Increased AT1 receptor expression and mRNA in kidney glomeruli of AT2 receptor gene-disrupted mice

Juan M. Saavedra1, Walter Häuser1, Gladys Ciuffo1, Giorgia Egidy1, Kwang-Lae Hoe1, Olaf Jöhren1, Takaaki Sembonmatsu2, Tadashi Inagami2, and Inés Armando1

1 Section on Pharmacology, National Institute of Mental Health, Bethesda, Maryland 20892; and 2 Vanderbilt University, Department of Biochemistry, School of Medicine, Nashville, Tennessee 37232


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The proposed feedback between angiotensin II AT2 and AT1 receptors prompted us to study AT1 receptor expression in kidneys of male AT2 receptor-gene disrupted mice (agtr2 -/y). In wild-type (agtr2 +/y) mice, AT1 receptor binding and mRNA is abundant in glomeruli, and AT1 receptor binding is also high in the inner stripe of the outer medulla. AT2 receptors are scarce, primarily associated to cortical vascular structures. In agtr2 -/y mice, AT1 receptor binding and mRNA were increased in the kidney glomeruli, and AT1 receptor binding was higher in the rest of the cortex and outer stripe of the outer medulla, but not in its inner stripe, indicating different cellular regulation. Although AT2 receptor expression is very low in male agtr 2 +/y mice, their gene disruption alters AT1 receptor expression. AT1 upregulation alone may explain the AT2 gene-disrupted mice phenotype such as increased blood pressure, higher sensitivity to angiotensin II, and altered renal function. The indirect AT1/AT2 receptor feedback could have clinical significance because AT1 antagonists are widely used in medical practice.

renin-angiotensin system; angiotensin II receptor types; gene-disrupted models


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ANGIOTENSIN II (ANG II), by stimulation of specific, discretely localized ANG II receptors, plays a crucial role in the modulation of renal function in mammals (31). ANG II receptors are classified into AT1 and AT2 types on the basis of their relative affinity for nonpeptidic-selective ligands (46) and molecular cloning (16, 19, 39). Most of the known actions of ANG II on the regulation of water and salt metabolism are dependent on stimulation of AT1 receptors (31, 46). In the kidney, stimulation of AT1 receptors by ANG II modulates both glomerular and tubular function including sodium retention, vasoconstriction of renal vessels, and decreased glomerular filtration rate (2).

AT1 receptors are present in large numbers in the adult mammalian kidney, with a major expression in the glomeruli, and lower levels in the renal cortical tubules, vasculature, medullar interstitial cells, and collecting ducts (1, 4, 6, 22, 23, 30, 36, 38, 40). Of the two AT1 receptor subtypes existing in rodents, AT1A and AT1B, the AT1A receptors predominate in the kidney (22). In adult rodents, kidney AT2 receptors were reported to be absent (4, 23, 41) or present at low levels (18, 38). Other studies reported a selective association of AT2 receptors in the adult kidney from different species, including humans, with vascular structures (8, 9, 10, 30, 49). The localized and restricted expression of AT2 receptors in association with renal arteries strongly suggested a function of AT2 receptors different from that of AT1 receptors, perhaps related to inhibition of angiogenesis and vasodilation (3, 34).

The availability of animal models with targeted disruption of specific genes provided an opportunity to further analyze the possible role of AT2 receptors. The targeted disruption of the mouse AT2 receptor gene significantly increased blood pressure and the sensitivity to the pressor action of ANG II, indicating an enhanced response to AT1 receptor stimulation (15, 17). We asked the question whether absence of AT2 receptor transcription could result in alterations in AT1 receptor binding or mRNA expression in selected areas of the kidney in this mouse model.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Mice were obtained form the Department of Biochemistry, Nashville University, and were kept under controlled conditions with free access to water and food, according to protocols approved by National Institute of Mental Health (NIMH) Animal Care and Use Committee. We produced the agtr2 gene-disrupted mice by the injection of agtr2 disrupted embryonic stem cells (E14-1) from the 129 Ola mouse line into blastocyts derived from C57BL/6 mice, as described previously (17). After the genotype of F2 heterozygous females (agtr2 -/+) was clearly confirmed, they were backcrossed with C57BL/6 wild-type males for three generations. Littermate gene-disrupted (agtr2 -/Y) and control wild-type (agtr2 +/Y) males were selected from the third backcross progeny to minimize the effect of differences in genetic background.

Mice were transported to NIMH, kept for 1 day under controlled conditions as above, and killed by decapitation between 10:00 AM and 11:00 AM. Kidneys were immediately removed, frozen in isopentane at -30°C, and stored at -80°C. For binding studies, sections (16 µm) were cut in a cryostat at -20°C, thaw-mounted on gelatin-coated slides, and dried overnight in a desiccator at 4°C. Sections were stored at -80°C until binding experiments were performed. Consecutive sections were used for ANG II receptor binding studies and in situ hybridization. Every tenth section was stained with hematoxylin and eosin to localize the structures expressing the binding or the receptor mRNA. For in situ hybridization experiments, sections were collected on silanated glass slides (Digene Diagnostics, Beltsville, MD) and stored at -80°C.

