Hypertension. 1997;30:859-867
(Hypertension. 1997;30:859-867.)
© 1997 American Heart Association, Inc.
Tissue Angiotensinogen Gene Expression Induced by Lipopolysaccharide in Hypertensive Rats
Nobuo Nyui;
Kouichi Tamura;
Satoshi Yamaguchi;
Masashi Nakamaru;
Tomoaki Ishigami;
Machiko Yabana;
Minoru Kihara;
Hisao Ochiai;
Naomichi Miyazaki;
Satoshi Umemura;
;
Masao Ishii
From the Second Department of Internal Medicine, Yokohama City (Japan)
University School of Medicine.
Correspondence to Satoshi Umemura, MD, Second Department of Internal Medicine, Yokohama City University School of Medicine, 3-9, Fukuura, Kanazawa-ku, Yokohama 236, Japan.
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Abstract
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Abstract There is now convincing evidence that various
tissues
express their own tissue renin-angiotensin system,
which may
be regulated independently of the systemic
renin-angiotensin
system. However, little information is
available on the regulation
of the tissue renin-angiotensin
system. We investigated the
regulation of tissue
angiotensinogen gene expression with respect
to the
development of hypertension. We measured basal and
lipopolysaccharide-stimulated
plasma
angiotensinogen concentrations by radioimmunoassay and
examined
the expression of tissue angiotensinogen by
Northern blot analysis
in spontaneously hypertensive rats (SHR)
and Wistar-Kyoto rats
(WKY) at 4 and 13 weeks of age. Basal plasma
angiotensinogen
concentration in SHR was comparable to that
in WKY at 4 weeks
of age and was significantly higher than that in WKY
at 13 weeks
of age. Lipopolysaccharide induced a significant
increase in
plasma angiotensinogen concentration in both
WKY and SHR at
4 and 13 weeks of age. At 4 weeks of age, the basal
levels of
angiotensinogen mRNA in the liver, fat, adrenal,
and aorta were
higher in WKY than in SHR. At 13 weeks of age, the basal
levels
of angiotensinogen mRNA in the fat, adrenal, aorta,
spleen,
and kidney were higher in WKY than in SHR, while that in the
liver
did not differ significantly between the two strains. At 4 weeks
of
age, pretreatment with lipopolysaccharide increased the
angiotensinogen
mRNA levels in the liver, fat, adrenal, and
aorta in both WKY
and SHR. At 13 weeks of age, pretreatment with
lipopolysaccharide
increased the angiotensinogen
mRNA levels in the liver, aorta,
and adrenal; decreased those in the
spleen; and had no effect
in the kidney in both WKY and SHR.
Interestingly, lipopolysaccharide
increased the
angiotensinogen mRNA level in fat only in SHR,
with no
effect in WKY, at 13 weeks of age. Lipopolysaccharide
stimulated
tumor necrosis factor-

mRNA expression in fat of WKY and
SHR,
and the increase in tumor necrosis factor-

mRNA level in SHR
was
significantly greater than that in WKY. Therefore, the increased
tumor
necrosis factor-

mRNA expression may be involved in the
increased
lipopolysaccharide-induced expression of
angiotensinogen gene
in fat of SHR at 13 weeks of age.
These data suggest that the
transcriptional and probably
posttranscriptional regulation
of angiotensinogen mRNA
differs between SHR and WKY, that the
regulation of
angiotensinogen gene expression is tissue-specific,
and
that the altered expression of the angiotensinogen gene
may
be involved in the development of hypertension.
Key Words: rats, inbred SHR angiotensinogen renin-angiotensin system lipopolysaccharide RNA, messenger tumor necrosis factor-
 |
Introduction
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Angiotensinogen
is an important component of the systemic RAS,
which participates in
blood pressure regulation and electrolyte
balance. The main source of
the renin substrate angiotensinogen
in plasma is the liver,
where angiotensinogen is synthesized
and secreted by the
constitutive pathway into the bloodstream.
