Am J Physiol Heart Circ Physiol 294: H1880–H1887, 2008.
First published February 22, 2008; doi:10.1152/ajpheart.01319.2007.
RNAi inhibition of mineralocorticoid receptors prevents the development
of cold-induced hypertension
Zhongjie Sun,1,2 Mahajoub Bello-Roufai,2 and Xiuqing Wang1,2
1
Department of Physiology, College of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma;
and 2Departments of Medicine and Physiology and Functional Genomics, College of Medicine, University of Florida,
Gainesville, Florida
Submitted 11 November 2007; accepted in final form 21 February 2008
short-hairpin RNA; aldosterone; blood pressure; adeno-associated
virus; cardiac hypertrophy; RNA interference
adverse effects on the human cardiovascular system. People who live in cold regions have a high
incidence of hypertension and related cardiovascular diseases
(3, 4, 6, 11, 13, 25, 36). Of the four seasons, the cold winter has
the highest mortality and morbidity from cardiovascular diseases (1, 24, 25, 36). Cold temperatures make hypertension
more severe in hypertensive patients and trigger myocardial
infarction and stroke (6, 7, 9, 13, 25, 36, 37). Chronic cold
exposure causes hypertension and cardiac hypertrophy in rats
within 1–3 wk (26, 27, 38, 39), namely cold-induced hypertension (CIH). CIH is a natural form of experimentally induced
COLD TEMPERATURES HAVE
Address for reprint requests and other correspondence: Z. Sun, Dept. of
Physiology, BMSB 662A, PO Box 26901, College of Medicine, Univ. of
Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., Oklahoma
City, OK 73034 (e-mail: [email protected]).
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hypertension that does not require surgery, an administration of
large doses of hormones or drugs, or genetic manipulation (26,
28). The characteristics of CIH are similar to those of human
essential hypertension (26, 28, 29). Therefore, it is important to
determine the mechanism of CIH. Previous studies from our
laboratory suggested that CIH may be associated with the
hyperactivity of the renin-angiotensin-aldosterone system
(RAAS) (27, 30 –33). A recent study from our laboratory
indicated that inhibition of the mineralocorticoid receptor
(MR) prevented the progression of CIH and renal damage (40).
However, it is not known whether the MR is involved in the
onset of CIH. The aim of this study was to determine the role
of the MR in the initiation and development of CIH. Although
pharmacological MR blockers such as spironolactone can inhibit the MR, they interact with androgen and progesterone
receptors, causing an unwanted side effect due to the lack of
specificity. These drugs are short lasting (⬍24 h). In addition,
eplerenone was found to increase serum triglycerides, cholesterol, alanine aminotransferase, ␥-glutamyl transpeptidase, creatinine, and uric acid (12). To overcome these limitations of
pharmacological MR blockers, we used the RNA interference
(RNAi) approach to achieve long-term and specific inhibition
of MR expression. We hypothesized that RNAi inhibition of
MRs attenuates or prevents the initiation and development
of CIH.
Our previous studies showed that the development of coldinduced cardiac hypertrophy is independent of high blood
pressure (BP) because the prevention or attenuation of CIH
does not attenuate cold-induced increases in heart weight (27,
32, 33). Thus cold-induced cardiac hypertrophy may be due to
endocrine changes associated with cold exposure. The cardiac
MR has been reported to be involved in the pathogenesis of
hypertrophy. Thus an additional aim of this study was to
determine whether the MR plays a role in the development of
cold-induced cardiac hypertrophy. We hypothesized that RNAi
inhibition of MRs attenuates cold-induced cardiac hypertrophy.
METHODS
Recombinant adeno-associated virus with short-hairpin small-interference RNA for MR and a scrambled sequence. The recombinant
adeno-associated virus (AAV) carrying short-hairpin small-interference RNA for MR (AAV.MRshRNA) or a scrambled sequence
(AAV.ControlshRNA) was constructed as described previously (40).
Briefly, the MRshRNA was designed using Dharmacon’s software,
The costs of publication of this article were defrayed in part by the payment
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in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0363-6135/08 $8.00 Copyright © 2008 the American Physiological Society
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Sun Z, Bello-Roufai M, Wang X. RNAi inhibition of mineralocorticoid receptors prevents the development of cold-induced hypertension.