ANG II receptor binding. Sar1-ANG II (Peninsula Laboratories, Belmont, CA) and CGP-42112 (Neosystems Laboratory, Strasbourg, France) were iodinated by New England Nuclear (Boston, MA) to a specific activity of 2,200 Ci/mmol.

Adjacent kidney sections were preincubated for 15 min at 22°C in 10-mM sodium phosphate buffer, pH 7.4, containing 120 mM NaCl, 5 mM EDTA, 0.005% bacitracin (Sigma, St. Louis, MO), and 0.2% protease-free BSA (Sigma), followed by incubation for 2 h at 22°C in fresh buffer, prepared as above with the addition of 50 µM Plummer's inhibitor (Calbiochem, La Jolla, CA), 100 µM phenylmethylsulfonyl fluoride (Sigma), 500 µM phenantrolin (Sigma), and 0.5 nM [125I]Sar1-ANG II. After an incubation for 120 min at 22°C the sections were washed four times for 1 min each in ice-cold 50 mM Tris-HCl buffer (pH 7.4), followed by a 30 s wash in ice-cold water, and dried under a stream of cold air.

Binding of [125I]Sar1-ANG II to AT1 receptors was determined in adjacent kidney sections as follows. Some sections were incubated with 0.5 nM [125I]Sar1-ANG II to determine total binding. Adjacent sections were incubated as above with the addition of 10-5 M losartan (DuPont-Merck, Wilmington, DE), to displace binding to AT1 receptors. Binding to AT1 receptors was calculated as the difference between total binding and the binding remaining in adjacent sections incubated in the presence of excess concentration of losartan. Similarly, binding of [125I]Sar1-ANG II to AT2 receptors was determined as the difference between total binding and binding in adjacent sections incubated in the presence of 10-6 M of PD-123319 (Parke-Davis, Ann Arbor, MI) to selectively displace binding to AT2 receptors. The concentrations of the AT1 and AT2 receptor-selective ligands were selected to give maximum-specific displacement (13). Nonspecific or background binding was determined by incubating consecutive sections with 10-6 M ANG II (Peninsula). The values remaining were subtracted from all determinations as described above. In the case of areas where we found binding only to AT1 receptors, the values obtained after displacement with excess concentrations of losartan were not significantly different from background values obtained by displacement with excess concentrations of unlabeled ANG II.

In addition, [125I]CGP-42112 binding was performed in another set of adjacent sections to confirm the presence or absence of AT2 receptors. At the concentrations used, [125I]CGP-42112 exclusively labels AT2, and not AT1, receptors (14). Buffers used in this assay had the same composition as those used for the binding with [125I]Sar1-ANG II. Tissue sections were preincubated for 15 min in incubation buffer followed by incubation for 120 min in fresh buffer containing 0.2 nM [125I]CGP-42112. To determine specific binding to AT2 receptors, consecutive sections were incubated in the presence of 10-6 M of PD-123319 to selectively displace binding to AT2 sites. Nonspecific binding was determined by incubating consecutive sections with 5 × 10-6 M of ANG II (Peninsula).

To further localize AT2 receptors histologically, [125I]CGP-42112 binding was performed in 6-µm thick kidney sections. Adjacent sections were stained with hematoxylin and eosin. After binding experiments, sections were fixed for 60 min in paraformaldehyde vapors at 80°C and dipped in photo emulsion. After exposure for 2 wk, sections were developed in Kodak D-19 developer for 3 min at 15°C, fixed for 4 min, and counter stained with hematoxylin and eosin.

To determine if the radio-labeled ligands could be significantly metabolized under the conditions of incubation, we analyzed aliquots of buffers obtained before and after incubation of kidney sections by reversed-phase high performance liquid chromatography as described earlier (13). No metabolism of the radio-labeled ligands was observed under the above-mentioned conditions (results not shown).

Quantitative receptor autoradiography. We exposed dry sections to Hyperfilm-3H (Amersham, Arlington Heights, IL) along with 16-µm sections of autoradiographic 125I microscales (Amersham) at 4°C. Films were developed in ice-cold D-19 developer (Eastman Kodak, Rochester, NY) for 4 min, fixed in Kodak rapid fixer for 4 min at 22°C, and rinsed in water for 15 min. We measured optical densities in the autoradiograms by computerized microdensitometry by using the NIH Image 1.6 analysis system (NIMH, Bethesda, MD). For quantitative autoradiography, we measured the optical densities separately in kidney glomeruli, the rest of the cortex, and outer and inner stripes of the outer medulla. The optical densities were related to the concentration of radioactivity present in the sections by comparison with the 125I microscales, and transformed to corresponding values of fmol/mg protein (37). These values should be considered as arbitrary units, because the ligand concentrations used are below saturation, and because the actual protein-tissue-protein concentration varies between the different regions of the kidney (37). We found similar differences between groups when values were calculated as optical densities before comparison to 125I standards.

