Androgen-dependent regulation of human
angiotensinogen expression in KAP-hAGT transgenic mice
Yueming
Ding and
Curt D.
Sigmund
Genetics Interdisciplinary Graduate Program, Departments of
Internal Medicine and Physiology and Biophysics, The University of Iowa
College of Medicine, Iowa City, Iowa 52242
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ABSTRACT |
We previously reported a novel
transgenic model expressing human angiotensinogen from the kidney
androgen-regulated protein promoter, and demonstrated sexually
dimorphic expression. Herein, we investigated the hormonal regulation
of this transgene. Testosterone increased transgene expression in
female mice in a dose- and time-dependent manner and was not detectable
3-days after treatment was halted. High doses of estrogen were required
to induce the transgene. Expression of transgene mRNA decreased after
castration of male transgenic mice. As in females, however, transgene
expression could be induced after administration of testosterone.
Flutamide, an androgen receptor antagonist, dose dependently blocked
transgene expression in males and blunted the induction caused by
testosterone in females. Neither testosterone nor estrogen altered the
proximal tubule cell-specific expression of the transgene. The data
suggest that the level of transgene expression in this model can be
controlled temporally and in magnitude by manipulating the levels of
androgen. The fortuitous androgen regulation of this transgene can be
used as a molecular "on-off" switch to control transgene expression and potentially manipulate blood pressure levels in this model.
renin-angiotensin system; hormonal regulation; inducible gene
expression; hypertension
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INTRODUCTION |
ANGIOTENSINOGEN
(AGT) has been shown to be modulated by many factors such as
glucocorticoids, estrogens, androgens, thyroid hormone, cytokines, and
ANG II (8, 19). These hormones can stimulate synthesis of
AGT in cultured cell lines, animal models, and humans (6, 10, 17,
18), and this induction is tissue specific. For instance,
androgen has been reported to increase kidney AGT mRNA levels, whereas
it has only a slight effect on liver AGT mRNA levels (11).
Similarly, estrogens can induce AGT expression in both kidney and liver
but not in aorta and adipose tissue (4, 13). The induction
of AGT by these hormones can be neutralized by antagonists such as
anti-glucocorticoid and anti-estrogen reagents, or by surgical
treatments such as adrenalectomy and thyroidectomy (2,
29). Most of the hormones are believed to regulate AGT
expression at the transcriptional level because their actions occur
rapidly and can be blocked by actinomycin D, a specific inhibitor of
transcription. Indeed, several hormonal response elements such as
glucocorticoid response element (GRE), estrogen response element (ERE),
thyroid hormone response element (TRE), and acute-phase response
element (APRE) have been identified in the 5' flanking region of the
human, rat, and mouse AGT genes (3). One of the GREs in
both the human and rat gene is essential for glucocorticoid induction,
and the other GRE can synergize this effect (12).
Like AGT, the kidney androgen-regulated protein (KAP) gene is
regulated by multiple hormones. The KAP gene encodes a protein of
unknown function and was initially identified by its detection as a
very abundant androgen-regulated mRNA in kidney (34).
Serial analysis of gene expression has revealed that KAP is the
second-most abundant mRNA in kidney (35). It is expressed
specifically in the epithelial cells of the proximal tubules although
different regions of the proximal tubule appear differentially
responsive to steroid hormones (5, 22, 36). For example,
androgens regulate the expression of KAP mRNA in epithelial cells of
the S1, S2, and S3 segments of the proximal tubule, whereas estrogen and thyroid hormone control the expression of KAP mRNA primarily in the
S3 segment (23, 32, 33). In female congenital thyroid hormone-deficient hyt/hyt mice, there is no KAP mRNA
expression, suggesting that thyroid hormones are required for its
expression in the kidney of females (32, 33). Males and
testosterone-induced females express KAP mRNA throughout the entire
proximal tubule. In contrast, females, castrated males, and androgen
receptor-deficient Tfm/Y mutant mice show KAP mRNA
exclusively in the S3 segment of the proximal tubule (24,
25). Similar to AGT, several hormone-response elements such as
ERE, TRE, and androgen response element (ARE) have also been identified
in the 5' flanking region of the KAP gene (28).
