartan ou pela expressão de receptores mito-AT2. A PA foi aproximadamente 13 ± 3 mmHg mais baixa nos camundongos PT-AT1-/- do que nos animais C57. A excreção (24 h) basal de Na+ (UNaV), K+ (UKV) e Cl- urinária (UClV) foi significativamente mais alta nos camundongos PT-AT1a-/- do que nos controles C57. As respostas pressoras frente à ANG II ocorreram de forma semelhante entre C57 e nocaute. Com relação as avaliações metabólicas, em animais C57, a ANG II promoveu a diurese, a ingestão de água, UNaV, UClV e UKV, sendo que a diurese foi atenuada pelo losartan sem alterar os níveis de ions. Já nos animais nocaute, a excreção basal de sódio foi maior do que nos animais C57, a infusão de ANG II promoveu diurese, mas menos que C57, aumentou da ingestão de água e promoveu UNaV, UClV e UKV, sendo que o losartan atenuou todas esses parâmetros. Não há diferenças significativas entre os sexos nas respostas pressoras e natriuréticas à ANG II em animais C57 e PT-AT1-/-. Com relação a deposição de colágeno, a lesão renal induzida por Ang II foi atenuado em camundongos PT-Agtr1a-/- comparado com C57. Em conjunto, temos in vitro que a ativação da ANG II intracelular ocorreu através da ação em ambos os receptores nas células mesangiais e na mitocôndria. E em in vivo, o presente estudo demonstra que a deleção dos receptores AT1a nos túbulos proximais do rim atenua hipertensão induzida por Ang II e lesão renal sem diferença significativa entre os sexos. Em conclusão, podemos afirmar que nos túbulos proximais, esta ação da ANG II na hipertensão e manutenção do equilíbrio hidroeletrolítico se dá principalmente através do AT1, já que na ausência deste receptor foi observada uma proteção renal frente aos efeitos deletérios da hipertensão.
Dysregulation of the renin angiotensin aldosterone system is one of the key factors in systemic arterial hypertension (SAH). Treatments AT1 receptor blocker, losartan, besides improve blood pressure (BP), also mitigate target organ damage, however in some patients, even after the treatment, still have any complications. Glomerular mesangial cells (MC) and the proximal tubule are targets of the action of angiotensin II (ANG II) and are associated with target organ damage in pathologies such as SAH. Increased ANGII release in MC can cause glomerular damage, promotes cell growth, hypertrophy, and fibrosis. We investigated the signaling of phosphorylated proteins and mitochondrial responses to paracrine or intracellular ANG II in murine MC (in vitro) and evaluated the renal function of the AT1 receptor in knockout mice of this receptor in the proximal tubule (in vivo). Methods: MC were cultivated in DMEM and Ham's F-12 medium, with 80% confluence, in absence of fetal bovine serum. Cells were treated with ANG II in the absence or presence of AT1 receptor inhibitors (losartan) and/or AT2 (PD 123319). The proteins p-PKCα, p-ERK1/2, p38, p65, and p50 NF-кB proteins were analyzed by Western Blotting (WB). The MC was transfected with adenoviral vectors encoding an ANG II intracellular fusion protein, the mito-ECFP-ANG II, treated with Losartan or double transfection with mito-GFP-AT2, for analysis of mitochondrial protein complexes I to V by WB. Knockout animals (PT-AT1-/- , PT) for the AT1 receptor in the proximal tubule and wild animals C57BL/6J (C57) of both sexes were divided into twelve groups (n=8/group). Animals were infused with ANG II via an osmotic pump for 2 weeks (40 ng/min, i.p.), treated simultaneously with losartan (20 mg/kg/day, v.o.) to study the roles of extrarenal and proximal tubule AT1. These animals had their BP verified via caudal plethysmography and submitted to a metabolic cage for the analysis of Na+, Cl- and K+ in the urine. Histological analysis of the left kidney of males was performed to observe collagen deposition. ANG II significantly increased the expression of p-ERK1/2, p-PKCα, p38, and NF-kb, and these responses were attenuated by losartan and/or PD123319. The mito-ECFP/ANG II increased the mitochondrial protein complex I to V, which was attenuated by losartan or by the expression of mito-AT2 receptors. BP was approximately 13 ± 3 mmHg lower in PT-AT1-/- mice than in C57 mice. Basal (24 h) excretion of Na+ (UNaV), K+ (UKV), and urinary Cl- (UClV) was significantly higher in PT-AT1a-/- mice than in C57 controls. The pressor responses to ANG II occurred similarly between C57 and knockout. On metabolic evaluations, in C57 animals, ANG II promoted diuresis, water intake, UNaV, UClV, and UKV, and diuresis was attenuated by losartan without changing ions levels. In the knockout animals, the basal excretion of sodium was higher than in the C57 animals, the infusion of ANG II promoted diuresis, but less than C57, increased water intake and promoted UNaV, UClV, and UKV, and losartan attenuated all these parameters. There are no significant gender differences in pressor and natriuretic responses to ANG II in C57 and PT-AT1-/- animals. The collagen deposition, ANG IIinduced kidney damage was attenuated in PT-Agtr1a-/- mice compared to C57. Together, we have in vitro that the activation of intracellular ANG II occurred through action on both receptors in mesangial cells and mitochondria. And in vivo, the present study demonstrates that the deletion of AT1a receptors in the proximal tubules of the kidney attenuates Ang II-induced hypertension and renal injury without significant difference between sexes. In conclusion, we can affirm that in the proximal tubules, this action of ANG II in hypertension and maintenance of electrolyte balance occurs mainly through AT1, since in the absence of this receptor, renal protection against the deleterious effects of hypertension was observed.