In situ hybridization histochemistry. In situ hybridization was performed by using 35S-labeled antisense and sense (control) riboprobes (21). A 478-bp EcoR I/SacI cDNA fragment of rat AT1A receptor (19, 39) showing 95 and 88% sequence homology to the mouse AT1A and AT1B receptor cDNA, respectively, and 99.4 and 92.5% amino acid homology to mouse AT1A and AT1B receptors, respectively (19, 39), was subcloned into the pBluescript II KS+ vector. Riboprobes were labeled by in vitro transcription by using an RNA labeling kit (Amersham). Because we used a riboprobe to the coding region of the rat AT1 receptor that has a very high homology between AT1A and AT1B-rodent subtypes, we did not determine the relative contribution of kidney AT1A or AT1B receptor subtypes in our experiments. However, over 95% of the renal AT1 receptors are of the AT1A subtype (22). Thus the mRNA data presented here predominantly reflects the regulation of the AT1A receptors.

Sections were fixed in 4% paraformaldehyde for 10 min, acetylated for 10 min in 0.1 M triethanolamine HCl, pH 8.0, containing 0.25% acetic anhydride, dehydrated in alcohols, and air dried. Each section was covered with 50-µl hybridization buffer containing 50% formamide, 0.3 M NaCl, 2 mM EDTA, 20 mM Tris, pH 8.0, 1 × Denhardt's solution, 10% dextran sulfate, 100 µg/ml salmon sperm DNA, 250 µg/ml yeast tRNA, 150 mM DTT, 0.1% SDS, and 40,000-counts per minute/µl sense or antisense probe. Sections were hybridized overnight at 54°C, treated with 40 µg/ml RNase A (Sigma, St. Louis, MO) for 30 min, and washed in sodium chloride/sodium citrate (SSC) with increasing stringency. After a final wash in 0.1 × SSC at 65°C for 60 min, sections were dehydrated through alcohols and exposed to Hyperfilm-3H (Amersham) along with 14C microscales (Amersham) for 7 days. Films were developed as described above. The intensities of hybridization signals in kidney glomeruli, the rest of the cortex, and outer and inner stripes of the outer medulla, were quantified as nCi/g tissue equivalent by measuring optical film densities by using the NIH Image 1.61 program. Data were calibrated with 14C microscales after subtraction of the values obtained in the same areas of adjacent sections hybridized with sense (control) probes (nonspecific hybridization). The values obtained represent arbitrary units, because the protein content or weight of the different kidney areas may be different (32). We found similar differences between groups when values were calculated as optical densities before comparison to 14C standards.

For cellular localization, slides were dipped in Kodak NTB2 photo emulsion, exposed for 4 wk, developed in Kodak D-19 developer for 3 min at 15°C, fixed for 4 min, and counterstained with hematoxylin and eosin (Fisher Scientific, Fair Lawn, NJ).

Statistical analysis. Results were expressed as means ± SE, calculated and analyzed by using GraphPad Prism (version 2.00) and Microsoft Excel (version 7.0a). Statistical analysis for values obtained from the displacement studies by using single concentrations of the displacers was performed by using a one-way ANOVA followed by post hoc analysis with the Newman-Keuls multiple comparison test. Mean ± SE values of wild-type and AT2 gene-disrupted mice were compared for significance by using unpaired Student's t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ANG II receptor subtype expression and mRNA in wild-type mice. We studied ANG II receptor subtype binding and mRNA in the kidney of male wild-type and AT2 gene-disrupted mice. Binding to AT1 receptors was, as expected, high and selectively localized in the kidney of wild-type mice. In wild-type mice, the highest levels of losartan-sensitive, PD-123319-insensitive, [125I]Sar1-ANG II binding to AT1 receptors were present on glomeruli (Fig. 1, A-E) and lower levels in the inner stripe of the outer medulla (Table 1). Binding to AT1 receptors was also present in the rest of the cortex and the outer stripe of the outer medulla (Table 1).


View larger version (86K):
[in this window]
[in a new window]
 
Fig. 1.   Autoradiography of ANG II receptor type binding in kidneys from male mice. Hematoxylin-eosin (H & E) staining [wild-type (A), AT2 gene deficient (F)] and autoradiographs with binding of 0.5 nM [125I]Sar1-ANG II in kidneys of wild-type (+/y) mice (B-E) and AT2 receptor gene-disrupted (-/y) mice (G-J) alone (B, G) or in the presence of 10-6 M ANG II (C, H), 10-5 M losartan (D, I), and 10-6 PD-123319 (E, J). Scale bar, 1 mm. Arrows point to losartan-sensitive [125I]Sar1-ANG II binding to AT1 receptors located on glomeruli. Arrowheads point to losartan-insensitive [125I]Sar1-ANG II binding to AT2 receptors that are absent in AT2 receptor gene-disrupted mice (I).


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   ANG II receptor AT1 binding and mRNA in mouse kidney

Emulsion autoradiography in wild-type mice revealed very high losartan-sensitive [125I]Sar1-ANG II binding in kidney glomeruli and low, diffuse losartan-sensitive binding throughout the whole renal cortex (Fig. 2, A-C).


