Polidoro Juliano Zequini, Rebouças Nancy Amaral, Girardi Adriana Castello Costa
Heart Institute (InCor), University of São Paulo Medical School, São Paulo, Brazil.
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
Front Physiol. 2021 May 14;12:642409. doi: 10.3389/fphys.2021.642409. eCollection 2021.
Adjustments in renal K excretion constitute a central mechanism for K homeostasis. The renal outer medullary potassium (ROMK) channel accounts for the major K secretory route in collecting ducts during basal conditions. Activation of the angiotensin II (Ang II) type 1 receptor (AT1R) by Ang II is known to inhibit ROMK activity under the setting of K dietary restriction, underscoring the role of the AT1R in K conservation. The present study aimed to investigate whether an AT1R binding partner, the AT1R-associated protein (ATRAP), impacts Ang II-mediated ROMK regulation in collecting duct cells and, if so, to gain insight into the potential underlying mechanisms. To this end, we overexpressed either ATRAP or β-galactosidase (LacZ; used as a control), in M-1 cells, a model line of cortical collecting duct cells. We then assessed ROMK channel activity by employing a novel fluorescence-based microplate assay. Experiments were performed in the presence of 10 M Ang II or vehicle for 40 min. We observed that Ang II-induced a significant inhibition of ROMK in LacZ, but not in ATRAP-overexpressed M-1 cells. Inhibition of ROMK-mediated K secretion by Ang II was accompanied by lower ROMK cell surface expression. Conversely, Ang II did not affect the ROMK-cell surface abundance in M-1 cells transfected with ATRAP. Additionally, diminished response to Ang II in M-1 cells overexpressing ATRAP was accompanied by decreased c-Src phosphorylation at the tyrosine 416. Unexpectedly, reduced phospho-c-Src levels were also found in M-1 cells, overexpressing ATRAP treated with vehicle, suggesting that ATRAP can also downregulate this kinase independently of Ang II-AT1R activation. Collectively, our data support that ATRAP attenuates inhibition of ROMK by Ang II in collecting duct cells, presumably by reducing c-Src activation and blocking ROMK internalization. The potential role of ATRAP in K homeostasis and/or disorders awaits further investigation.
肾脏钾排泄的调节是钾稳态的核心机制。肾外髓质钾（ROMK）通道是基础状态下集合管中主要的钾分泌途径。已知在钾饮食限制的情况下，血管紧张素II（Ang II）激活1型受体（AT1R）会抑制ROMK活性，这突出了AT1R在钾保留中的作用。本研究旨在探讨AT1R结合伴侣——AT1R相关蛋白（ATRAP）是否会影响集合管细胞中Ang II介导的ROMK调节，如果是，则深入了解潜在的机制。为此，我们在M-1细胞（一种皮质集合管细胞的模型细胞系）中过表达了ATRAP或β-半乳糖苷酶（LacZ；用作对照）。然后，我们采用一种基于荧光的新型微孔板分析法评估ROMK通道活性。实验在存在10 μM Ang II或溶剂的情况下进行40分钟。我们观察到，Ang II在LacZ中显著抑制了ROMK，但在过表达ATRAP的M-1细胞中没有。Ang II对ROMK介导的钾分泌的抑制伴随着ROMK细胞表面表达的降低。相反，Ang II不影响转染了ATRAP的M-1细胞中ROMK的细胞表面丰度。此外，过表达ATRAP的M-1细胞对Ang II的反应减弱伴随着酪氨酸416处c-Src磷酸化的降低。出乎意料的是，在用溶剂处理的过表达ATRAP的M-1细胞中也发现磷酸化c-Src水平降低，这表明ATRAP也可以独立于Ang II-AT1R激活下调这种激酶。总体而言，我们的数据支持ATRAP减弱了集合管细胞中Ang II对ROMK的抑制，可能是通过降低c-Src激活和阻止ROMK内化。ATRAP在钾稳态和/或疾病中的潜在作用有待进一步研究。
