©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Angiotensin II Type 1a Receptor-deficient Mice with Hypotension and Hyperreninemia (*)

(Received for publication, May 15, 1995)

Takeshi Sugaya (1) (4),   Shin-ichiro Nishimatsu (1) Keiji Tanimoto (1) Eriko Takimoto (1) Toshiyuki Yamagishi (1) Kenkichi Imamura (3) Saori Goto (3) Kazunori Imaizumi (4) , Yutaka Hisada (4) Akio Otsuka (4) Hiromi Uchida (4) Masaki Sugiura (4) Katsuhiro Fukuta (3) Akiyoshi Fukamizu (1)(§),   Kazuo Murakami (1) (2)

From the (1)Institute of Applied Biochemistry, (2)Tsukuba Advanced Research Alliance, University of Tsukuba, and (3)National Institute of Animal Health, Tsukuba, Ibaraki 305, Japan and (4)Lead Generation Research Laboratories, Tanabe Seiyaku Co., Ltd., Kashima, Osaka 532, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Angiotensin (AT) II, the bioactive octapeptide in the renin-angiotensin system that plays a key role in cardiovascular homeostasis, exerts its multiple effects through the different types of AT receptors, AT1a, AT1b, and AT2. Previously, we showed chronic hypotension in angiotensinogen (the precursor of AT)-deficient mice and a dramatic increase in renin mRNA levels in its kidney, but it remains unclear which types of AT receptors regulate the blood pressure and renin gene expression. In order to elucidate the physiological roles of AT1a receptor, we generated mutant mice with a targeted replacement of the AT1a receptor loci by the lacZ gene. In the heterozygous mutant mice, the strong lacZ staining was found in the glomerulus and juxtaglomerular apparatus of the renal cortex, which coincided with that of the signals detected by in situ hybridization. Chronic hypotension was observed in the heterozygous and homozygous mutant mice, with 10 and 22 mm Hg lower systolic blood pressure, respectively, than that of wild-type littermates. Both the levels of renin mRNA in the kidney and plasma renin activity were markedly increased only in the homozygous mutant mice. These results demonstrated that an AT1a-mediated signal transduction pathway is, at least in part, involved in the regulation of blood pressure and renin gene expression.


INTRODUCTION

The renin-angiotensin system (RAS), (^1)composed of renin, angiotensinogen, angiotensins, and angiotensin-converting enzyme, is believed to be one of the most important regulators of cardiovascular homeostasis(1) . A potent vasoconstrictor octapeptide angiotensin (AT) II (AII), the bioactive product of RAS, exerts numerous physiological responses through its specific receptors located on the plasma membrane. Pharmacological studies have revealed that there are two different types of AII receptors, AT1 and AT2, in mammalians and that the above responses are mainly mediated through AT1 receptors(2) . In rodents, AT1 receptors are situated as the two isoforms, designed AT1a and AT1b, at two different loci, which are 94% identical at the amino acid level and pharmacologically indistinguishable from each other(3) .

Our previous studies illustrated that the overproduction of AII in transgenic mice carrying both the human renin gene and the human angiotensinogen gene (Tsukuba hypertensive mice) leads to a sustained increase in blood pressure (4) and that mice with a null mutation of the angiotensinogen gene display chronic hypotension and a dramatic increase in renin mRNA levels in the kidney, due to the complete loss of angiotensin signals(5) . These clearly show that RAS plays an indispensable role in the maintenance of blood pressure under physiological conditions. However, it has not yet been determined which types of AT receptors are involved in the regulation of blood pressure and renin gene expression.

In the present study, to clarify the functional importance of AT1a-mediated signal transduction pathways and the cellular localization of the receptor expression, we generated mutant mice in which the AT1a receptor gene was disrupted by replacing with the beta-galactosidase (lacZ) gene such that lacZ activity is under the transcriptional control of the endogenous AT1a locus. We demonstrated that the AT1a receptor localized to glomerulus and juxtaglomerular apparatus of renal cortex and the homozygous mutant mice display hypotension and hyperreninemia.


EXPERIMENTAL PROCEDURES

Gene Targeting and Generation of Mutant Mice

A genomic DNA phage library from C57BL/6 mouse was screened with a 416-bp human AT1 cDNA, which corresponds to the portion from the first methionine to the third transmembrane region as a probe. After screening of 5 10^5 phages, we isolated two clones encoding a complete open reading frame of the AT1a gene confirmed by sequencing(6) .

