(Received for publication, May 15, 1995)
From the
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.
The renin-angiotensin system (RAS), ()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 -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.
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 (-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.
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.
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.
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.