View larger version (179K):
[in this window]
[in a new window]
 
Fig. 2.   Emulsion autoradiography of binding to AT1 and AT2 receptors in kidneys from male mice. Emulsion autoradiographs of binding of 0.5 nM [125I]Sar1-ANG II to glomeruli of kidneys from AT2 receptor gene-disrupted (-/y) mice in the absence (B) and presence of 10-5 M losartan (C). In addition, [125I]CGP-42112 binding is shown in vascular structures of kidneys from wild-type (+/y) mice (E). A and D: histology of B and E, respectively. A and D: arrows point to glomeruli. D and E: arrowheads point to a blood vessel. Scale bars, 60 µm. Scale bar in A applies to A-C. Scale bar in D applies to D and E.

AT1 receptor mRNA was also detected in the kidney of male wild-type mice. As it was the case with AT1 receptor binding, AT1 receptor mRNA was higher in the glomeruli, and lower in the rest of the cortex and in the outer and inner stripes of the outer medulla (Table 1 and Fig. 3, A and B). Low levels of AT1 mRNA expression were located in the renal vasculature (Fig. 3C).


View larger version (66K):
[in this window]
[in a new window]
 
Fig. 3.   Autoradiography of ANG II receptor subtype mRNA in kidneys from male mice. Film (A and B) and emulsion (C) autoradiograms after in situ hybridization by using an AT1 receptor-specific riboprobe. A: wild-type mice. B: AT2 gene-disrupted mice. Note the presence of AT1 receptor mRNA in glomeruli from wild-type mice (arrow in C). C: arrowhead points to a blood vessel. Scale bar in A is 2 mm (also applies to B). Scale bar in C is 250 µm.

In some cortical structures of wild-type mice, of different shape and size than the glomeruli, we found high-losartan-insensitive, PD-123319-sensitive [125I]Sar1-ANG II binding, indicative of AT2 receptors (Fig. 1D). To confirm the presence of AT2 receptors in the cortical structures distinct from glomeruli, we used AT2 receptor-selective [125I]CGP-42112 binding (Fig. 4). All [125I]CGP-42112 binding was displaced by ANG II (Fig. 4B) and by the AT2 receptor-specific ligand PD-123319 (Fig. 4D) but not by the AT1 receptor-specific ligand losartan (Fig. 4C). Specific [125I]CGP-42112 binding sites were detected in the kidney cortex and, as determined by emulsion autoradiography, were associated only with vascular structures, in particular the arcuate arteries (Fig. 2, D and E). Emulsion autoradiographic analysis of other structures in the kidney cortex, and in particular the kidney glomeruli, did not reveal accumulation of silver grains above background levels.


View larger version (82K):
[in this window]
[in a new window]
 
Fig. 4.   Autoradiography of binding to AT2 receptors in kidneys from male mice. Binding of 0.2 nM [125I]CGP-42112 in kidneys of wild-type (+/y) mice (A-D) and AT2 receptor gene-disrupted (-/y) mice (E-H) alone (A, E) and in the presence of 5 × 10-6 ANG II (B, F), 10-5 M losartan (C, G), and 10-6 PD-123319 (D, H). Arrowheads point to specific binding of [125I]CGP-42112 to AT2 receptors. Scale bar, 1 mm (A-H).

ANG II AT1 receptor expression and mRNA in AT2 gene-disrupted mice. As expected, the AT2 receptor gene-disrupted male did not express losartan-insensitive, PD-123319-sensitive (AT2) [125I]Sar1-ANG II binding sites (Fig. 1, F-J) or AT2 receptor-selective [125I]CGP-42112 binding (Fig. 4, E-H) in the renal cortex or in other areas of the kidney.

A remarkable difference in ANG II AT1 receptor binding studied with quantitative autoradiography, and mRNA, as determined by in situ hybridization, was noted in AT2 gene-disrupted mice compared with wild-type controls. In AT2 receptor gene-disrupted mice, both [125I]Sar1-ANG II binding to AT1 receptors and AT1 receptor mRNA were higher compared with values in wild-type mice. Significant increases in AT1 receptor binding (~125%) (Table 1, Fig. 1, G and J) and AT1 mRNA (~50%) (Table 1, Fig. 3, B and C) were found in glomeruli.

Binding was significantly increased in the rest of the renal cortex (3-fold) and in the outer stripe of the outer medulla (~90%). In these areas there was a tendency toward increased expression of AT1 mRNA, a ~45% increase in the rest of the renal cortex, and about a ~55% increase in the outer stripe of the outer medulla (Table 1).

In the rest of the renal cortex and the outer stripe of the outer medulla of AT2 gene-disrupted mice, AT1 receptor expression was also significantly higher (Table 1). In these areas there was increased expression of AT1 mRNA, but the results did not achieve statistical significance (Table 1). Conversely, no significant differences were found in AT1 receptor binding or mRNA in the inner stripe of the outer medulla between wild-type animals and AT2 gene-deficient male mice (Table 1).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We report that gene disruption of the ANG II AT2 receptor results in marked and selective alterations on the protein and mRNA expression of the renal AT1 receptors in the male mouse.

