ARTICLE |
Correspondence to: Jörg Peters, Dept. of Pharmacology, University of Heidelberg, INF 366, 69120 Heidelberg, Germany. E-mail: Joerg.Peters@urz.uni-heidelberg.de
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Summary |
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We studied regulation of the AT2 receptor by investigating the effect of bilateral nephrectomy (bNX) in SpragueDawley rats. The expression of aldosterone synthase (CYP11B2) and AT2 receptor mRNA was detected by nonradioactive in situ hybridization. AT2 receptor mRNA was detected in cells of the first two or three subcapsular cell layers of the zona glomerulosa (ZG) and in the medulla of sham-operated animals. After bNX, the number and area of distribution of AT2 receptor-positive cells increased in the ZG. This was associated with an enlargement of the steroidogenic active ZG and with reduced proliferation rate (sham 5.9 ± 0.9%; bNX 2.4 ± 0.2%; p<0.02). Infusion of angiotensin II (ANG II; 200 ng/kg/min SC for 56 hr) to bNX rats did not reverse the effect of nephrectomy on the distribution of AT2 receptor expression, although mRNA levels per cell were reduced compared to NX alone. ANG II infusion decreased proliferation rate further (0.4 ± 0.07%; p<0.001). In the adrenal medulla after bNX, decreased expression of the AT2 receptor was associated with increased proliferation (2.6 ± 0.2% vs 6.6 ± 0.5%). These results demonstrate differential regulation of the AT2 receptor in the adrenal gland and suggest that expression of the AT2 receptor is involved in regulating proliferation and differentiation in the ZG and medulla. (J Histochem Cytochem 49:649656, 2001)
Key Words: AT2 receptor, adrenal gland, nephrectomy, CYP11B2, reninangiotensin system
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Introduction |
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The effects of ANG II on a given cell type depend on the presence or absence of AT1 or AT2 receptors. These two main classes of angiotensin receptors belong to the seven transmembrane domain receptor family and consist of 359 amino acids for the AT1 receptor and 363 amino acids for the AT2 receptor. They share a homology of 3235% amino acids only (
We were interested in the regulation of AT2 receptor expression under conditions that increase the number of aldosterone synthase-expressing cells by differentiation processes rather than by proliferation. We chose bilateral nephrectomy (bNX) as an appropriate model. BNX increases aldosterone production and ZG size more prominently than sodium depletion, but we supposed this was unlikely to be accomplished by proliferation because an enlarged ZG is already present within a very short period of time, i.e., 2 days (
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Materials and Methods |
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Animals, Nephrectomy, and Hemodialysis
Adult male SpragueDawley rats weighing 400520 g were housed under alternating 12-hr light and dark cycles at a constant temperature between 20 and 22C. They were fed a standard laboratory chow [Ssniff (R/M-H); Soest, Germany) and had free access to tapwater. All animal experiments were in accordance with international ethical rules and appreciated by a governmental ethical commitee.
Under ketamine/xylazine anesthesia (75 mg/kg and 6 mg/kg bw), animals were either bilaterally nephrectomized (n=12) or sham-operated (n=6). They received chronic indwelling catheters into the femoral artery and vein and an intraperitoneally located osmotic minipump. Hemodialysis was performed every day after surgery as described previously (
Tissue Preparation
Rats were anesthetized with ketamine/xylazine (75 mg/kg and 6 mg/kg bw, respectively). After cannulation of the abdominal aorta just below the renal arteries, a retrograde perfusion was performed with 2% freshly prepared paraformaldehyde in PBS, pH 7.4, for 90 sec at a pressure level of 220 mmHg and for 90 sec at a pressure level of 170 mmHg. This was followed by a perfusion with 18% sucrose in PBS, adjusted to 800 mOsm/kg for another 3 min at the same pressure level. Adrenal glands were removed, gently sliced, mounted on small pieces of styrofoam, and quickly snap-frozen in liquid nitrogen-cooled isopentane.
Preparation of Riboprobes and In Situ Hybridization (ISH)
Full-length rat AT2 receptor cDNA (2.8 kb) was initially adapted and subcloned into the pcDNAI vector as described in the original report (
A specific probe of CYP11B2 and CYP11B1 was generated by PCR from a full-length cDNA, which had been prepared from the rat adrenal cDNA.