To construct a targeting vector for the AT1a gene (Fig.1A), an NcoI site was created around the nucleotide sequences including the translation initiation codon of the gene by the site-directed mutagenesis method using polymerase chain reaction (PCR), and then the NcoI and PmaCI fragment of the gene was replaced with the lacZ cassette(7) . The neomycin phosphotransferase (neo) gene cassettes possessing the promoter and polyadenylation signal from pMC1neoPoly(A) (Stratagene) were placed downstream of the lacZ gene in the opposite orientation. The 810-bp XhoI/NcoI fragment and the 4.0-kb PmaCI/BamHI fragment of the AT1a gene were included upstream and downstream of these cassettes, respectively. At the 3` terminus of the homologous region, the herpes simplex virus thymidine kinase gene was inserted to negatively select by Ganciclovir (GANC, Syntex) for random integrations. The TT2 ES cells, derived from an F1 embryo between C57BL/6 and CBA mice, were grown on embryonic fibroblast feeder cells as described elsewhere(8) . Following electroporation of cells with 20 µg/ml linearized targeting vector (Bio-Rad gene pulser at settings of 400 V and 125 microfarads), cells were selected in 225 µg/ml G418 and 0.5 mg/ml GANC. Homologous recombination in ES cells was checked by PCR using the recombination-specific primer set. ES clones positive for PCR analysis were further analyzed by Southern blot analysis; after digestion of genomic DNA with BamHI, size separation in a 0.8% agarose gel, and transfer to nylon membrane, generation of an 8.0-kb wild-type and a 6.8-kb mutant fragment was identified by probes A and B. The absence of additional random integrations of the targeting construct was confirmed by hybridization with a neo probe (probe C).


Figure 1: Targeted disruption of the AT1a gene by homologous recombination in ES cells and mice. A, structure of the targeting vector and partial restriction map of the AT1a gene locus before and after targeting event. The intronless open reading frame (ORF) is shown as a closedbox, and lacZ (beta-galactosidase gene), neo (neomycin phosphotransferase gene), HSV-tk (herpes simplex virus-thymidine kinase gene), and pBSK (Bluescript KS(-)) are shown as openboxes, respectively. To construct the targeting vector, a 1.4-kb fragment including the translation initiation codon of the AT1a gene was replaced with lacZ and neo cassettes. neo was placed in the opposite orientation downstream of lacZ. The position of the probes used for Southern blot analysis (closedbar) and expected fragment sizes after BamHI digests of genomic DNA are also shown. The restriction sites used are: B, BamHI; E, EcoRI; P, PmaCI; X, XhoI; and N, NcoI. B, Southern blot analysis of ES cell DNA. Genomic DNAs extracted from wild-type and targeted ES cell clones were digested with BamHI, electrophoresed, and blotted. The hybridization probes were: A and B, probes located outside and inside the targeting vector, respectively; and C, neo probe. Other restriction enzymes were used to confirm the homologous nature of the recombination (data not shown). C, Southern blot analysis of representative litter derived from a heterozygous intercross. Genomic DNAs isolated from tails of wild-type (+/+), heterozygous (+/-), and homozygous (-/-) mutant mice were digested with BamHI, electrophoresed, and blotted. Fragments obtained from wild-type (8.0 kb) and targeted alleles (6.8 kb) were detected by probe A in two lines.



Chimeric mice were generated by injecting the ES cells into ICR 8-cell embryos(8) . Chimeric males with greater than 50% agouti coat color were bred to ICR females, and germline transmission of the mutant allele was identified by Southern blot analysis of tail DNA from F1 offspring with agouti coat color. The heterozygous mice were interbred to obtain mice homozygous for the AT1a gene disruption.

Histological Analysis

For in situ hybridization, samples were immediately frozen at -60 °C and cut into 15-µm cryostat sections. The 490-bp SacI/PmaCI fragment and the 520-bp SacI/SpeI fragment, which correspond to 3`-untranslated regions of AT1a and AT1b, respectively, were used as the subtype-specific probes to prevent the cross-hybridization. Each subtype-specific region for AT1a and AT1b was subcloned into pBluescript II (Stratagene). Antisense and sense probes were made by in vitro transcription in the presence of digoxigenin-labeled dUTP. In situ hybridization using the digoxigenin-labeled probes was performed as described previously(9) . For lacZ staining, samples were fixed in a solution containing 2% paraformaldehyde, 0.2% glutaraldehyde, and 0.02% Nonidet P-40 in phosphate-buffered saline for 60 min at room temperature and treated in 30% sucrose for 12 h at 4 °C. 10-µm frozen sections were stained at 37 °C for 1-3 h in a solution containing 0.4 mg/ml Bluo-Gal (Life Technologies, Inc.), 3 mM K(3)Fe(CN)(6), 3 mM K(4)Fe(CN)(6), and 1 mM MgCl(2) in phosphate-buffered saline.