First, we analyzed the distribution of AT1 receptor binding and mRNA in the kidney of wild-type male mice. The highest numbers of AT1 receptors and highest AT1 mRNA expression occur in the glomeruli, significant AT1 receptor binding, and mRNA in the inner stripe of the outer medulla, and lower binding and receptor mRNA in the rest of the kidney cortex. These results are in agreement with previous data from other mammalian species and with the demonstration of AT1 receptors, not only in glomeruli but also in proximal and distal tubules, medullar interstitial cells, and the vasculature (1, 4, 6-10, 22, 23, 30, 36, 38, 40, 48). In addition, we detected low levels of AT1 receptor binding, and significant levels of AT1 receptor mRNA, in the outer stripe of the outer medulla of wild-type mice. This indicates that some AT1 receptors may be located, in the mouse, in medullar structures that do not express AT1 receptors in the rat (47).

We found a clear expression of AT2 receptor binding in the kidney of the wild-type mice, a finding that again contrasts with the reported absence of AT2 receptors in adult rats (1, 4, 5). The number of AT2 receptors in the adult mouse kidney was much lower than that of AT1 receptors, and followed a different pattern of localization. AT2 receptor binding was restricted to very selective cortical areas associated with renal vessels. This is in agreement with the reported localization of AT2 receptors in renal vessels of other species, including humans (8, 9, 10, 30, 48). Under the conditions of our assay, we could not find AT2 receptor binding in kidney glomeruli and interstitial cells, as proposed with immunocytochemical techniques (38). If present, these receptors might be expressed in low amounts below the sensitivity of our assay.

Thus our analysis of ANG II receptor subtypes in the adult mice indicated that both receptors were clearly expressed, that the AT1 receptor predominated, and that the localization of the receptor subtypes occurred, at least for the most part, with exception of the vasculature, in separate renal structures.

Next, we studied ANG II receptor subtype expression and mRNA in AT2 gene-disrupted mice. In the kidney of adult AT2 gene-disrupted mice there was no PD-123319-sensitive, losartan-insensitive binding and no [125I]CGP 42112, AT2-selective binding. The total absence of AT2 receptor binding in the AT2 gene-disrupted mouse was expected, because AT2 receptors are encoded by a single gene (16, 19, 39), and confirmed the identity of AT2 receptor binding sites with the cloned AT2 receptor.

The main finding in our study was the demonstration of a significant increase in AT1 receptor expression and mRNA in the kidney of adult AT2 gene-disrupted mice. Ours is a clear example of how the gene disruption of one receptor can alter the expression of another receptor. This is particularly remarkable considering that the expression of renal AT2 receptors in wild-type mice is very low, compared with that of AT1 receptors. Our results further support the hypothesis that the level of expression of the AT2 receptors inversely correlates with that of AT1 receptors. Earlier studies demonstrated that the cardiac specific over-expression of AT2 receptors attenuates the AT1-mediated pressor and chronotropic effects of ANG II (29). In addition, AT2 receptors are highly expressed in the fetal kidney, and their numbers decrease during gestation in parallel with a concomitant increase in AT1 receptor expression (4).

Activation of AT1 and AT2 receptors in wild-type animals results in effects different, and in most cases opposite; and stimulation of AT2 receptors was postulated to limit the response of AT1 receptors to ANG II (17). Stimulation of kidney AT1 receptors decreases cortical and medullar blood flow and decreases urine flow and natriuresis, the principal renal effects of ANG II, shifting pressure natriuresis to the right (11). Conversely, in wild-type animals, stimulation of AT2 receptors was reported to increase pressure natriuresis (26) and their stimulation results in renal vasodilatation, probably as a consequence of production of cGMP, (42), nitric oxide (43), and prostaglandin F2 alpha (44), and increased arachidonic acid release (20). AT1 receptor blockade produces vasodilatation, diuresis and natriuresis, and this was proposed to be due to unopposed AT2 receptor stimulation (3, 33, 35, 42). The evidence of a physiological role of renal AT2 receptors, however, is controversial. AT2 receptor blockade was reported to potentiate the ANG II-induced PGE2 production after AT1 receptor stimulation (42) and prevents the effect of ANG II on the vasculature (24). However, other studies indicate that chronic blockade of AT2 receptors in wild-type adult animals does not alter the response to AT1 receptor stimulation by ANG II (33).

In mice lacking AT2 receptor expression there is increased sodium retention, rightward shift in pressure natriuresis and diuresis, reduction in cortical and medullar blood flow, increased blood pressure, and enhanced hypertensive response to ANG II administration (12, 15, 17). The phenotype of the AT2 receptor gene-disrupted mouse may be related to the upregulation of the AT1 receptor, rather than negatively reflecting the actions of the AT2 receptor (12, 45). It is also possible that the AT2 receptor gene-disrupted phenotype could be the result of a lost balance between AT1 and AT2 receptors, with a shift toward predominance of AT1-related effects. In addition, higher AT1 receptor transcription and expression could explain the enhanced fibrosis after ureteral occlusion in AT2 gene-deficient mice (25, 28), because kidney fibrosis induced by ANG II is AT1 receptor dependent (27).