1 A
physiologically inactive decapeptide, Ang I, is
released
from angiotensinogen by renin and further
hydrolyzed by ACE
to produce the octapeptide Ang II, which mediates
vasoconstriction
and aldosterone secretion through specific
interactions with
Ang II receptors present on vascular smooth
muscle and adrenal
glands, respectively.
2 Regulatory
mechanisms for the synthesis
and secretion of hepatic
angiotensinogen have been extensively
investigated in
physiological and
pathophysiological alterations.
Inflammation,
3 nephrectomy,
4 and treatment
with glucocorticoids,
5 thyroid
hormones,
6
estrogens,
7 or Ang II
8 stimulate hepatic
synthesis
and secretion of angiotensinogen, increase
circulating levels
of angiotensinogen, and consequently
elevate the activity of
the circulating RAS.
9 Recently,
the existence of the tissue
RAS has been established. Similar to the
systemic RAS, the activity
of most of this system is implicated in
cardiovascular regulation.
However, in contrast to the
circulating system, the tissue RAS
may be regulated by local factors,
although definitive evidence
supporting this claim is lacking. There is
a strong correlation
between blood pressure and plasma
angiotensinogen.
10 11 Several
studies showed
that PRA in SHR is either similar to or lower
than that in normotensive
rat strains.
12 13 14 Moreover, plasma
Ang II levels are not
elevated in SHR.
13 The tissue RAS may
play an important
role in hypertension and functions of the
heart, adrenal gland, kidney,
blood vessel wall, and adipose
tissue. Recently, several experimental
disease states have been
shown to be associated with changes in
angiotensinogen gene
expression in affected organs. For
example, an increase in angiotensinogen
gene expression was
seen in the heart after myocardial infarction
15 or in the
kidney exposed to arterial hypertension.
16
Recent
studies using cultured hepatocytes
17 18 19
and transgenic animals
20 21 22 reported that the 5' flanking
region of the angiotensinogen
gene might contain
cis-acting regulatory elements. One crucial
DNA control
element, located between -531 and -557 in the rat
gene 5' to the
transcription start site, contains the sequence
5'-AGTTGGGATTTCCCAACC-3',
which we have called the
APRE.
23 The APRE is a TNF-

inducible
enhancer that
confers TNF-

induction onto an inert minimal promoter.
The APR,
initiated experimentally by intraperitoneal LPS
injection
and effected by the production of TNF-

, is a
potent inducer
of hepatic angiotensinogen expression. Past
studies have demonstrated
an immune component in the pathogenesis of
systemic hypertension
in both humans and animal models, including the
SHR.
24 25 The
SHR has been shown to exhibit thymocytotoxic
autoantibodies
26 and impaired T- and B-cell
function.
27 28 Thymic tissue obtained
from normotensive
rats and implanted in the SHR reverses the
T-cell dysfunction and
attenuates the subsequent development
of hypertension.
29
These results suggest a correlation between
immunological abnormalities
and the development of hypertension
in SHR. In the present study we
examined the regulation of angiotensinogen
gene expression
by activation of the angiotensinogen gene APRE
in various
tissues of SHR early in the developmental phase of
hypertension (4
weeks of age) and when hypertension was established
(13 weeks of age)
and compared findings with those in WKY.
 |
Methods
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Animals
Male WKY and SHR were purchased from the Disease Model
Cooperative
Research Association (Kyoto, Japan) at 4 and 13 weeks of
age.
They were housed two to three animals per cage and maintained
under
controlled conditions of light, temperature, and humidity. All
animals
had free access to tap water and rat chow containing 0.3%
NaCl.
The rat chow was purchased from Oriental Kobo Kogyo.
Blood Pressure and Body Weight Measurements
SBP was measured by photoelectric volume oscillometry using an
automated tail-cuff sphygmomanometer (UR-5000, Ueda) at 4 and 13 weeks
of age.30 The reported values are averages of 10 to 15
consecutive measurements.
