ATRAP (AT1R Associated Protein) Role on Angiotensin II-Mediated NHE3 Activity Modulation

Angiotensin II (AngII) is one of the most important modulators of fluid transport at proximal tubule, playing a considerable role in the regulation of Na+/H+ exchanger isoform 3 (NHE3), which is responsible for the major part of transcelular Na+ transport at this nephron segment. Such regulatory mechanism is mediated by AngII receptor type 1 (AT1R), a G protein-coupled receptor (GPCR). Considering that the recently identified AT1R interacting protein called ATRAP seems to act as a negative regulator of this receptor, this work aimed to address, by overexpression experiments, how ATRAP affects NHE3 regulation mediated by AngII/AT1R in a proximal tubule cell line (OKP). The NHE3 activity was evaluated by measuring intracellular pH (pHi) recovery after previous acidification with ammonium chloride pulse. Besides control groups previously treated or not with AngII (10−10M for 50 minutes), the experimental groups – cells overexpressing one of the recombinant proteins LacZ/myc-His, LacZ-V5-His, AT1aR-myc-His and ATRAP-V5-His – were also treated with AngII. Our results showed, as expected, a significant faster pHi recovery rate in OKP cells treated with AngII (6,544±0,8425 min in untransfected cells; 3,059±0,4659 min in untransfected cells with AngII; 2,784±0,6104 and 2.641±0,3911 min in LacZ/myc-His and LacZ/V5-His transfected cells with AngII, respectively). Despite our initial hypothesis, we did not observe a faster pHi recovery rate in OKP cells overexpressing AT1R-myc-His (3,026±0,5445 min), what could be due to lack of expression of functional recombinant proteins, since the protein size on Western blot was lower than expected, or consequence of redundant effect on intracellular signaling. On the other hand, ATRAP overexpression decreased pHi recovery rate in comparison with the other experimental groups treated with AngII (4,423±0,9867 min). Our results support the hypothesis that ATRAP plays an inhibitory effect on Ang/AT1R modulation of NHE3.