Transgenic mice containing the human angiotensinogen (hAGT)
gene driven by the KAP promoter (KAP-hAGT) exhibit sexually dimorphic expression of the transgene specifically in renal proximal tubule cells
(9). In male transgenic mice, renal expression of the transgene is constitutively active. In contrast, in female transgenic mice, KAP-hAGT mRNA was undetectable under baseline conditions but
could be markedly induced by administration of testosterone. The
purpose of the present study was to assess the ease with which the KAP
promoter could be hormonally regulated in male and female KAP-hAGT
transgenic mice. KAP-hAGT expression was examined in females treated
with testosterone or estrogen, and in males treated with the
androgen-receptor antagonist flutamide or after castration. Because
double transgenic mice containing the human renin and KAP-hAGT
transgenes are hypertensive, our long-term goal of the present study is
to develop a "molecular switch" to control the blood pressure of
hREN/KAP-hAGT mice by manipulating the levels of KAP-hAGT expression
(7).
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METHODS |
Transgenic mice and animal husbandry.
KAP-hAGT single transgenic mice were generated and characterized as
previously described (9). All mice were maintained by
backcross breeding to C57BL/6J mice. Transgenic mice were identified by
PCR amplification of DNA isolated from tail biopsies as described previously (9). All mice were fed standard mouse chow
(Teklad LM-485) and water ad libitum unless otherwise indicated. Care of mice met or exceeded the standards set forth by the National Institutes of Health in the Guidelines for the Care and Use of Laboratory Animals (NIH publication 86-23, revised 1985). All procedures were approved by the University of Iowa Animal Care and Use
Committee. Experimental mice were killed by CO2 asphyxiation.
Administration of drugs and surgery.
All experiments were performed in duplicate. Representative results are
shown. Female mice were treated with testosterone or estrogen by
administration of a testosterone or estrogen pellet. The testosterone
pellet (catalog no. A-151, Innovative Research of America, Sarasota,
FL) contains 5 mg testosterone continuously released for 21 days.
Different dosages of estrogen pellets (catalog no. A-121, Innovative
Research of America) were used: 1, 5, 10, 25, 75, and 200 mg. They were
also designed for continuous release for 21 days. Mice were first
anesthetized with metofane (0.1 ml in an inhalation chamber), and the
pellet was implanted subcutaneously in the back and tunneled to the
nape of the neck with a 10-gauge trocar. The incision was sutured, and
the mice were allowed to recover on a heating pad. The whole procedure
took <5 min. Mice treated with a testosterone pellet or sham-operated
control mice were killed at the indicated time point after
implantation. Mice treated with an estrogen pellet or their
sham-operated control mice were killed 8 days after implantation.
Testosterone powder (Steraloids, Wilton, NH) was dissolved in sesame
oil (Sigma) to make a 10 mg/ml final stock solution. Sonication was
used to fully dissolve the steroid. For the dose-response study, female
mice were anesthetized with metofane and injected subcutaneously with different doses of liquid testosterone made from the stock solution (10 mg/ml) or with vehicle. After daily injection of testosterone liquid or
vehicle for 1-5 days, the mice were killed. For the time course of
testosterone induction and decay of KAP-hAGT mRNA, female KAP-hAGT
transgenic mice were subcutaneously injected with liquid testosterone
(50 µg · g body
wt
1 · day
1) for 1-5 days.
These mice were killed at different days post-testosterone treatment.
Flutamide powder (Sigma) was dissolved in a 1:1 (vol/vol) mixture of
absolute ethanol and sesame oil. For male mice, different doses of
liquid flutamide or ethanol/oil vehicle were injected subcutaneously.