There is a recent report (12) of increased AT1 receptor mRNA in the whole kidney of AT2 gene-disrupted mice. Our findings of increased AT1 receptor binding indicate that an actual increase in AT1 receptor expression occurs in AT2 receptor gene-disrupted mice. In addition we demonstrate here that upregulation of renal AT1 receptors predominantly occurs in cortical structures, the kidney glomeruli, and probably in the tubular epithelium, and in the outer stripe of the outer medulla. In AT2 receptor gene-disrupted mice, there are no changes in AT1 receptor expression in the inner stripe of the outer medulla. Thus the AT1 upregulation and the AT2-AT1 feedback in this model are structure and cell specific. Although the expression of glomerular and cortical interstitial AT1 receptors may depend on AT2 receptor expression, the AT1 receptor expression in the inner stripe of the outer medulla does not. We conclude from our results that AT1 receptors in renal glomeruli, interstitial, and medullar cells are differentially regulated.

Last, our findings further support the hypothesis of a crosstalk or feedback between AT1 and AT2 receptor subtypes. It appears that AT1 receptors for the most part do not coexist with AT2 receptors in the same renal cells. For this reason, the mechanism of feedback is probably indirect, through modifications in kidney function related to the absence of AT2 receptors, and the upregulation of renal AT1 receptors.

In our model, when alterations in the transcription and expression of AT2 receptors are present from birth, renal AT1 receptor expression and consequently renal function, are profoundly affected in the adult. Because AT1 receptor antagonists are widely used in the treatment of cardiovascular disease, the crosstalk/feedback between AT1 and AT2 receptors has significant pathophysiological and clinical relevance.


    ACKNOWLEDGEMENTS

W. Haüser was supported by a grant from ASTRA GmbH, Germany.


    FOOTNOTES

Address for reprint requests and other correspondence: J. M. Saavedra, Section on Pharmacology, National Institute of Mental Health, Center Dr. MSC 1514, Bldg. 10, Rm. 2D-57, Bethesda, MD 20892 (E-mail: Saavedrj{at}irp.nimh.nih.gov).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5 April 2000; accepted in final form 29 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Allen, AM, Zhuo J, and Mendelsohn FAO Localization of angiotensin AT1 and AT2 receptors. J Am Soc Nephrol 10: S23-S29, 1999[ISI][Medline].

2.   Ardaillou, R. Angiotensin II receptors. J Am Soc Nephrol 10: S30-S39, 1999[ISI][Medline].

3.   Arima, S, Endo Y, Yaoita H, Omata K, Ogawa S, Tsunoda K, Abe M, Takeuchi K, Abe K, and Ito S. Possible role of P-450 metabolite of arachidonic acid in vasodilator mechanism of angiotensin II type 2 receptor in the isolated microperfused rabbit afferent arteriole. J Clin Invest 100: 2816-2823, 1997[Abstract/Free Full Text].

4.   Ciuffo, GM, Viswanathan M, Seltzer AM, Tsutsumi K, and Saavedra JM. Glomerular angiotensin II receptor subtypes during development of rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 265: F264-F271, 1993[Abstract/Free Full Text].

5.   Correa, FMA, Viswanathan M, Ciuffo GM, Tsutsumi K, and Saavedra JM. Kidney angiotensin II receptors and converting enzyme in neonatal and adult Wistar-Kyoto and spontaneously hypertensive rats. Peptides 16: 19-24, 1995[ISI][Medline].

6.   de Gasparo, M, and Levens NR. Pharmacology of angiotensin II receptors in the kidney. Kidney Int 46: 1486-1491, 1994[ISI][Medline].

7.   Gasc, JM, Shanmugam S, Sibony M, and Corvol P. Tissue-specific expression of type 1 angiotensin II receptor subtypes. An in situ hybridization study. Hypertension 24: 531-537, 1994[Abstract].

8.   Gibson, RE, Thorpe HH, Cartwright ME, Frank JD, Schorn TW, Bunting PB, and Siegl PK. Angiotensin II receptor subtypes in renal cortex of rats and rhesus monkeys. Am J Physiol Renal Fluid Electrolyte Physiol 261: F512-F518, 1991[Abstract/Free Full Text].

9.   Goldfarb, DA, Diz DI, Tubbs RR, Ferrario CM, and Novick AC. Angiotensin II receptor subtypes in the human renal cortex and renal cell carcinoma. J Urol 151: 208-213, 1994[ISI][Medline].

10.   Gröne, HJ, Simon M, and Fuchs E. Autoradiographic characterization of angiotensin receptor subtypes in fetal and adult human kidney. Am J Physiol Renal Fluid Electrolyte Physiol 262: F326-F331, 1992[Abstract/Free Full Text].

11.   Gross, V, Lippoldt A, Schneider W, and Luft FC. Effect of captopril and angiotensin II receptor blockade on pressure natriuresis in transgenic (mRen2) 24 rats. Hypertension 26: 471-479, 1995[Abstract/Free Full Text].

12.   Gross, V, Schunck WH, Honeck H, Milia AF, Kargel E, Walther T, Bader M, Inagami T, Schenider W, and Luft FC. Inhibition of pressure natriuresis in mice lacking the AT2 receptor. Kidney Int 57: 191-202, 2000[ISI][Medline].