The sense (5'-GGGATGTCATCTCTGAGTATG-3') and antisense (5'-GATTGCTGTCGTGTCAAC-3') primers used give rise to a fragment of 182 bp, corresponding to nucleotides 779961 of CYP11B1, and a fragment of 185 bp, corresponding to nucleotides 779964 of CYP11B2 (
ISH has been described in detail previously (
The specificity of the ISH signal obtained was verified by parallel incubation with antisense and sense riboprobes on alternate sections. Throughout all experiments, sense probes did not produce any detectable signal. Antisense riboprobes for CYP11B2 and CYP11B1 were used on alternate sections as single probes and were applied simultaneously for double hybridization.
Immunohistochemistry
Immunohistochemistry was performed (n=3 per group) according to the method described previously (
Fresh frozen sections 7 µm thick were transfered onto silane-coated glass slides and air-dried for 20 min. All incubation steps were performed at room temperature. Sections were incubated with blocking solution (2% BSA, 0.15% Triton in PBS, pH 7.4) for 1 hr. Then the slides were incubated for 3 hr either with the first antibody, Ki-67 diluted 1:100 in 2% BSA, PBS, pH 7.4 or with normal rabbit serum diluted 1:100, as a negative control on alternate sections. They were washed three times with PBS for 5 min and then incubated in the dark for 50 min with Cy3-conjugated AffiniPure donkey antirabbit IgG (Dianova) diluted 1:600 in PBS. After three washing steps in PBS for 5 min, slides were covered with bicarbonate-buffered 66% glycine, pH 8.6. Throughout all experiments, no staining of nuclei was observed with the control normal rabbit serum.
Representative sections were photographed with a Polivar 2 (Firma Reichert Jung; Vienna, Austria), visualized with Richardson staining solution, and photographed again in the same position to identify the zonal localization of positive cells. A ratio of positively stained proliferating cells to the total number of cells was obtained by counting each 9001000 cells of the ZG/ZI and 788800 cells of the medulla per rat.
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Results |
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AT2 Receptor Expression
AT2 receptor mRNA was observed in the adrenal gland of sham-operated animals mainly in the medulla (Fig 1A). The staining in the medulla was not homogeneous, although almost all cells in the medulla were positive with various degree of intensity. The intensity of the signal in the ZG was far weaker than in the medulla and was restricted to the cells of the first two or three cell layers subcapsularly. Staining was observed neither in the adrenal capsule, demonstrating specificity of the reaction, nor in the zona fasciculata or zona reticularis. CYP11B2 mRNA was detected in the first two or three subcapsular cell layers of the ZG only (Fig 1B).
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Compared to sham-operated rats (Fig 2A), bNX led to a marked increase in the expression of AT2 receptor mRNA in the ZG (Fig 2C). The staining in the ZG became so intense that maximal staining was accomplished after only 6 hr of color reaction, whereas in sections of control rats the signal was even less intense after 48 hr. There was not only an increase per cell but also a change in the pattern of distribution. Concomitantly with the enlargement of the steroidogenic active ZG (from two or three to five or six cell layers), as defined by the expression of CYP11B2 mRNA (Fig 2B and Fig 2D), the area of cells expressing AT2 receptor mRNA increased similarly from two or three to five to seven layers of cells. However, in contrast to the homogeneous distribution of CYP11B2 in bNX animals, distribution of AT2 receptor mRNA was not homogeneous. Only a few clusters of cells were found to express the AT2 receptor gene. ANG II infusion to bNX rats, although suppressing much of the increase of AT2 receptor mRNA, did not prevent the spread of AT2 receptor expression over an area of five to seven cell layers (Fig 2E).
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In contrast to the increase of AT2 receptor expression in the ZG by bNX, this condition led to a suppression of the receptor in the medulla (Fig 3A and Fig 3B). Only a few single cells still expressed AT2 receptor mRNA to a significant level after bNX. No marked changes were observed between ANG II- or NaCl-infused nephrectomized rats with regard to AT2 receptor expression in the medulla (data not shown).
