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AT1R‐Associated Protein (ATRAP) Inhibits Angiotensin II‐Mediated Downregulation of ROMK Channels in Collecting Duct Cells

Polidoro, Juliano Zequini ; Reboucas, Nancy Amaral ; Girardi, Adriana Castello Costa

The FASEB journal, 2018-04, Vol.32 (S1), p.620.18-620.18 [Periódico revisado por pares]

The Federation of American Societies for Experimental Biology

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  • Título:
    AT1R‐Associated Protein (ATRAP) Inhibits Angiotensin II‐Mediated Downregulation of ROMK Channels in Collecting Duct Cells
  • Autor: Polidoro, Juliano Zequini ; Reboucas, Nancy Amaral ; Girardi, Adriana Castello Costa
  • É parte de: The FASEB journal, 2018-04, Vol.32 (S1), p.620.18-620.18
  • Descrição: Although aldosterone has been classically considered the main hormone modulating net electrolyte transport in distal segments, many studies demonstrated that angiotensin II (AngII) and its type 1 receptor (AT1R) also regulate different channels and transporters at distal nephron segments, which could explain at least in part the different electrolyte handling that takes place at distal tubule and collecting ducts during hypovolemia and hypokalemia. Moreover, several reports have shown that a specific partner of AT1R, AT1R‐associated protein (ATRAP), plays an inhibitory role on AT1R‐mediated signaling and cellular responses in different tissues including kidney. Neverthelless, it is not fully understood ATRAP involvement in AngII‐induced regulation of transporters and channels in nephron, particularly in AngII‐mediated inhibition of ROMK channels observed under hypokalemic conditions. In this study, by using protein overexpression strategy in M‐1 cells, a mouse cell line of cortical collecting ducts, we aimed to address whether, and if so, how ATRAP modulates ROMK activity regulation mediated by AngII. We took advantage of a recent developed fluorescent method for assessing ROMK activity (FluxOR™ II Green Potassium Ion Channel Assay), based on thalium‐sensitive dyes and thalium ion‐influx mediated by ROMK. As expected for a collecting duct model, ROMK‐mediated thalium influx was significantly inhibited after treating them with angiotensin II during 30 minutes in different concentrations (10−6 to 10−12 M) in comparison with vehicle‐treated group. Furthermore, by pretreating cells during 30 minutes with 10 μM VU591, a highly specific ROMK inhibitor, we confirmed that ROMK provides the major thalium entry route in these cells, since ~88% of thalium‐induced fluorescence increase was abolished by this inhibitor (29,960 ± 0,978% in vehicle group vs. 3,658 ± 1,742% in VU591 group [p< 0.001; n=3]). When we separately overexpressed in these cells a control protein (beta‐galactosidase [LacZ]) and mouse ATRAP protein, we observed that, after a previous 30 minutes treatment with AngII (10−11M), fluorescence enhancement induced by thalium influx was significantly elevated in ATRAP‐overexpressing cells compared to control group (25.65 ± 2.27% vs. 8.12 ± 0.52% of fluorescence increase [p < 0.001; n = 5]). Accordingly, preliminary results of total ROMK protein levels, at control conditions and after a longer exposure to AngII (10−7 M during 16 hours), showed that ROMK expression tended to be increased in ATRAP group and that AngII tended to decrease channel expression in both LacZ and ATRAP groups (1.000 ± 0.072 in LacZ group; 0.872±0.137 in AngII‐treated LacZ group; 1.349±0.192 in ATRAP group; 1.099+0.144 in AngII‐treated ATRAP group [p=0.1487; n=6]). Further experiments are planned to solve this question. Collectively, our results support the hypothesis that ATRAP counterregulates angiotensin II‐induced inhibition of ROMK activity and suggest that downregulation of ROMK expression could be one of its underlying mechanisms. Support or Funding Information CAPES and FAPESP This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
  • Editor: The Federation of American Societies for Experimental Biology
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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