Angiotensina (1-7) e Angiotensina II: indutoras de translocação de aquaporinas 2 transfectadas em células LLC-PK1

• Ano de defesa: 2016
• Autor(a) principal: Nogueira, Marie Doki [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Aquaporin-2; Angiotensin- (1-7); Angiotensin II; Sistema renina angiotensina; Imunofluorescência; Aquaporina-2; Angiotensina-(1-7); Angiotensina II
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=3906078; https://repositorio.unifesp.br/handle/11600/46253

Introduction: When there is an increase in osmolality or a decrease in plasma volume, the hypothalamic receptors stimulate the pituitary to secrete the antidiuretic hormone arginine vasopressin (AVP). This hormone is recognized as the physiological stimulator of the translocation of AQP2 from cytoplasmic vesicles to the apical pole of the principal cells of the collecting duct, increasing water reabsorption until the osmotic equilibrium is reached. In addition to AVP, it has been shown that in primary IMCD cells, Ang II also has the ability to modulate AQP2 and this occurs through the AT1 receptor (AT1R), however, there are no reports on the ability of Ang- (1-7) to modulate the AQP2. Objectives: The aim of this study was to evaluate whether Ang (1-7) and Ang II would be able to translocate AQP2 present in LLC-PK1 cells, transfected with AQP2 (LLC-PK1-AQP2 in the presence or absence of the Losartan, PD-123319 and D-Ala-A779, antagonists of AT1R, AT2R and MasR respectively. Materials and Methods: The presence of AQP1 and AQP2, recombinant protein and microfilaments were visualized by immunofluorescence. The same method was used to observe the translocation of AQP2 present in LLC-PK1-AQP2 cells before and after treatment with Ang- (1-7) and Ang II in the presence or absence of Losartan, PD-123319 and D-Ala- A779. Phosphorylation of AQP2-S256 and S261 was analyzed by Western blotting at 2, 5, 15 and 30 minutes of treatment with Ang- (1-7) and Ang II in the presence or absence of Losartan, PD-123319 and D-Ala -A779. Gene modulation, after long-term stimuli, for 6, 12 and 24 hours with Ang- (1-7) and Ang II was analyzed by RT-PCR. For statistical analysis, one way Anova tests were used followed by Dunnet's multiple comparison test. Results: Our study showed: 1) Presence of AQP1 and AQP2 vesicles, both perinuclear and on the plasma membrane in LLC-PK1-AQP2 CT cells. 2) Presence of recombinant AQP2 in the plasma membrane of cells stimulated with AVP. 3) Change in microfilament organization in response to AVP, Ang- (1-7) and Ang II). 4) Ang- (1-7) and Ang II, were able to translocate the AQP2 vesicles from the cytoplasm to the plasma membrane. 5) Pre-treatments with Losartan, D-Ala-A779 and PD-123319, modulated the response of AQP2 and the phosphorylation levels obtained with Ang- (1-7) and Ang II. 6) Gene responses to AQP2 were statistically significant in the treatments of 6 h with AVP and in 24 h with Ang- (1-7). Conclusions: 1) For the first time, it has been shown that Ang- (1-7) is able to induce the translocation of AQP2 from cytoplasmic vesicles to the plasma membrane. 2) Ang- (1-7), appears to have a more effective effect on the increase in S256 phosphorylation and decrease in phosphorylation of S261 from AQP2 than Ang II. 3) There was a reorganization of actin microfilaments after stimulation with AVP, Ang- (1-7) and Ang II suggesting that these microfilaments are important in the targeting of AQP2 vesicles. 4) The previous inhibition with Losartan, D-ALA- (A779) and PD-123319 showed that Ang- (1-7) and Ang II can act through other receptors besides the specific ones, suggesting the formation of dimers or oligomers between these receptors. 5) The mixture of the three antagonists totally blocked the AQP2 translocation. 6) Ang- (1-7) increased the gene expression of AQP2 in 24h, suggesting to be a late gene inducer, compared to AVP.