13.   Häuser, W, Jöhren O, and Saavedra JM. Characterization and distribution of angiotensin II receptor subtypes in the mouse brain. Eur J Pharmacol 348: 101-114, 1998[ISI][Medline].

14.   Heemskerk, FMJ, and Saavedra JM. Quantitative autoradiography of angiotensin II AT2 receptors with [125I]CGP 42112. Brain Res 677: 29-38, 1995[ISI][Medline].

15.   Hein, L, Barsh GS, Pratt RE, Dzau VJ, and Kobilka BK. Behavioral and cardiovascular effects of disrupting the angiotensin II type-2 receptor gene in mice. Nature 377: 744-747, 1995[ISI][Medline].

16.   Ichiki, T, Herold CL, Kambayashi Y, Bardhan S, and Inagami T. Cloning of the cDNA and the genomic structure of the mouse angiotensin II type 2 receptor. Biochim Biophys Acta 1189: 247-250, 1994[ISI][Medline].

17.   Ichiki, T, Labosky PA, Shiota C, Okuyama S, Imagawa Y, Fogo A, Niimura F, Ichikawa I, Hogan BLM, and Inagami T. Effects on blood pressure and explorative behaviour of mice lacking angiotensin II type-2 receptor. Nature 377: 748-750, 1995[ISI][Medline].

18.   Ichiki, T, and Inagami T. Expression, genomic organization and transcription of the mouse angiotensin II type 2 receptor gene. Circ Res 76: 693-700, 1995[Abstract/Free Full Text].

19.   Iwai, N, Yamano Y, Chaki S, Konishi F, Bardhan S, Tibbetts C, Sasaki K, Hasegawa M, Matsuda Y, and Inagami T. Rat angiotensin II receptor: cDNA sequence and regulation of the gene expression. Biochem Biophys Res Commun Commun 177: 299-304, 1991[ISI][Medline].

20.   Jacobs, LS, and Douglas JG. Angiotensin II type 2 receptor subtype mediates phospholipase A2-dependent signaling in rabbit proximal tubular epithelial cells. Hypertension 28: 663-668, 1996[Abstract/Free Full Text].

21.   Jöhren, O, Inagami T, and Saavedra JM. AT1A, AT1B and AT2 angiotensin II receptor subtype gene expression in rat brain. Neuroreport 6: 2549-2552, 1995[ISI][Medline].

22.   Kakinuma, Y, Fogo A, Inagami T, and Ichikawa I. Intrarenal localization of angiotensin II type 1 receptor mRNA in the rat. Kidney Int 43: 1229-1235, 1993[ISI][Medline].

23.   Kakuchi, J, Ichiki T, Kiyama S, Hogan BLM, Fogo A, Inagami T, and Ichikawa I. Developmental expression of renal angiotensin II receptor genes in the mouse. Kidney Int 47: 140-147, 1995[ISI][Medline].

24.   Levy, BI, Benessiano J, Henrion D, Caputo L, Heymes C, Duriez M, Poitevin P, and Samuel JM. Chronic blockade of AT2-subtype receptors prevents the effect of angiotensin II on the rat vascular structure. J Clin Invest 98: 418-425, 1996[Abstract/Free Full Text].

25.   Li, WG, Chen WM, Ye YZ, Ichiki T, and Inagami T. Long-term effects of angiotensin II type 2 (AT2) receptor null mutation on renal fibronectin deposition and PA/PAI-1 regulation. J Am Soc Nephrol 9: 502A, 1998.

26.   Lo, M, Liu KL, Lantelme P, and Sassard J. Subtype 2 of angiotensin II receptors controls pressure-natriuresis in rats. J Clin Invest 95: 1394-1397, 1995[ISI][Medline].

27.  Lombardi DM, Viswanathan M, Vio CP, Saavedra JM, Schwarz SM, and Johnson RJ. Renal and vascular injury induced by exogenous angiotensin II is AT1 receptor dependent. Nephron in press.

28.   Ma, J, Nishimura H, Fogo A, Kon V, Inagami T, and Ichikawa I. Accelerated fibrosis and collagen deposition develop in the renal interstitium of angiotensin type 2 receptor null mutant mice during ureteral obstruction. Kidney Int 53: 937-944, 1998[ISI][Medline].

29.   Masaki, H, Kurihara T, Yamaki A, Inomata N, Nozawa Y, Mori Murasawa S, Kizima K, Maruyama K, Horiuchi M, Dzau VJ, Takahashi H, Iwasaka T, Inada M, and Matsubara H. Cardiac-specific overexpression of angiotensin II AT2 receptor causes attenuated response to AT1 receptor-mediated pressor and chronotropic effects. J Clin Invest 101: 527-535, 1998[Abstract/Free Full Text].

30.   Matsubara, H, Sugaya T, Murasawa S, Nozawa Y, Mori Y, Masaki H, Maruyama K, Tsutumi Y, Shibasaki Y, Moriguchi Y, Tanaka Y, Iwasaka T, and Inada M. Tissue-specific expression of human angiotensin II AT1 and AT2 receptors and cellular localization of subtype mRNAs in adult human renal cortex using in situ hybridization. Nephron 80: 25-34, 1998[ISI][Medline].