Dysregulation of the renin angiotensin aldosterone system is one of the key factors in systemic arterial hypertension (SAH). Treatments AT1 receptor blocker, losartan, besides improve blood pressure (BP), also mitigate target organ damage, however in some patients, even after the treatment, still have any complications. Glomerular mesangial cells (MC) and the proximal tubule are targets of the action of angiotensin II (ANG II) and are associated with target organ damage in pathologies such as SAH. Increased ANGII release in MC can cause glomerular damage, promotes cell growth, hypertrophy, and fibrosis. We investigated the signaling of phosphorylated proteins and mitochondrial responses to paracrine or intracellular ANG II in murine MC (in vitro) and evaluated the renal function of the AT1 receptor in knockout mice of this receptor in the proximal tubule (in vivo). Methods: MC were cultivated in DMEM and Ham's F-12 medium, with 80% confluence, in absence of fetal bovine serum. Cells were treated with ANG II in the absence or presence of AT1 receptor inhibitors (losartan) and/or AT2 (PD 123319). The proteins p-PKCα, p-ERK1/2, p38, p65, and p50 NF-кB proteins were analyzed by Western Blotting (WB). The MC was transfected with adenoviral vectors encoding an ANG II intracellular fusion protein, the mito-ECFP-ANG II, treated with Losartan or double transfection with mito-GFP-AT2, for analysis of mitochondrial protein complexes I to V by WB. Knockout animals (PT-AT1-/- , PT) for the AT1 receptor in the proximal tubule and wild animals C57BL/6J (C57) of both sexes were divided into twelve groups (n=8/group). Animals were infused with ANG II via an osmotic pump for 2 weeks (40 ng/min, i.p.), treated simultaneously with losartan (20 mg/kg/day, v.o.) to study the roles of extrarenal and proximal tubule AT1. These animals had their BP verified via caudal plethysmography and submitted to a metabolic cage for the analysis of Na+, Cl- and K+ in the urine. Histological analysis of the left kidney of males was performed to observe collagen deposition. ANG II significantly increased the expression of p-ERK1/2, p-PKCα, p38, and NF-kb, and these responses were attenuated by losartan and/or PD123319. The mito-ECFP/ANG II increased the mitochondrial protein complex I to V, which was attenuated by losartan or by the expression of mito-AT2 receptors. BP was approximately 13 ± 3 mmHg lower in PT-AT1-/- mice than in C57 mice. Basal (24 h) excretion of Na+ (UNaV), K+ (UKV), and urinary Cl- (UClV) was significantly higher in PT-AT1a-/- mice than in C57 controls. The pressor responses to ANG II occurred similarly between C57 and knockout. On metabolic evaluations, in C57 animals, ANG II promoted diuresis, water intake, UNaV, UClV, and UKV, and diuresis was attenuated by losartan without changing ions levels. In the knockout animals, the basal excretion of sodium was higher than in the C57 animals, the infusion of ANG II promoted diuresis, but less than C57, increased water intake and promoted UNaV, UClV, and UKV, and losartan attenuated all these parameters. There are no significant gender differences in pressor and natriuretic responses to ANG II in C57 and PT-AT1-/- animals. The collagen deposition, ANG IIinduced kidney damage was attenuated in PT-Agtr1a-/- mice compared to C57. Together, we have in vitro that the activation of intracellular ANG II occurred through action on both receptors in mesangial cells and mitochondria. And in vivo, the present study demonstrates that the deletion of AT1a receptors in the proximal tubules of the kidney attenuates Ang II-induced hypertension and renal injury without significant difference between sexes. In conclusion, we can affirm that in the proximal tubules, this action of ANG II in hypertension and maintenance of electrolyte balance occurs mainly through AT1, since in the absence of this receptor, renal protection against the deleterious effects of hypertension was observed.
Descrição
Citação
LEITE, Ana Paula de Oliveira. O papel dos receptores AT1 na hipertensão induzida por Angiotensina II: análise do perfil de ativação das vias de sinalização e dos complexos mitocondriais em células mesangiais; e avaliação da resposta pressórica e natriurética - de camundongos knockout para receptores AT1 no túbulo proximal. São Paulo, 2021. 74 p. Tese (Doutorado em Nefrologia) – Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2021.