After daily injection for 4 days, the mice were killed. For female
mice, a testosterone pellet (5 mg testosterone designed for 21-day
release) was first subcutaneously implanted. After 4 days of
testosterone treatment, these mice were subcutaneously injected with
liquid flutamide (4 g). After 4 days of daily injection, the mice were killed.
Male mice were anesthetized with metofane and cleaned at the scrotum
with ethanol. A 1-cm median incision was made at the tip of the
scrotum. The testes lying in the sacs can be seen by placing pressure
on the lower abdomen. A 5-mm incision was made into each sac, and the
testis, epididymis, vas deferens, and spermatic blood vessels were
pulled out. A single ligature was placed around the spermatic blood
vessels and vas deferens. The testis and epididymis were removed by
severing the blood vessels and vas deferens distal to the ligature. The
remaining vas deferens was pushed back into the sac, and the incision
was sutured. The castrated mice were put on a heating pad to recover.
The whole surgery lasted <10 min. Mice were killed 1-7 days after
castration. Some castrated mice were subcutaneously implanted with a
testosterone pellet (5 mg testosterone designed for 21-day release) 8 days after castration and were killed 4 days later.
Analysis of gene expression.
Northern blot analysis was used to examine the expression of hAGT and
KAP in kidneys of KAP-hAGT transgenic mice. Kidney samples were removed
from mice, frozen on dry ice, and stored in
80°C. Total kidney RNA
was isolated as previously described (9). Total kidney RNA
(20 µg) was separated by 1.5% agarose gel and transferred to a
supportive nitrocellulose membrane. RNA blots were hybridized with a
32P-labeled antisense hAGT RNA probe transcribed from a
partial cDNA that was derived from exon 2 of the hAGT gene at
nucleotides 302-819 relative to the transcription start site or a
32P-labeled antisense KAP RNA probe transcribed from a
partial cDNA clone encompassing coordinates 93-521. Most blots
were performed in duplicate.
An RNase protection assay (RPA) was performed to quantify the
expression of hAGT in the kidney of KAP-hAGT transgenic mice. A
Hyb-Speed RPA kit (Ambion) was used according the manufacturer's protocol using 20 µg total kidney RNA. The full-length probes for
hAGT and mouse
-actin genes are nucleotides 630 and 330, respectively, and the expected protected fragments are nucleotides 518 and 250, respectively. The RPA bands were quantified by using a
Molecular Dynamics Storm 820 PhosphorImager system and the ImageQuant Version 4.0 software provided by the manufacturer. hAGT mRNA expression was normalized relative to mouse
-actin in each RPA reaction.
For immunohistochemistry analysis, mice were perfused with PBS buffer
in the circulation immediately after they were killed by
CO2 asphyxiation. The fixing solution (4%
paraformaldehyde, 0.5% glutaraldehyde, 100 mM sodium phosphate buffer,
pH 7.4) was then perfused to replace PBS and to fix tissues. Kidneys
were isolated, fixed in the same solution for 2 h, and immersed in 30% sucrose solution overnight at 4°C. Kidneys were frozen in OCT on
dry ice and sectioned at 8-10 µm. Slides were first rinsed with
Superblock (Pierce) for 5 min and incubated with 0.1% Triton X-100 in
Superblock for 10 min at room temperature. After permeabilization by
Triton X-100, slides were then incubated with rabbit anti-hAGT primary
antibody diluted 1:1,000 in 0.1% Triton X-100 in Superblock overnight
at 4°C. Slides were then washed with PBS for 10 min and incubated
with secondary indocarbocyanine-labeled donkey anti-rabbit antibody
diluted 1:500 in PBS at 37°C for 2 h. Slides were washed again
with PBS and mounted with a coverslip. Confocal microscopy was
performed with a Bio-Rad MRC-1024 Hercules laser scanning confocal
microscope equipped with a Kr/Ar laser.
