Harrison-Bernard L M, Navar L G, Ho M M, Vinson G P, el-Dahr S S
Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA.
Am J Physiol. 1997 Jul;273(1 Pt 2):F170-7. doi: 10.1152/ajprenal.1997.273.1.F170.
Molecular and functional studies have suggested that AT1 receptors are present in most nephron segments, yet direct demonstration of AT1 at these sites is lacking. The present study was performed to determine the intrarenal localization of the AT1 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 AT1 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 AT1 was observed in mesangial cells and podocytes. Macula densa cells stained positively. Similar localization of the AT1 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 AT1 receptor is more widely distributed along the nephron than previously described and includes renal vascular smooth muscle and proximal and distal epithelial sites.
分子和功能研究表明，大多数肾单位节段都存在血管紧张素Ⅱ1型（AT1）受体，但缺乏在这些部位的直接证据。本研究旨在利用单克隆抗肽（氨基酸残基8 - 17）抗体（6313/G2）确定成年雄性Sprague-Dawley大鼠肾内AT1受体的定位。对肾脏蛋白提取物进行的蛋白质印迹分析显示，有一条主要的41 kDa免疫反应带，与推导的cDNA序列分子量相对应。为了确定最佳固定条件，将肾脏组织浸入Bouin氏液、10%缓冲福尔马林或4%多聚甲醛中进行固定。通过用免疫原性肽序列预吸附抗体来证明免疫染色的特异性。在近端小管刷状缘和基底外侧膜中可见明显的AT1免疫染色。此外，远端小管、皮质和髓质集合管以及肾动脉血管系统均表现出特异性免疫反应。在系膜细胞和足细胞中观察到肾小球AT1染色。致密斑细胞呈阳性染色。使用三种组织固定方法获得了相似的AT1受体定位，尽管在Bouin固定的组织中血管和肾小球染色强度最高。本研究表明，AT1受体在肾单位中的分布比先前描述的更广泛，包括肾血管平滑肌以及近端和远端上皮部位。