RNA Isolation and Northern Blot Analysis

Total RNA was isolated from the kidneys of six independent age-matched mice using ISOGEN (NipponGene) based on the acid guanidinium thiocyanate/phenol/chloroform extraction method(10) . Ten micrograms of RNA were denatured with formamide, separated by electrophoresis, and transferred to a nylon membrane. The 490-bp SacI/PmaCI fragment that corresponds to 3`-untranslated regions of AT1a was used as the AT1a receptor-specific probe. Probes for mouse renin and glyceraldehyde-3-phosphate dehydrogenase were previously described(11) .

Measurement of Plasma Renin Activities

Blood samples were withdrawn from wild-type (+/+), heterozygous (+/-), and homozygous mutant (-/-) mice (88-94 days). Blood was collected into ice-cold microcentrifuge tubes containing EDTA, which were then immediately centrifuged in order to isolate the plasma fraction. The plasma renin activity was measured by radioimmunoassay. The concentration of inactive renin was determined by subtraction of angiotensin I cleaved by the plasma renin with or without the treatment of proteinase activations as described previously(12) .

Measurement of Blood Pressure

Wild-type (+/+), heterozygous (+/-), and homozygous mutant (-/-) mice at the age of 56-62 days were used for blood pressure measurement. The systolic and diastolic blood pressure was measured by a programmable sphygmomanometer (BP-200, Softron, Japan) using the tail cuff method as described previously(4) . Statistical analysis for comparison of blood pressure was performed by using Student's t test. A value of p < 0.05 was considered significant. Results are expressed as mean ± S.E.


RESULTS AND DISCUSSION

Generation of AT1a Receptor-deficient Mice

To generate a null mutation of the AT1a gene, we designed a targeting vector that would replace the complete AT1a coding region with the promoterless lacZ gene (Fig.1A). After electroporation of TT2 cells with the targeting vector, homologous recombination was confirmed by PCR and Southern blot hybridization analyses (Fig.1B). Three independent cell lines out of 330 G418 and GANC-resistant cells had undergone homologous recombination at the AT1a locus. These clones were injected into ICR 8-cell embryos to generate chimeric mice, and two clones gave rise to germline transmission. After confirmation of the transmission of the mutations into germ cells, the heterozygous mice were intercrossed to produce homozygous offspring, and the mutation at AT1a receptor loci was detected by genomic Southern analysis of tail DNA (Fig.1C). Of the 396 offspring analyzed, 82 (21%) were homozygous for the disrupted allele, and 121 (31%) were wild type, indicating normal embryonic development of the homozygous mutant mice. In the following study, to make equivalence in the effects of other gene backgrounds except for the AT1a gene, we used these intercrossed littermates of heterozygous mice for further physiological experiments.

To confirm if the lacZ gene would be inserted in-frame starting from the translational initiation codon of the AT1a gene and expressed under the control of the regulatory element of that gene, we compared the lacZ staining with the signals of specified expression of AT1a mRNA. In the heterozygous mice, the strong lacZ staining was identified in the glomerulus and juxtaglomerular apparatus of the renal cortex (Fig.2, A and E). The localization of lacZ staining in the heterozygous mice coincided with that of the signals obtained by in situ hybridization using the antisense cRNA probe for the AT1a-specific region in the wild-type (Fig.2B) and the heterozygous mice (data not shown). No signals using the antisense probe specific for the AT1b region (Fig.2C) or the sense probe for the AT1a-specific region (Fig.2D) were detected in the heterozygous mice. These results are in good agreement with those found by in situ hybridization studies using AT1a-specific S-labeled riboprobes (13, 14) and indicate the regulated lacZ expression under control of the endogenous AT1a promoter.


Figure 2: lacZ staining and in situ hybridization of AT1a receptor in renal cortex from wild-type and heterozygous mutant mice. The lacZ staining in heterozygous mutant mice was observed in glomeruli, juxtaglomerular apparatus, and proximal tubules (A). Especially, the strong signals were obtained in mesangial cells and afferent and efferent arterioles (D). The localization of the lacZ staining in the heterozygous mice coincided with that of the signals obtained by in situ hybridization using the antisense cRNA probe for the AT1a-specific region in the wild-type (B) and the heterozygous mice (data not shown). No signal using the antisense probe for the AT1b-specific region (C) or the sense probe for the AT1a-specific region (D) was detected in glomeruli of the heterozygous mice. Bars = 25 µm.



Hypotension

We previously showed that the systolic blood pressure in the angiotensinogen-deficient mice decreased to 66% of that in wild-type mice(5) . To test the effect of AT1a gene disruption on blood pressure, we measured blood pressure of the AT1a-deficient mice by a tail cuff method. A significant decrease in both the systolic and diastolic blood pressure was observed in the heterozygous and homozygous mutant mice with 10 and 22 mm Hg lower systolic blood pressure, respectively, than that of the wild-type littermates, and the systolic blood pressure in the homozygous mutant mice decreased to 77% of that in the wild-type mice (Fig.3). The decreased blood pressure was also observed in mice with AT1a receptor mutation (15) .