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RESULTS |
Our previous results show that the KAP-hAGT transgene is
constitutively expressed in the kidney of adult male mice. To further study transgene expression in males, we examined the time course of
KAP-hAGT expression after birth and during the first few weeks of life
(Fig. 1). Human AGT expression was very
low from newborn to 3 wk of age but dramatically increased at 4 wk of
age, reaching a maximum between 6 and 8 wk of age. This temporal
pattern of expression of KAP-hAGT is similar to that of the endogenous
KAP gene and parallels serum testosterone levels in rodents, suggesting strong androgen regulation (1, 20).

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Fig. 1.
Transgene expression during postnatal development.
Representative Northern blots showing expression of the transgene
(KAP-hAGT), endogenous kidney androgen-regulated protein (KAP), and 18S
rRNA in kidney from adult (A) male (M) and female (F) and newborn (N)
and 1-, 2-, 3-, 4-, 6-, and 8-wk-old male mice. Shown is one of 2 Northern blots run with independent samples from different mice.
Identical results were obtained on both blots. Each blot was also run
in duplicate.
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We performed a dose-response to testosterone in female KAP-hAGT mice to
determine the range of transgene induction possible by exogenous
testosterone. Transgene expression was gradually increased by daily
injection (3 days) of testosterone within the concentration ranging
from 5 to 100 µg/g body wt (Fig.
2A). An increase was also
observed when the dose of testosterone was held constant (50 µg · g body wt
1 · day
1)
and different times of injection were examined (Fig. 2B).
Human AGT expression reached a maximum after 5 days. Endogenous KAP mRNA also increased in a dose- and time-dependent manner although the
level of induction was less than the transgene. In addition to
testosterone, the endogenous KAP gene is estrogen responsive (23). High doses of estrogen (1.2-9.5 mg/day) induced
the expression of the transgene and endogenous KAP gene in female
KAP-hAGT mice (Fig. 3).

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Fig. 2.
Transgene expression in response to testosterone.
Expression of the transgene (KAP-hAGT), endogenous KAP, and 18S
rRNA was examined in kidney from adult male (M) and female (F) mice.
A: female mice were treated with the indicated dose of
testosterone (µg/g body wt, injected liquid) each day for
3-consecutive days. B: female mice were treated
with 50 µg/g testosterone each day (injected liquid) for the
indicated number of consecutive days. Identical samples were subjected
to RNAse protection, normalized to expression of endogenous mouse
-actin, and analyzed on a PhosphorImager. Relative expression of
KAP-hAGT (%) in each lane compared with the control male (lane
1) is shown. Similar results were obtained with a second set of
independent samples.
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Fig. 3.
Transgene expression in response to estrogen. Expression
of the transgene (KAP-hAGT), endogenous KAP, and 18S rRNA was examined
in kidney from adult male (M) and female (F) mice. Female mice were
treated with the indicated dose of 17 -estradiol as subcutaneous
pellets (mg/day for 21-day release) for 4 days. Similar results were
obtained with duplicate samples.
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We next considered that the most useful application for a regulated
promoter such as KAP would be if transgene expression could be
repressed as well as induced. We therefore examined the time course of
transgene mRNA decay once androgen administration was discontinued. One
group of female mice was treated for 1-5 days with 50 µg · g body wt
1 · day
1 testosterone
as above, and a second group was treated for 5 days (daily injection)
and then allowed to recover from testosterone treatment for 1-8
days. As above, KAP-hAGT mRNA was induced to a maximal level after 5 days of treatment (Fig. 4A). Expression of transgene mRNA decreased steadily after testosterone was
discontinued (by 32, 65, 85, and 90%, respectively, for days 1-4) and could not be detected 5 days after testosterone
treatment was halted. A graphic representation of the data is shown in
Fig. 4B. In contrast, the endogenous KAP mRNA retained its
baseline expression level after testosterone treatment was halted.
These data indicate that the KAP promoter employed in this transgene is
1) androgen-responsive, 2) much more dependent on
androgen than the endogenous KAP promoter, and 3) can be
turned both "on" and "off" by manipulating androgen levels.