LPS Treatment
LPS (Escherichia coli, Sigma Chemical Co) in a dose
of 100 µg/kg was injected
intraperitoneally into 6 WKY or SHR at 4 weeks of
age and 15 WKY or SHR at 13 weeks of age. As controls, vehicle was
injected intraperitoneally into 6 WKY or SHR at 4
weeks of age and 15 WKY or SHR at 13 weeks of age. For measurement of
plasma RAS and analysis of angiotensinogen mRNA
expression, 6 hours after LPS stimulation all rats were decapitated.
For the analysis of TNF-
mRNA, 1 hour after LPS stimulation
all rats were decapitated. The liver, aorta, adrenal, kidney, spleen,
and fat were removed and immediately frozen in liquid nitrogen. Fats
were isolated from epididymal fat pads.
Biochemical Assays
PRA was measured by radioimmunoassay.31 Briefly, 50
µL plasma was incubated for 1 hour at 37°C with 5 µL
8-hydroxyquinoline, 5 µL dimercaprol, 25 µL Na2EDTA
(4%), and 165 µL Tris acetate buffer (0.1 mol/L, pH 7.4)
containing 0.1% lysozyme, and the generated Ang I was measured with
the Renin Riabead Ang I kit (Dainabot Co, Ltd). For measurement of
plasma angiotensinogen (p-angiotensinogen)
concentration, 100 µL plasma was incubated for 5 hours at 37°C with
5 µL 8-hydroxyquinoline, 5 µL dimercaprol, 25 µL
Na2EDTA, 50 µL rat kidney renin, and 65 µL Tris acetate
buffer containing lysozyme, and the generated Ang I was measured by
radioimmunoassay. The p-angiotensinogen concentration was
expressed as picomoles Ang I equivalents per milliliter. Plasma Ang II
(p-Ang II) concentration was determined by a specific radioimmunoassay,
using an antibody to Ang II kindly provided by Dr Kazuaki Shimamoto
(Sapporo [Japan] Medical College).32
RNA Isolation and Analysis
Northern blot analysis was performed essentially as
described previously.33 34 Total RNA from tissues was
extracted with the guanidinium thiocyanate/cesium chloride
centrifugation method.35 RNA concentration
was determined by ultraviolet spectrophotometry. Each RNA sample (20
µg) was denatured with 1 mol/L glyoxal and 50% dimethyl
sulfoxide, electrophoresed on a 1.2% agarose gel, and transferred to
nylon membranes (GeneScreen Plus, DuPontNew England Nuclear). Filters
were prehybridized for 30 minutes at 60°C in a solution consisting of
1% sodium dodecyl sulfate, 1 mol/L NaCl, and 10%
dextran sulfate. Hybridization proceeded for 16 hours at 60°C in the
same solution containing 300 µg/mL denatured salmon sperm DNA
and 1x106 cpm/mL of the 32P-labeled rat
angiotensinogen cDNA probes36 or TNF-
cDNA
probes.37 Filters were washed twice with 2x SSC (1x
SSC=0.15 mol/L NaCl, 0.015 mol/L sodium citrate) for 5
minutes at room temperature, twice with 2x SSC and 1% sodium
dodecyl sulfate for 30 minutes at 60°C, and twice with 0.1x
SSC for 15 minutes at room temperature. Dried filters underwent
autoradiography at -70°C with an intensifying
screen. Expression of angiotensinogen mRNA or TNF-
mRNA
was measured with a Fujix Bio-Imaging Analyzer (BAS 2000, Fuji
Photo Film) and normalized to the signal generated by probing for the
constitutively expressed GAPDH gene.38 A 1089-bp
Aat IAat II cDNA fragment of the rat
angiotensinogen gene, which was kindly provided by Drs
Shigetada Nakanishi (Kyoto University) and Hiroaki Ohkubo (Kumamoto
University), was used as a hybridization probe.36 The
TNF-
cDNA probe was kindly provided Asahi Chemical Industry. The
GAPDH cDNA probe used was a generous gift of Dr Ray Wu (Cornell
University, Ithaca, NY).38
Statistical Analysis
For the statistical analysis of differences among
groups, the unpaired Student's t test was used. All
quantifiable data are expressed as mean±SE.
