Expression of neuronal type nitric oxide synthase and renin in the juxtaglomerular apparatus of angiotensin type-1a receptor gene-knockout mice

Abstract

Angiotensin type-1a (AT1a) receptor gene-knockout (AT1a-/-) mice exhibit chronic hypotension and renin overproduction. In the kidneys of AT1a-/- mice, the activity of neuronal type nitric oxide synthase (N-NOS) was histochemically detected by nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase (NADPHd) reaction combined with N-NOS immunohistochemistry. The localization of renin was detected by immunohistochemistry and the results were analyzed morphometrically. The levels of N-NOS and renin mRNA in the renal cortical tissue were determined by reverse transcription-PCR and Northern blot analysis, respectively. In the renal sections from wild-type mice, NADPHd activity and N-NOS immunoreactivity were localized to the discrete region of the macula densa in contact with the parent glomerulus. In contrast, N-NOS-positive macula densa cells were distributed beyond the original location of the macula densa, occasionally extending to the opposite side of the distal tubules. The mean number of N-NOS positive macula densa cells was significantly increased in AT1a-/- mice (186 per 100 glomeruli) compared with wild-type mice (65 per 100 glomeruli). AT1a-/- mice showed 1.4-times higher N-NOS mRNA levels in the renal cortical tissues than wild-type mice. The plasma renin activity was significantly higher in AT1a-/- mice (205.5 +/- 26.1 ng/ml/hr) than in wild-type mice (8.0 +/- 0.2 ng/ml/hr). The renin-positive areas per glomerulus and renal renin gene expression were 12-times and 2.6-times higher in AT1a-/- mice than in wild-type mice, respectively. These abnormalities, however, were less remarkable in AT1a-/- mice compared with angiotensinogen-knockout mice. When AT1a-/- mice were fed a high-salt diet, the signal intensity of the NADPHd reaction and the number of positively-stained macula densa cells were significantly decreased. The levels of renal cortical N-NOS mRNA were also suppressed by the treatment. Dietary salt loading produced a parallel decrease in plasma renin activity, renal renin-immunoreactive areas, and the levels of renin mRNA without affecting systemic blood pressure. These results provide evidence for the possible involvement of N-NOS at the macula densa in the increased renin production in AT1a-/- mice.

Renal Renin-Angiotensin System

“…This concept is supported by recent evidence that the superoxide dismutase mimetic, tempol, restores the reduced bioavailability of nNOS-derived NO in spontaneously hypertensive rats [38]. In addition, the expression of the nNOS gene and protein in the macula densa are upregulated in angiotensinogen-gene-knockout mice [39] and AT1 receptor-deficient mice [40]. Interestingly, the enhanced ability of nNOS-derived NO to counteract the TGF-mediated afferent arteriolar constriction observed in AT1 receptor-deficient mice [41], suggests that the modulation of TGF responses by Ang II is partially due to decreased activity of macula densa nNOS.…”

Alterations in Renal Endothelial Nitric Oxide Synthase Expression by Salt Diet in Angiotensin Type-1a Receptor Gene Knockout Mice

“…Total RNA was isolated from the renal cortex by the acid guanidinium thiocyanate-phenol-chloroform extraction method (16); 0.3 g of the sample RNA was mixed with 10 U/l SuperScript III reverse transcriptase (Invitrogen, Ontario, Canada), 25 ng/ml oligo(dT) [12][13][14][15][16][17][18] , 0.5 mM dNTP, and 5 mM dithiothreitol. The mixture was incubated at 50°C for 50 min, heated to 70°C for 15 min to terminate the RT reaction, and then incubated with 0.1 U/ml RNase H at 37°C for 20 min.…”

“…Kidneys were fixed in 0.1 M phosphate buffer (pH 7.4) containing 4% paraformaldehyde and 0.2% picric acid for 24 h, as described previously (12,17,18). The samples were cryoprotected with phosphate buffer containing 30% sucrose for 24 h and then frozen in isopentane cooled by liquid nitrogen.…”

“…We have used these mice as a model system to study the regulation of the renin angiotensin system (RAS) and related systems (11). We have found that homozygous mutant mice exhibit chronic hypotension and morphologic abnormalities in the renal vasculature (11,12). Moreover, we recently demonstrated that intrarenal arteries of At1aϪ/Ϫ mice show irregular arrangements of vascular smooth muscle cells and that these cells have a proliferative phenotype (13).…”

Interstitial Fibroblast-Like Cells Express Renin-Angiotensin System Components in a Fibrosing Murine Kidney

“…Polyclonal rabbit anti-AT 1 R antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), anti-AT 2 R antibodies, 19 anti-REN antibodies (a generous gift from K. Murakami, University of Tsukuba, Tsukuba, Japan), 20 and anti-ACE antibodies (a generous gift from P. Corvol, College de France, France) 21 were used as the primary antibodies for detection of the RAS components. For phenotyping interstitial fibroblast-like cells (FbLCs), polyclonal goat anti-vimentin (VIM) antibodies (Sigma, St. Louis, MO), rabbit anti-FSP1 antibodies, 22 and anti-TGF-␤1 antibodies (LC, which recognizes intracellular TGF-␤1), 13 and fluorescein isothiocyanate (FITC)-conjugated monoclonal mouse anti-␣-smooth muscle actin (␣-SMA) antibody (Sigma) were used.…”