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Fig. 4.
Decay of transgene expression after withdrawal of
testosterone. Expression of the transgene (KAP-hAGT), endogenous KAP,
and 18S rRNA was examined in kidney from adult male (M; lane
1) and female mice (all other lanes). A: female mice
were treated daily with 50 µg/g testosterone for 0-5 days
[increasing ramp (top left), injected liquid]. Another
group of female mice was treated daily with 50 µg/g testosterone for
5 days and then left untreated for 1-8 days [decreasing ramp
(top right)]. B: quantification of KAP-hAGT
after RNase protection assay of the same samples shown in A.
Filled bar, baseline level of KAP-hAGT mRNA in males. The time course
matches the blot in A. Identical results were obtained with
a second set of independent samples.
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To determine whether transgene expression could be similarly
manipulated in males, we examined its response to castration and
readdition of testosterone, and its response to androgen receptor antagonism. Transgene mRNA gradually diminished with castration and was
below the limit of detection 4 days after castration (Fig. 5B). KAP expression also
decreased with castration but remained at easily detectable levels
throughout the experiment. Testosterone administration to castrated
male mice caused a superinduction of transgene expression above the
baseline observed in normal males (Fig. 5A). Daily injection
of the androgen-receptor antagonist flutamide for 4 days caused a
dose-dependent decrease in transgene mRNA (Fig.
6A). In fact, flutamide
treatment (2 g/day) reduced transgene mRNA levels to undetectable
levels. Moreover, administration of flutamide (4 g/day) blunted the
increase in transgene expression caused by testosterone treatment of
female mice (Fig. 6B).

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Fig. 5.
Transgene expression in response to castration.
Expression of the transgene (KAP-hAGT), endogenous KAP, and 18S rRNA
was examined in kidney from adult male mice. A: male mice
underwent either sham (M), castration (C), or castration followed by
administration of a testosterone pellet (T). Expression was analyzed
8-days after castration. Testosterone (5 mg pellet designed for 21-day
release) was administered for 4-days, 8-days after castration.
B: male mice underwent either sham (M) or castration, and
expression was analyzed 1-7 days after castration. Identical
samples were subjected to RNAse protection, normalized to expression of
endogenous mouse -actin, and analyzed on a PhosphorImager. Relative
expression of KAP-hAGT (%) in each lane compared with the control male
(lane 1 of each panel) is shown below the KAP-hAGT Northern
blot.
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Fig. 6.
Transgene expression in response to flutamide. Expression of the
transgene (KAP-hAGT), endogenous KAP, and 18S rRNA was examined in
kidney from adult male (M) and female (F) mice. A: male mice
were treated with either daily vehicle (V) or flutamide at the
indicated dose (g) for 4 days. B: female mice were treated
with either vehicle (V), testosterone (T; 5-mg pellet designed for
21-day release) for 4 days or were first treated with testosterone for
4 days followed by daily injection of flutamide (4 g) daily for 4 days
(4+T).
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Finally, cell-specific expression of hAGT protein was examined by
immunohistochemistry (Fig. 7).
Fluorescent signals specific to hAGT protein were restricted to the
proximal tubule cells in KAP-hAGT males. No signals were observed in
controls, suggesting that the antibody was specific to hAGT. As
expected, there was no positive staining in KAP-hAGT females.
Importantly, hAGT protein was localized to the proximal tubule cells in
KAP-hAGT females treated with estrogen (9.5 mg/day, F+E) or
testosterone (0.24 mg/day, F+T). Consistent with our Northern assay and
RPAs, a stronger signal was observed in KAP-hAGT females treated with
testosterone than estrogen. The results indicate that hormonal
treatment does not alter the cell-specificity of KAP-hAGT expression,
thus validating the use of estrogen, androgen, and androgen-receptor
antagonists as "molecular switches" to regulate the expression of
this and other KAP promoter-controlled transgenes.