 |
Results
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Characteristics and LPS-Induced Change in Plasma RAS in WKY
and SHR
SBP, body weight, and wet tissue weights of kidney and heart
in
WKY and SHR are summarized in the Table

.
The SBP of SHR at
4 weeks of age was statistically higher than that of
age-matched
WKY (
P<.0001) and increased further at 13 weeks
of age compared
with that of WKY (
P<.0001). Concomitant
with the blood pressure
increase, the ratio of wet tissue weight of
heart to body weight
in SHR became significantly greater at 13 weeks of
age. No such
difference was observed between WKY and SHR with respect
to
the ratio of wet tissue weight of kidney to body weight at 4
and 13
weeks of age. As shown in Fig 1

, basal
PRA and p-Ang
II in SHR were comparable to values in age-matched WKY.
In SHR,
LPS induced a significant increase in p-Ang II at 13 weeks of
age,
but in WKY the change was not significant. On the other hand,
LPS
induced no significant change in PRA in either WKY or SHR
at both 4 and
13 weeks of age. Basal p-angiotensinogen concentration
in
SHR was significantly higher than in WKY at 13 weeks of age,
and LPS
induced a significant increase in p-angiotensinogen
concentration
in both WKY and SHR at both 4 and 13 weeks of age. SBP in
basal
conditions was significantly correlated with
p-angiotensinogen
concentration (
r=.611,
P<.001).

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Figure 1. Plasma levels of renin (PRA),
angiotensinogen (p-ATNG), and Ang II (p-Ang II) in
untreated rats (-) and rats 6 hours after administration of 100 µg
LPS per kilogram body weight (+) in WKY and SHR at 4 (left) and 13
(right) weeks of age. Values are mean±SE (n=6-12).
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Tissue-Specific and LPS-Induced Expression of
Angiotensinogen mRNA in WKY and SHR
The angiotensinogen gene is expressed mainly in the
liver, but it is also expressed in the fat, adrenal, aorta, kidney, and
spleen. As shown in Fig 2
, basal liver
angiotensinogen mRNA levels in WKY were significantly
higher than those in SHR at 4 weeks of age and comparable to those in
SHR at 13 weeks of age. As shown in Figs 3
, 4
, and 5
,
the expression of basal angiotensinogen mRNA in fat,
adrenal, and aorta was suppressed in SHR compared with WKY at 4 and 13
weeks of age. As shown in Fig 6
, basal
angiotensinogen mRNA levels in spleen and kidney were lower
in SHR than in WKY at 13 weeks of age. A previous study reported that
liver angiotensinogen mRNA levels rapidly increased during
the first 5 hours and reached a maximal level at 5 to 10 hours after
LPS administration.39 Therefore, we examined the
expression of angiotensinogen mRNA 6 hours after LPS
administration. Treatment with LPS for 6 hours increased
angiotensinogen mRNA levels in liver, adrenal, and aorta in
both WKY and SHR at 4 and 13 weeks of age (Figs 2
, 4
, and 5
). LPS
decreased angiotensinogen mRNA levels in spleen, and had no
effect on levels in the kidney in both WKY and SHR at 13 weeks of age
(Fig 6
). Although we also examined basal and LPS-induced
angiotensinogen mRNA expression in the spleen and kidney at
4 weeks of age, we could not detect angiotensinogen mRNA
expression in the basal and LPS-induced state (data not shown). LPS
increased angiotensinogen mRNA level in fat in both WKY and
SHR at 4 weeks of age. However, at 13 weeks of age, LPS increased the
angiotensinogen mRNA level only in SHR, with no effect in
WKY (Fig 3
).