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Zonal Changes and Proliferation Indices
In sham-operated rats, CYP11B2 mRNA was detected in cells of the first two or three subcapsular cell layers only, with no crossreaction to CYP11B1, which was detected only in the ZF and ZR, decreasing gradually centripetally. A few single cells in the medulla were positive for CYP11B1 as well, reflecting the presence of steroidogenic cells in the medulla. The steroidogenic undifferentiated zone remained free of staining in the double ISH of CYP11B1 and CYP11B2 of control adrenals (Fig 4A).
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After bNX, the cells of the ZG were markedly hypertrophic. Between ZG and ZF, a zone with cells free of staining could no longer be detected (Fig 4B. Only single cells remained negative for both probes, but unstained cells were also found deeper within the zona fasciculata. No difference was observed between bNX- and bNX/ANG II-treated groups (Fig 4B and Fig 4C). The capsule remained free of staining.
In the ZG/ZI, the rate of proliferation was higher in sham-operated animals than in bNX animals (5.86 ± 0.93% vs 2.41 ± 0.24%; p<0.02). The proliferation rate was even lower in bNX/ANG II than in both other groups (0.41 ± 0.07%; p<0.001).
In contrast, in the adrenal medulla the proliferation rate increased in bNX (from 2.6 ± 0.2% in sham-operated rats to 6.6 ± 0.5% in bNX; p<0.05). No significant difference was observed between bNX and bNX/ANG II (6.6 ± 0.5% vs 4.7 ± 1.1%; n.s.).
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Discussion |
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Taking advantage of nonradioactive ISH with the use of specific riboprobes, it is possible not only to define the different zones of the adrenal cortex but also to localize the expression of a given gene to single cells. Under control conditions, we found AT2 receptor mRNA predominantly within the medulla, as expected, and to a lesser degree in subcapsular cells of the zona glomerulosa.
Some authors reported the presence of AT2 receptors not only in the zona glomerulosa but also in the zona fasciculata by use of immunhistochemistry (
Little information is available about the regulation of the AT2 receptor in the adult rat adrenal gland. After a low-sodium diet, an increase of AT2 receptor mRNA has been observed in the ZG by Northern blotting analysis (
It is noteworthy that the appearance of this receptor after bNX is associated with growth processes in the adrenal cortex of adult rats. Thus far, most studies focusing on the AT2 receptor were carried out in vitro using bovine adrenocortical cells or medullary PC12W cells. It has been shown that the AT2 receptor exerts an antiproliferative effect and even promotes apoptosis, depending on the cell type (
In the adrenal cortex, a role for the AT1 receptor in modulating growth processes needs to be considered. The AT1R is known to increase proliferation. Reduced AT1R activation due to reduced levels of circulating ANG II and reduced levels of AT1AR could well account for the decreased proliferation observed. However, any activation of AT1R by additionally infused ANG II to bNX rats should have resulted in higher proliferation rates compared with vehicle-infused nephrectomized rats. Instead, a further inhibition was observed. This rules out the possibility that inhibition of proliferation is a result of diminished number of activated AT1R. In contrast, our data strengthen the hypothesis of an inhibitory effect of the AT2 receptor on proliferation. They do not, however, allow the conclusion that the AT2 receptor causes the NX-induced growth. Certainly, other factors, such as increased potassium levels, should be independently involved (
The adrenal changes that lead to a rise in aldosterone production observed after bNX are different from those observed with other stimulatory conditions. For instance, the enlargement of the ZG following sodium depletion is mainly due to proliferation and the undifferentiated zone migrates centripetally (
In contrast to AT2 receptor regulation under a low-sodium diet, which leads to an increased expression in the ZG and the medulla (
In contrast to the observations in the outer cortex, in the medulla we found a reduced level of AT2 receptor mRNA after bNX. At first view this was rather unexpected, because bNX is known to increase levels of catecholamines (
The model of bilateral nephrectomy may represent a useful tool to further study the function of the AT2 receptor in the zona glomerulosa and medulla and also to elucidate the derangement in catecholamine release observed in uremic patients.
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Acknowledgments |
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Supported by Grant Pe 366/3-3 from the Deutsche Forschungsgemeinschaft and by the Forschungsfond der Fakultät für Klinische Medizin Mannheim of the University of Heidelberg.
We wish to thank Prof Wilhelm Kriz (Department of Anatomy and Cell Biology I, University of Heidelberg) for kindly providing facilities for histological analysis.
Received for publication July 5, 2000; accepted December 5, 2000.
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