Flow Cytometric Analysis of At1a Receptor Protein Expression in Renal Cortical Endosomes in Angiotensin Ii-Induced Hypertensive Rats
The positive feedback regulation of angiotensinogen and AT1 receptor expression in renal cortical cells by angiotensin II (Ang II) may play an important role in augmenting intrarenal Ang II formation/accumulation or in enhancing tubular reabsorptive responses in Ang II-dependent hypertension. The aim of the present study was to determine the AT1A receptor expression in rat renal cortical endosomes and intermicrovillar clefts during Ang II-induced hypertension using flow cytometry. Adult male Sprague-Dawley rats were treated with either vehicle (n=8) or chronic Ang II infusion via an osmotic minipump (n=8; 80 ng/min, s.c.) for 13 days. Rats receiving chronic Ang II infusion developed hypertension progressively over 12 days (Control: 120 ± 9 mmHg; Ang II: 191 ± 17 mmHg; p<0.05). To quantitate the expression of the AT1A receptor in isolated/purified renal cortical endosomes and intermicrovillar clefts, AT1A receptor antibody binding curves were performed using a rabbit polyclonal antibody to the cytosolic tail of the AT1A receptor. AT1A receptor binding was significantly increased by 40%-60% in both renal endosomes (Control: 115.2 ± 5.4 fluorescence units; Ang II: 160.9 ± 17.6 fluorescence units, p<0.05) and intermicrovillar clefts (Control: 26.4 ± 4.8 fluorescence units; Ang II: 44.7 ± 8.6 fluorescence units, p<0.05) in the Ang II-induced hypertensive rats. No significant difference was observed in basolateral membranes between the groups. These results indicate that increased expression of AT1A receptors in renal cortical endosomes and intermicrovillar clefts may promote trafficking of Ang II into the intracellular endosomal compartment and enhance tubular reabsorption during Ang II-induced hypertension.