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Figure 2. Top, Northern blot analysis of total RNA (20
µg) from liver at 4 (left) and 13 (right) weeks of age of untreated
rats (-) and rats at 6 hours after administration of 100 µg LPS per
kilogram body weight (+) in WKY and SHR. Upper blots show
angiotensinogen (ATNG) mRNA; lower blots show GAPDH mRNA.
Markers for 28S and 18S rRNA are indicated by solid arrowheads. Bottom,
Relative angiotensinogen mRNA levels.
Angiotensinogen mRNA levels were measured with a BAS 2000
image analyzer normalized to the signal generated by probing
for GAPDH gene expression and are expressed relative to levels achieved
with RNA from tissues of untreated WKY. Values are mean±SE
(n=6-12).
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Figure 3. Top, Northern blot analysis of total RNA (20
µg) from fat at 4 (left) and 13 (right) weeks of age of untreated
rats (-) and rats 6 hours after administration of 100 µg LPS per
kilogram body weight (+) in WKY and SHR. Upper blots show
angiotensinogen (ATNG) mRNA; lower blots show GAPDH mRNA.
Markers for 28S and 18S rRNA are indicated by solid arrowheads. Bottom,
Relative angiotensinogen mRNA levels.
Angiotensinogen mRNA levels were measured with a BAS 2000
image analyzer normalized to the signal generated by
probing for GAPDH gene expression and are expressed relative to
levels achieved with RNA from tissues of untreated WKY. Values are
mean±SE (n=6-12).
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Figure 4. Top, Northern blot analysis of total RNA (20
µg) from adrenal at 4 (left) and 13 (right) weeks of age of untreated
rats (-) and rats 6 hours after administration of 100 µg LPS per
kilogram body weight (+) in WKY and SHR. Upper blots show
angiotensinogen (ATNG) mRNA; lower blots show GAPDH mRNA.
Markers for 28S and 18S rRNA are indicated by solid arrowheads. Bottom,
Relative angiotensinogen mRNA levels.
Angiotensinogen mRNA levels were measured with a BAS 2000
image analyzer normalized to the signal generated by probing
for GAPDH gene expression and are expressed relative to levels achieved
with RNA from tissues of untreated WKY. Values are mean±SE
(n=6-12).
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|

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Figure 5. Top, Northern blot analysis of total RNA (20
µg) from aorta at 4 (left) and 13 (right) weeks of age (right) of
untreated rats (-) and rats 6 hours after administration of 100 µg
LPS per kilogram body weight (+) in WKY and SHR. Upper blots show
angiotensinogen (ATNG) mRNA; lower blots show GAPDH mRNA.
Markers for 28S and 18S rRNA are indicated by solid arrowheads. Bottom,
Relative angiotensinogen mRNA levels.
Angiotensinogen mRNA levels were measured with a BAS 2000
image analyzer normalized to the signal generated by probing
for GAPDH gene expression and are expressed relative to levels achieved
with RNA from tissues of untreated WKY. Values are mean±SE
(n=6-12).
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Figure 6. Top, Northern blot analysis of total RNA (20
µg) from spleen (left) and kidney (right) at 13 weeks of age of
untreated rats (-) and rats 6 hours after administration of 100 µg
LPS per kilogram body weight (+) in WKY and SHR. Upper blots show
angiotensinogen (ATNG) mRNA; lower blots show GAPDH mRNA.
Markers for 28S and 18S rRNA are indicated by solid arrowheads. Bottom,
Relative angiotensinogen mRNA levels.
Angiotensinogen mRNA levels were measured with a BAS 2000
image analyzer normalized to the signal generated by probing
for GAPDH gene expression and are expressed relative to levels achieved
with RNA from tissues of untreated WKY. Values are mean±SE
(n=12).