31.   Matsusaka, T, and Ichikawa I. Biological functions of angiotensin and its receptors. Annu Rev Physiol 59: 395-412, 1997[ISI][Medline].

32.   Miller, JA. The calibration of 35S or 32P with 14C-labeled brain paste or 14C-plastic standards for quantitative autoradiography using LKB Ultrofilm or Amersham Hyperfilm. Neurosci Lett 121: 211-214, 1991[ISI][Medline].

33.   Muller, C, Endlich K, and Helwig JJ. AT2 antagonist-sensitive potentiation of angiotensin II-induced constriction by NO blockade and its dependence on endothelium and P450 eicosanoids in rat renal vasculature. Br J Pharmacol 124: 946-952, 1998[Abstract].

34.   Munoz-Garcia, R, Maeso R, Rodrigo E, Navarro J, Ruilope LM, Casal MC, Cachofeiro V, and Lahera V. Acute renal excretory actions of losartan in spontaneously hypertensive rats: role of AT2 receptors, prostaglandins, kinins and nitric oxide. J Hypertens 13: 1779-1784, 1995[ISI][Medline].

35.   Munzenmaier, DH, and Greene AS. Opposing actions of angiotensin II on microvascular growth and arterial blood pressure. Hypertension 27[part 2]:: 760-765, 1996[Abstract/Free Full Text].

36.   Navar, LG, Harrison-Bernard LM, Wang CT, Cervenka L, and Mitchell KD. Concentrations and actions of intraluminal angiotensin II. J Am Soc Nephrol 10: S189-S195, 1999[ISI][Medline].

37.   Nazarali, AJ, Gutkind JS, and Saavedra JM. Calibration of [125I]-polymer standards with [125I]-brain paste standards for use in quantitative receptor autoradiography. J Neurosci Methods 30: 247-253, 1989[ISI][Medline].

38.   Ozono, R, Wang ZQ, Moore AF, Inagami T, Siragy HM, and Carey RM. Expression of the subtype 2 angiotensin (AT2) receptor protein in rat kidney. Hypertension 30: 1238-1246, 1997[Abstract/Free Full Text].

39.   Sasamura, H, Hein L, Krieger JE, Pratt RE, Kobilka BK, and Dzau VJ. Cloning, characterization, and expression of two angiotensin receptor (AT-1) isoforms from the mouse genome. Biochem Biophys Res Commun 185: 253-259, 1992[ISI][Medline].

40.   Sechi, LA, Grady EF, Griffin CA, Kalinyak JE, and Schambelan M. Distribution of angiotensin II receptor subtypes in rat and human kidney. Am J Physiol Renal Fluid Electrolyte Physiol 262: F236-F240, 1992[Abstract/Free Full Text].

41.   Shanmugan, S, and Sandberg K. Ontogeny of angiotensin receptors. Cell Biol Int 20: 169-176, 1996[ISI][Medline].

42.   Siragy, HM, and Carey RM. The subtype-2 (AT2) angiotensin receptor regulates renal cyclic guanosine 3',5'-monophosphate and AT1 receptor-mediated prostaglandin E2 production in conscious rats. J Clin Invest 97: 1978-1982, 1996[Abstract/Free Full Text].

43.   Siragy, HM, and Carey RM. The subtype 2 (AT2) angiotensin receptor mediates renal production of nitric oxide in conscious rats. J Clin Invest 100: 264-269, 1997[Abstract/Free Full Text].

44.   Siragy, HM, and Carey RM. The subtype 2 angiotensin receptor regulates renal prostaglandin F2 alpha formation in conscious rats. Am J Physiol 27: R1103-R1107, 1997.

45.   Siragy, HM, Inagami T, Ichiki T, and Carey RM. Sustained hypersensitivity to angiotensin II and its mechanism in mice lacking the subtype-2 (AT2) angiotensin receptor. Proc Natl Acad Sci USA 96: 6506-6510, 1999[Abstract/Free Full Text].

46.   Timmermans, PB, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JA, and Smith RD. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev 45: 205-251, 1993[ISI][Medline].

47.   Wisden, W, and Morris BJ. In situ hybridization with synthetic oligonucleotide probes. In: In Situ Hybridization Protocols for the Brain, edited by Wisden W, and Morris BJ.. New York: Academic, 1994, p. 9-34.

48.   Zhuo, J, Alcorn D, Allen AM, and Mendelsohn FAO High resolution localization of angiotensin II receptors in rat renal medulla. Kidney Int 42: 1372-1380, 1992[ISI][Medline].

49.   Zhuo, J, Dean R, MacGregor D, Alcorn D, and Mendelsohn FAO Presence of angiotensin II AT2 receptor binding sites in the adventitia of human kidney vasculature. Clin Exp Pharmacol Physiol 3: S147-S153, 1996.


Am J Physiol Renal Fluid Electrolyte Physiol 280(1):F71-F78