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Molecular mechanisms involved in modulation of AT1R by ATRAP and associated proteins and effects on renal tubular ion transport


Angiotensin receptor type I (AT1R) activation by angiotensin II (AngII) induces complex mechanisms of intracellular signaling, which result in increase of NaCl and H2O reabsorption in renal tubules. These effects are important in developing and maintenance of arterial hypertension. Our goal with the present study is elucidate new aspects of the AT1R functioning, with special interest on the role of the AT1R/ATRAP (AT1R Associated Protein) association. ATRAP has been viewed as a negative regulator of AT1R, which neutralizes many effects of AngII on smooth muscle vascular cells, cardiac myocytes, and, possibly, on renal tissue. We intend to investigate the effects of the AT1R/ATRAP interaction on AT1R mediated signaling, dependent and independent of G proteins, and on internalization and degradation of the AT1 receptor, in the proximal tubule cell line of opossum, OKP, using over expression or knockdown of ATRAP. Since the Na+/H+ exchanger NHE3 is the main route for Na+ reabsorption in proximal tubules, we will analyze the expression and activity of this transporter in response to AngII in cells with over-expression or knockdown of ATRAP. In HEK293 cell line, we will analyze the role of ATRAP on AT1R interaction with ²-arrestin 1 and 2, with Rab5, Rab4, Rab11, and with Hsc70.One of the important signaling pathways down regulated by ATRAP is the AngII-AT1R/calcineurin/NFAT, and we will investigate the effect of ATRAP on the CAML (Ca2+ modulating cyclophilin ligand) mediated change in intracellular Ca2+ and on Ca2+ influx through store operated channels (SOC/Orai). We intend to analyze the possible interaction of CAML with the complex Orai-STIM1-RACK1 and the role of ATRAP on this interaction.AngII is also an important regulator of ion transport in distal convoluted tubules (DCT), and over-expression of ATRAP in DCT attenuates the arterial hypertension induced by AngII. On the other hand, calcineurin inhibitors, cyclosporine A (CsA) and tracolimus, induce hypertension and hyperkalemia, which are reversed by thiazide diuretics, same clinical symptoms observed in patients with WNK4 gene mutation. WNK4 is a key kinase in regulation of ion transport in DCT, and the expression of this protein is increased by AngII and CsA. All considering, we intend to investigate the mechanisms involved in modulation of WNK4 expression in a DCT cell line by Ang II and CsA, and the role of ATRAP on these regulatory process.The methods we intend to use are: cloning and expression of recombinant fusion proteins; siRNA; intracellular pH and Ca2+ measurements with fluorescent probes; BRET and FRET for protein interactions; western immunoblotting; immune precipitation; confocal microscopy. (AU)