Figure 3: Comparison of blood pressures between mutant mice. Statistical differences were analyzed by Student's t test. Symbols: *, p = 0.0551 and**, p = 0.0002 versus +/+ (systolic blood pressure (SBP)); #, p = 0.1914 and ##, p = 0.0029 versus +/+ (diastolic blood pressure (DBP)). +/+, wild-type mice; +/-, heterozygous mutant mice; -/-, homozygous mutant mice. Values shown are mean (n = 7) ± S.E.



Hyperreninemia

Renin production is under the direct negative control of AII, as has been suggested by infusion of suppressive doses of AII in the renal artery(16) , the effect of AII on renin release from the isolated perfused kidney(17) , and the effect of AII on prorenin secretion in cultured juxtaglomerular cells(18) . In our previous study, a drastic increase in renin mRNA levels was found in the kidney of mice with the targeted disruption of angiotensinogen gene, due to the lack of a negative feedback regulation by AII(5) . Taken together, one could expect that the shut-off of AT1a receptor-mediated signalings may elicit an up-regulation of the renin gene expression in the AT1a receptor-deficient mice. To test this possibility, we first confirmed the levels of expression of AT1a receptor in the kidney RNA from the wild-type, heterozygous, and homozygous mice by Northern blotting (Fig.4A). The hybridization signals of AT1a receptor appeared in the heterozygous mice at approximately half the levels present in the wild type, whereas the homozygous mice had no detectable transcripts. A duplicate blot was analyzed with a glyceraldehyde-3-phosphate dehydrogenase probe to confirm that the RNA sample was intact. These results indicated that the AT1a receptor mRNA was completely absent from the homozygous mutant mice.


Figure 4: Northern blot analysis (A) and plasma renin activity (B) of the mutant mice. A, 10 µg of total RNA, isolated from the kidneys of six independent age-matched adult mutant mice, were hybridized with either a 3`-untranslated AT1a-specific probe, renin probe, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. B, plasma renin activity. Statistical differences were analyzed by Student's t test. Values shown are mean (n = 5) ± S.E. Symbols: #, p = 0.0135 versus +/+ (active renin); , p = 0.0019 versus +/+ (inactive renin); +/+, wild-type mice; +/-, heterozygous mutant mice; -/-, homozygous mutant mice.



We next examined if expression of the renin gene was affected by the null mutation of AT1a receptor. Northern blot analysis of kidney RNA from wild-type, heterozygous, and homozygous mutant mice revealed that the level of renin mRNA markedly increased in the homozygous mutant mice in comparison with that in the heterozygous mutant and wild-type mice (Fig.4A). Consequently the plasma concentration of active renin increased 7-8-fold in the homozygous mutant mice compared with that found in the heterozygous mutant and wild-type mice, and most of its plasma renin activity was detected as an active form (Fig.4B). These results indicated that the homozygous mice display hyperreninemia and suggested that the function of AT1a is dominant regarding the regulation of renin gene expression because AII can suppress it even in the heterozygous mice.

In conclusion, we generated a line of mutant mice in which the AT1a receptor gene was disrupted by replacing with the lacZ gene. In the heterozygous mutant mice, the strong lacZ staining was identified in the glomerulus and juxtaglomerular apparatus of the renal cortex. Both the expression levels of renin mRNAs in the kidney and plasma renin activity were not significantly different between the heterozygous mutant and wild-type mice but were markedly increased in the homozygous mutant mice. On the contrary, a significant decrease in blood pressure was observed in the heterozygous and homozygous mutant mice. These results demonstrated that an AT1a-mediated signal transduction pathway is, at least in part, involved in the regulation of blood pressure and renin gene expression. Our mice with the replacement of AT1a receptor gene by the lacZ under the control of AT1a endogenous promoter should be useful to further investigate the specific function of AT1a receptor, such as the developmental expression of this gene and the precise identification of the AT1a production site in the central nervous systems under pharmacological conditions.


FOOTNOTES

*
This work was supported by grants from the Ministry of Education, Science, and Culture of Japan and Special Research Project on Circulation Biosystems at the University of Tsukuba. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Inst. of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan. Tel.: 81-298-53-6599; Fax: 81-298-53-4605.

^1
The abbreviations used are: RAS, renin-angiotensin system; AT, angiotensin; AII, angiotensin II; bp, base pair(s); kb, kilobase(s); ES, embryonic stem; PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Drs. Shinichi Aizawa, Takeshi Yagi, Yumiko Saga, Yasuhide Furuta, Yuzo Kadokawa, and Fumihiro Sugiyama for technical advice. We also thank Drs. Hitoshi Miyazaki, Kyoko Isahara, and Eisei Noiri for helpful discussion.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.