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LPS-Induced Expression of TNF-
mRNA in WKY and SHR
In most tissues, the response of angiotensinogen gene
expression to LPS was comparable in WKY and SHR. The only exception was
fat tissue at 13 weeks of age. Recent studies showed that TNF-
is
expressed in adipose tissue,40 and another study suggested
that TNF-
was involved in the induction of
angiotensinogen gene expression by
inflammation.41 Thus, we examined LPS-induced TNF-
mRNA
expression in fat and liver of WKY and SHR at 13 weeks of age. First,
we examined the expression of TNF-
mRNA 6 hours after LPS
administration. However, we could not detect TNF-
mRNA in basal and
LPS-stimulated levels in fat and liver of WKY and SHR at 13 weeks of
age (data not shown). Previous study reported that the maximal
induction of TNF-
mRNA is 1 hour after LPS
administration37 ; therefore, we examined the expression of
TNF-
mRNA 1 hour after LPS administration. As shown in Fig 7
, we could not detect basal TNF-
mRNA
in both WKY and SHR in liver and fat. LPS stimulated TNF-
mRNA
expression in the fat of WKY and SHR, and the induction of TNF-
mRNA
level in SHR was significantly greater than that in WKY.
 |
Discussion
|
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In the present study, basal p-angiotensinogen
levels did not
differ between SHR and WKY at 4 weeks of age, although
SHR had
a higher SBP than WKY. At 13 weeks of age, SBP in SHR elevated
farther,
and basal p-angiotensinogen levels increased
significantly in
SHR to a level higher than that in WKY (Fig 1

).
Furthermore,
SBP was significantly correlated with
p-angiotensinogen concentration
(
r=.611,
P<.001) in WKY as well as in SHR. On the other hand,
SBP
showed no relationship with PRA and p-Ang II. These results
confirm our
previous study using WKY and SHR at 6 and 14 weeks
of
age,
42 and we speculate that the increase in
p-angiotensinogen
levels in SHR at 13 weeks of age promotes
further development
of hypertension as well as secondary changes in
response to
hypertension rather than being the primary pathogenic
mechanism
of hypertension. Although at 13 weeks of age basal
p-angiotensinogen
concentration in SHR was significantly
higher than that in WKY,
basal p-Ang II concentration was comparable in
both strains.
In addition, basal PRA in SHR was comparable to that in
WKY.
A previous study showed that plasma ACE activity in WKY at 4
and
18 weeks of age was higher than that in age-matched SHR.
43
The results of the present study suggested that the basal plasma
ACE
activity in WKY is higher than that in SHR. Therefore, basal
p-Ang
II in SHR may be comparable to that in WKY.
Previous studies reported that treatment with 100 µg/kg LPS in
Wistar rats significantly increased p-angiotensinogen
during the first 12 hours.39 44 In the present study,
injection of LPS produced a significant increase in
p-angiotensinogen concentration in both WKY and SHR at 4
and 13 weeks of age. On the other hand, LPS induced a significant
increase in Ang II in SHR but no significant change in WKY at 13 weeks
of age. The main factors limiting the rate of Ang II production
are angiotensinogen, PRA, and ACE. LPS induced no
significant changes in PRA in either WKY or SHR. It was reported that
the regulation of ACE in vascular tissues differs between SHR and WKY
and that the level of ACE activity in plasma and vascular tissue may be
regulated in a different manner within a given rat
strain.43 Therefore, the finding that pretreatment with
LPS did not alter p-Ang II level in WKY despite an increase in
p-angiotensinogen level may be due to differences in the
regulation of plasma ACE activity between SHR and WKY at 13 weeks of
age.