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Fig. 7.
Cell-specific expression of the transgene. Confocal
immunofluorescence images of kidney from nontransgenic (C ), untreated
female (F), estrogen-treated female (F+E), testosterone-treated female
(F+T), and male (M) transgenic mice are shown. Top row:
low-power micrographs (×4). Bottom row: high-power (×20)
micrographs. Staining is in bright orange.
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DISCUSSION |
The major finding of the present study is that the expression of
the KAP-hAGT transgene is androgen dependent and is markedly responsive
to both increases or decreases in androgen. In addition, high doses of
estrogen can also stimulate KAP-hAGT expression in female mice. These
data suggest that the level of KAP promoter activity in transgenic mice
can be controlled both temporally and in magnitude by manipulating the
levels of androgen. Consequently, the fortuitous androgen regulation of
this promoter can be used as a molecular "on-off" switch to control
transgene expression in this model.
The KAP promoter contains elements with homologies to consensus
sequences for regulatory motifs such as an ERE, ARE, and TRE; and the
gene is stimulated by androgen, estrogen, and thyroid hormone
(23). The KAP-hAGT transgene used in our study contains 1,542 bp of the KAP promoter fused to a 10.3-kb hAGT genomic clone encompassing exons II-V and including 1) a 70-bp segment
derived from the 3' end of intron I, 2) introns II-IV, and
3) the native 1.4-kb 3' flanking sequence containing the
poly(A) sites. The promoter contains an ARE at position
39, an ERE at
1189, two half-palindromic ERE sites at
604 and
264, and a TRE at
609 (28). In the present study, we indicate that the
KAP-hAGT transgene, like the endogenous KAP gene, is responsive to
androgen. However, although qualitatively similar, the induction of
transgene expression by testosterone in KAP-hAGT mice is quantitatively
much greater than that of the endogenous KAP gene. After androgen
treatment, hAGT expression was increased by 40-fold, but KAP expression
was only increased 4-fold. Moreover, the baseline level of transgene expression in females is much lower than baseline KAP mRNA, and the
decrease in transgene mRNA caused by castration is much greater than
KAP mRNA. Therefore, whereas the KAP gene is androgen responsive, the
transgene is androgen dependent.
One potential explanation for high-level induction of the transgene in
response to androgen is that the 10.3-kb hAGT genomic clone used in our
construct contains AREs or androgen sequences involved in
androgen-mediated increases in transgene mRNA stability. Indeed, the
hAGT gene itself is strongly androgen responsive in kidney
(37). Thus the high magnitude of induction caused by testosterone might be due to combined effects from AREs or other similar sequences present in both the KAP promoter and within the hAGT
gene. Interestingly, the identification of two enhancer elements in the
vicinity of hAGT exon V has been reported (26, 27).
Alternatively, the lower baseline levels of transgene mRNA may involve
thyroid hormones, as several studies suggest that thyroid hormones are
necessary to maintain basal KAP expression. In thyroid
hormone-deficient hyt/hyt mice, KAP gene expression follows
a pattern similar to that observed for the KAP-hAGT transgene (32, 33). In untreated hyt/hyt females, KAP
expression is undetectable but is restored to normal levels by thyroid
hormone administration. Male hyt/hyt mice have normal
baseline KAP expression, which is diminished to undetectable levels by
castration. The 1,542-bp KAP promoter used in the present study
contains a putative TRE at
609 and two TGACC motifs at
604 and
264, which have been described as sequences able to bind thyroid
hormone receptors in other genes (30). Nevertheless, it is
not clear whether these TRE sequences are sufficient for thyroid
hormone to exert its complete response. It remains possible that
elements responsive to thyroid hormone exist further 5' of
1542 or
within the KAP gene itself.
Increasing estrogen levels above normal caused an increase in
endogenous KAP expression but only at high doses (23).
