Immunohistochemical localization of ANG II AT(1) receptor in adult rat kidney using a monoclonal antibody

Molecular and functional studies have suggested that AT(1) receptors are present in most nephron segments, yet direct demonstration of AT(1) at these sites is lacking. The present study was performed to determine the intrarenal localization of the AT(1) receptor utilizing a monoclonal anti-peptide (amino acid residues 8-17) antibody (6313/G2) in adult male Sprague-Dawley rats. Western blot analysis of kidney protein extracts showed a predominant 41-kDa immunoreactive band corresponding to the molecular weight of the deduced cDNA sequence. To determine optimal fixation conditions, kidney tissues were immersion fixed in Bouin's solution, 10% buffered Formalin, or 4% paraformaldehyde. Specificity of immunostaining was documented by preadsorption of the antibody with the immunogenic peptide sequence. Prominent AT(1) immunostaining was visualized in the proximal tubule brush-border and basolateral membranes. In addition, distal tubules, cortical and medullary collecting ducts, and the renal arterial vasculature exhibited specific immunoreactivity. Glomerular staining for AT(1) was observed in mesangial cells and podocytes. Macula densa cells stained positively. Similar localization of the AT(1) receptor was obtained using the three tissue fixation methods, although the intensity of vascular and glomerular staining was highest in Bouin-fixed tissues. The present study demonstrates that the AT(1) receptor is more widely distributed along the nephron than previously described and includes renal vascular smooth muscle and proximal and distal epithelial sites.