In the present study, although the expression of basal
angiotensinogen mRNA levels in all tissues including the
liver was suppressed in SHR compared with WKY at 4 weeks of age, basal
p-angiotensinogen levels were not different between SHR and
WKY at 4 weeks of age. Furthermore, although the expression of basal
angiotensinogen mRNA levels in most tissues was suppressed
in SHR compared with WKY, there was no significant difference in the
liver at 13 weeks of age. In contrast, basal
p-angiotensinogen level was higher in SHR than WKY at 13
weeks of age. These discrepancies between angiotensinogen
mRNA levels and p-angiotensinogen are consistent
with previous studies.42 45 All available evidence
indicates that p-angiotensinogen is secreted only
constitutively and cannot be stored within secretory
granules.46 This implies that angiotensinogen
production is controlled mostly at the transcriptional and
posttranscriptional levels of the angiotensinogen gene.
Consequently, the discrepancy between the basal mRNA level and basal
plasma concentration may be due to differences between SHR and WKY in
transcriptional or posttranscriptional regulation of
angiotensinogen mRNA. For example, glucocorticoids, despite
their strong effect on transcription of the angiotensinogen
gene, may have opposing effects on translation of its
mRNA.47 In contrast to glucocorticoids, estrogens and
thyroid hormones affect both angiotensinogen secretion and
liver angiotensinogen mRNA expression to a comparable
extent. In addition, Ang II increases the synthesis of
angiotensinogen in hepatocytes by stabilizing
angiotensinogen mRNA.5 In previous study,
Lodwick et al45 reported no difference in the level of
circulating angiotensinogen between 25-week-old SHR and WKY
on either a normal or high salt diet, whereas they found a higher level
of angiotensinogen mRNA in WKY liver compared with SHR
liver. Since plasma renin concentration was different between the
strains, they suggested that a relatively higher consumption of
angiotensinogen in WKY may at least partly account for the
similar p-angiotensinogen. Therefore, the discrepancy
between p-angiotensinogen concentration and liver
angiotensinogen mRNA levels at 4 and 13 weeks of age in the
present study may be due to altered translational machinery of
angiotensinogen mRNA in the liver and a difference in the
metabolism rate of angiotensinogen in the
circulation and/or peripheral tissues. However, the exact
mechanism should be examined by future study.
The 5' flanking region of the angiotensinogen gene contains
cis-acting regulatory elements. Analysis of this
region of rat and human genes has shown consensus sequences for a
glucocorticoid response element, a thyroid response element, an
estrogen response element, and APRE. Most previous studies testing the
effect of glucocorticoids in vivo have used doses about 100 times
higher than that required to induce hypercorticism. Administration of
corticosterone at doses that induced clear signs of hypercorticism did
not stimulate p-angiotensinogen.48 These data
make it unlikely that glucocorticoids act as primary regulators of
angiotensinogen. Rather, glucocorticoids may function as
permissive factors that facilitate the actions of other agents, such as
interleukins. In contrast, the APR, initiated experimentally by
intraperitoneal administration of bacterial LPS, is
a potent inducer of hepatic angiotensinogen
expression.23 39 In previous studies, initiation of
systemic inflammation by a single dose of
intraperitoneal LPS resulted in a fivefold increase
in hepatic steady-state mRNA levels at 3 hours23 and a
threefold increase in p-angiotensinogen concentration at 8
hours.49 In the LPS-induced APR, the
macrophage-derived cytokines TNF-
and
interleukin-6 are likely to be the activators of hepatic
angiotensinogen gene expression.41 50 51 LPS
in the bloodstream rapidly binds to the serum protein, referred to as
lipopolysaccharide binding protein (LBP).52
Complexes of LPS and LBP bind macrophage surface protein
CD14.53 Circulating cytokines are secreted from
activated macrophages. The cytokines