Like endogenous KAP expression, there was no induction of transgene expression in KAP-hAGT females administered estrogen at the doses lower
than 0.48 mg/day. Only at high doses (1.2-9.5 mg/day) was the
induction of hAGT expression observed, suggesting the KAP promoter may
not respond to physiological doses of estrogen but can be stimulated by
high doses of estrogen. Indeed, ovariectomy of mice does not eliminate
KAP gene expression and, in fact, results in a slight increase
(23), further suggesting estrogen in physiological doses
may have no effect on the induction of the KAP promoter. Because
estrogen has only a modest stimulatory action on hAGT, it is clear that
it will not be a useful tool to activate the transgene.
The strong androgen dependence of the KAP-hAGT transgene motivated our
long-term goal of developing an inducible hypertension model in which
blood pressure levels could be manipulated by exogenous hormone. The
role of the renin-angiotensin system in the regulation of blood
pressure and sodium and water homeostasis is well recognized. AGT is
the only known substrate for the enzyme renin. Because the level of AGT
in humans is close to the Michaleis-Menten constant value
Km for renin (16), AGT levels can
dictate the activity of the RAS, and its upregulation may cause an
increase in blood pressure. There is compelling evidence that
perturbations in AGT synthesis result in changes in RAS activity and
blood pressure. First, one haplotype of AGT shows a strong correlation
with plasma AGT and hypertension (15, 16). Next, injection
of antisense AGT oligodeoxynucleotides decreases plasma AGT, ANG-II,
and blood pressure in the spontaneously hypertensive rat
(14). Transgenic mice containing both hREN and
hAGT exhibit high plasma AGT, ANG II, and chronic hypertension
(21). Finally, plasma AGT levels and blood pressure were
found to increase progressively in mice containing zero to four copies
of the AGT locus (31). Thus we can use these mice as
models of inducible hypertension by manipulating hAGT expression in
them. We reported that male transgenic mice containing both KAP-hAGT
and hREN exhibit chronic hypertension, and blood pressure in females
can be increased in response to androgen treatment (7).
Testosterone caused an increase in blood pressure in female double
transgenic mice, 19 mmHg after 1 day, 31 mmHg after 2 days, and peaking
at 40 mmHg (149 ± 4 mmHg) after 3 days. Testosterone did not
cause any change in blood pressure in the control group. Thus altering
androgen levels in hREN/KAP-hAGT double transgenic mice provides
temporal control over the onset and duration of hypertension. Such an
inducible model would provide a major advantage over presently existing
transgenic models of hypertension, in which the transgenes are
activated at birth and are expressed throughout life, and provide an
opportunity to examine the physiological consequences of hypertension
on organ function and end-organ damage in mice with varied exposure to hypertension.
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ACKNOWLEDGEMENTS |
We acknowledge the outstanding technical assistance of Kelly
Andringa, Patricia Lovell, Lucy Robbins, and Norma Sinclair for generation and genotyping of KAP-hAGT transgenic mice, Deborah Davis
for assistance with timed breedings, and Henry Keen and Robin Davisson
for reviewing the PhD dissertation resulting in this manuscript.
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FOOTNOTES |
Funds in support of this work were obtained from the National Heart,
Lung, and Blood Institute (HL-55006 and HL-48058). C. D. Sigmund
was an Established Investigator of the American Heart Association. Y. Ding was a predoctoral fellow of the American Heart Association Iowa
(Heartland) Affiliate. Transgenic mice were generated and maintained at
the University of Iowa Transgenic Animal Facility, which is supported,
in part, by the College of Medicine and the Diabetes and Endocrinology
Research Center. DNA sequencing was performed at the University of Iowa
DNA Core Facility.
Address for reprint requests and other correspondence: C. D. Sigmund, Dept. of Internal Medicine and Physiology and Biophysics, 2191 Medical Laboratory, The Univ. of Iowa College of Medicine, Iowa
City, Iowa 52242 (E-mail: curt-sigmund{at}uiowa.edu).
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.
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