Interacting molecule of AT1 receptor, ATRAP, is colocalized with AT1 receptor in the mouse renal tubules

Abstract

The renin-angiotensin system in the kidney plays a critical role in the regulation of renal hemodynamics and sodium handling through the activation of vascular, glomerular and tubular angiotensin II type 1 (AT1) receptor-mediated signaling. We previously cloned a molecule that specifically bound to the AT1 receptor and modulated AT1 receptor signaling in vitro, which we named ATRAP (for AT1 receptor-associated protein). The purpose of this study is to analyze the renal distribution of ATRAP and to examine whether ATRAP is co-expressed with the AT1 receptor in the mouse kidney. We performed in situ hybridization, Western blot analysis, and immunohistochemistry to investigate the expression of ATRAP mRNA and protein in the mouse kidney. The results of Western blot analysis revealed the ATRAP protein to be abundantly expressed in the kidney. Employing in situ hybridization and immunohistochemistry, we found that both ATRAP mRNA and the protein were widely distributed along the renal tubules from Bowman's capsules to the inner medullary collecting ducts. ATRAP mRNA was also detected in the glomeruli, vasculature, and interstitial cells. In all tubular cells, the ATRAP protein colocalized with the AT1 receptor. Finally, we found that the dietary salt depletion significantly decreased the renal expression of ATRAP as well as AT1 receptor. These findings show ATRAP to be abundantly and broadly distributed in nephron segments where the AT1 receptor is expressed. Furthermore, this is the first report demonstrating a substantial colocalization of ATRAP and AT1 receptor in vivo.

Atrap Deficiency Increases Arterial Blood Pressure and Plasma Volume

“…BASIC RESEARCH www.jasn.org the aorta as determined by Western blotting and no Atrap expression in the vasculature of the kidney as determined by immunostaining. 19 In the kidney, the organ with the highest Atrap expression, Atrap was expressed predominantly in the proximal tubule. This finding is in agreement with another renal localization study 19 ; however, unlike this recent study, we did not find significant tubular Atrap expression in segments other than the proximal tubule, although scattered single cells of the thick ascending limb and the distal convoluted tubule seemed to express Atrap.…”

“…This result is in agreement with a recent study that addressed the tissue distribution of Atrap by Western blotting. 19 Co-staining with the vascular smooth muscle cell marker SM22 revealed virtually no Atrap expression in blood vessels. This observation is congruent with a report that indicated very low levels of Atrap expression in Figure 9.…”

“…After reverse transcription, real-time PCR was performed for Atrap, AT1 receptor, and ␤-actin using a Light-Cycler system (Roche, Mannheim, Germany). For immunohistochemistry assays, an anti-mouse Atrap antibody was generated in rabbits according to standard protocols using a 13-amino acid immunizing peptide (amino acids 148 through 161 of the C-terminal tail of the Atrap protein 19 ). Commercial antibodies were used for detection of SM22, calbindin, TammHorsfall glycoprotein (Santa Cruz Biotechnology, Santa Cruz, CA), megalin (gift from Dr. Bachmann, Charite, Berlin, Germany) and renin (Davids Biotechnologie, Regensburg, Germany).…”

Effect of Olmesartan on Tissue Expression Balance Between Angiotensin II Receptor and Its Inhibitory Binding Molecule

“…5,6,12 The anti-AT1R antibody (sc-1173) was purchased from Santa Cruz Biotechnology, Inc. 5 Western blot analysis was performed as described previously. 5,6 Briefly, the cell extracts (20 g per lane) or rat tissue extracts (10 g per lane for liver and kidney and 20 g per lane for heart) were used for electrophoresis. Membranes (Millipore) were incubated with either (1) anti-ATRAP antibody or (2) anti-AT1R antibody and subjected to enhanced chemiluminescence (Amersham Biosciences).…”

“…3,4 We showed that ATRAP is expressed in many tissues such as the kidney, heart, and liver. 5 Previous studies showed that ATRAP exerts its inhibitory action on AT1R signaling by promoting a constitutive internalization of AT1R and decreasing the cell surface AT1R number. 6,7 In this study, we examined whether the expression of ATRAP is regulated in a tissue-specific manner in spontaneously hypertensive rats (SHR).…”