interleukin-6 and TNF-
bind to specific receptors, activating second
messenger signaling cascades that ultimately control the nuclear
expression of the two transcription factor families that bind to the
APRE. The p65 subunit of nuclear factor-
B (Rel A)/nuclear
factor-
B1 (NF-
B1) is a transcription factor complex associated
with inhibitory protein (I
B). Rel A/NF-
B1 is released
from inhibitors in response to cytokine signaling
events, allowing it to translocate into the nucleus and stimulate
transcription of the angiotensinogen
gene.41 50 54 55 56
Pretreatment with LPS increased angiotensinogen mRNA levels
in the liver, aorta, and adrenal in both WKY and SHR at 4 and 13 weeks
of age, decreased levels in the spleen, and had no effect on levels in
the kidney in both WKY and SHR at 13 weeks of age. This indicates that
LPS-mediated regulation of angiotensinogen gene expression
is tissue-specific. After pretreatment with LPS,
angiotensinogen mRNA levels in most tissues in SHR, with
the exception of fat, were similar to those in WKY at 4 and 13 weeks of
age. In fat, LPS increased angiotensinogen mRNA in both WKY
and SHR at 4 weeks of age, whereas LPS increased
angiotensinogen mRNA in SHR but not in WKY at 13 weeks of
age. Angiotensinogen is expressed abundantly in adipose
tissue, and angiotensinogen gene expression increases
during adipogenic differentiation.57 A previous study
showed that angiotensinogen gene expression in adipocytes
was nutritionally regulated and that blood pressure was modulated by
fasting and refeeding in a manner that paralleled the adipocyte
angiotensinogen mRNA level, irrespective of no change in
either the hepatic angiotensinogen mRNA level or
p-angiotensinogen concentration.58
Several inflammatory mediators, including TNF-
, interleukin-6, and
interleukin-1, were induced by LPS stimulation. This complexity of
factors participates in the regulation of angiotensinogen.
Previous studies reported that TNF-
and interleukin-6
activate angiotensinogen gene
expression.41 50 51 In contrast, interleukin-1 decreased
the angiotensinogen secretion and
angiotensinogen gene expression in
hepatocytes.51 In particular, local
production of TNF-
may have important roles in regulating
lipolysis, insulin sensitivity, and angiotensinogen
expression in the tissue RAS in fat deposits.41 59 60 61 In
the present study, although LPS induced TNF-
mRNA in both WKY
and SHR, the induction of TNF-
mRNA level in SHR was significantly
greater than that in WKY. These results suggest that the enhanced
expression of TNF-
gene in fat may be involved in the LPS-mediated
activation of the fat angiotensinogen gene in SHR at 13
weeks of age. Many studies suggest that immunological mechanisms may
contribute to the development of hypertension in
humans24 25 and animals.28 The differences
that we observed between SHR and WKY may involve immunological
abnormalities in SHR, and regulation of angiotensinogen
gene expression in fat may play a part in the development of
hypertension.
In conclusion, our data indicate that the LPS-mediated regulation of
angiotensinogen gene expression is tissue-specific. Our
results also suggest that altered angiotensinogen gene
expression in fat is involved in the development of hypertension in
SHR.
 |
Selected Abbreviations and Acronyms
|
|---|
| ACE |
= |
angiotensin-converting enzyme |
| Ang I, II |
= |
angiotensin I, II |
| APR |
= |
acute phase response |
| APRE |
= |
acute phase response element |
| LPS |
= |
lipopolysaccharide |
| PRA |
= |
plasma renin activity |
| RAS |
= |
renin-angiotensin system |
| SBP |
= |
systolic blood pressure |
| SHR |
= |
spontaneously hypertensive rat(s) |
TNF- |
= |
tumor necrosis factor- |
| WKY |
= |
Wistar-Kyoto rat(s) |
|
 |
Acknowledgments
|
|---|
This study was supported in part by grants from the Ministry
of
Education, Science, Sports, and Culture of Japan; the Uehara
Memorial
Foundation; and the Yokohama Foundation for Advancement
of Medical
Science.
Received October 7, 1996;
first decision November 6, 1996;
accepted March 17, 1997.
 |
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