Am J Physiol Heart Circ Physiol 294: H1880–H1887, 2008. First
published February 22, 2008; doi:10.1152/ajpheart.01319.2007.—The
objective was to determine whether the mineralocorticoid receptor
(MR) plays a role in the initiation and development of cold-induced
hypertension (CIH) by testing the hypothesis that the RNA interference (RNAi) inhibition of the MR attenuates CIH. The recombinant
adeno-associated virus (AAV) carrying a short-hairpin small-interference RNA for MR (MRshRNA) or a scrambled sequence (ControlshRNA) was constructed. Six groups of albino mice were used (6
mice/group). Three groups were exposed to cold (6.7°C), whereas the
remaining three groups were kept at room temperature (RT; warm) as
controls. In each temperature condition, three groups received an
intravenous injection of MRshRNA, ControlshRNA, or virus-free
PBS, respectively, before exposure to cold. The viral complexes
(0.35 ⫻ 1011 particles/mouse, 0.5 ml) or PBS (0.5 ml) was delivered
into the circulation via the tail vein. The blood pressure (BP) of the
mice treated with ControlshRNA or PBS increased significantly
during exposure to cold, whereas the BP of the cold-exposed
MRshRNA-treated mice did not increase and remained at the level of
the control group kept at RT. Thus AAV delivery of MRshRNA
prevented the initiation of CIH. MRshRNA significantly attenuated
cardiac and renal hypertrophy. MRshRNA decreased the cold-induced
increase in MR protein expression to the control level in the hypothalamus, kidneys, and heart, indicating an effective prevention of the
cold-induced upregulation of MR. RNAi inhibition of MR resulted in
significant decreases in the plasma level of norepinephrine, plasma
renin activity, and plasma level of aldosterone in cold-exposed mice.
MR played a critical role in the initiation and development of CIH.
AAV delivery of MRshRNA may serve as a new approach for the
prevention of cold-induced hypertension.
MR AND COLD-INDUCED HYPERTENSION
AJP-Heart Circ Physiol • VOL
MR expression was normalized with the expression of glyceraldehyde-3-phospshate dehydrogenase (GAPDH), which was served as an
internal control. Briefly, equal volumes of mixtures (30 ␮l and 3
␮l/sample for MR and GAPDH, respectively) were loaded for all
groups. Membranes were blocked overnight at 4°C before incubation
with primary antibodies. For MR, the membranes were incubated with
rabbit polyclonal anti-MR primary antibody (dilution, 1:500; Santa
Cruz Biotechnology) overnight and then with goat anti-rabbit secondary antibody (dilution 1:2,000; Santa Cruz Biotechnology) for 2 h. For
GAPDH, the membranes were incubated with mouse monoclonal
anti-GAPDH primary antibody (dilution 1:30,000; Santa Cruz Biotechnology) for 1 h and then with goat anti-mouse secondary antibody
for 2 h. Enhanced chemiluminescence was added to the membranes
and exposed to photosensitive films. The films were imaged by using
a Bio-Rad transilluminator. Protein band intensities were quantified
using Quantity One version 4.1.1.1 c system (Bio-Rad, Hercules, CA).
AAV capsid protein expression was similarly analyzed using antiAAV antibodies (VP1, VP2, and VP3; Strategen).
IHC analysis of MR expression. After a 24-h fixation with 4%
paraformaldehyde, the heart and kidneys were incubated overnight in
70% ethanol at 4°C and processed for paraffin embedding. Sections (5
␮m) were used for the immunostaining of MR. Brains were dehydrated in 20% sucrose for 48 h before embedding with optimal cutting
temperature. For immunostaining, the sections were treated for 30 min
with 0.25% Triton X-100 for antigen retrieval, 5 min with peroxidaseblocking solution (Dako), and 15 min with protein blocker (Biocare).
The sections were then incubated with polyclonal goat anti-MR
primary antibody (dilution 1:100; Santa Cruz Biotechnology) for 1 h
and then with a donkey anti-goat secondary antibody (dilution 1:200;
Santa Cruz Biotechnology) for 30 min. The sections were examined
and photographed using a Zeiss Axioplan 2 photomicroscope coupled
with a digital color camera. In the hypothalamus, the MR expression
level was evaluated by calculating the percentage of MR-positive
neurons in 150 neurons in each of three different areas.
Statistical analysis. The data for BP and BW were analyzed by a
repeated-measures two-way ANOVA followed by one-way ANOVA.
The remaining data were analyzed by a two-way ANOVA (main
factors, temperature and treatment). The Newman-Keuls procedure
was used to assess the significance of difference between means.
Significance was set at a 95% confidence limit.
RESULTS
Effect of AAV delivery of MRshRNA on BP and BW. BP did
not differ significantly among the six groups during the control
period at RT (Fig. 1A). No significant difference in BP was
found between any two groups at 24 h after gene delivery. The
BP of the ControlshRNA- and PBS-treated groups increased
significantly within 4 days of exposure to cold. The BP of these
two groups continued to increase thereafter and reached a
plateau (⬇140 mmHg) by 3 wk of exposure to cold. No
significant difference in BP was found between these two
groups throughout the experiment. In contrast, the BP of the
MRshRNA-treated group did not increase significantly and
remained at the level of the PBS-warm group during exposure
to cold. One dose of MRshRNA kept the BP at the control level
for at least 32 days (length of the study; Fig. 1A). MRshRNA
or ControlshRNA did not significantly change BP in mice
maintained at RT (warm).
Despite the observed differences in BP, all animals grew at
approximately the same rate (Fig. 1B). No significant difference in BW was found between any two groups at any time
points (P ⬎ 0.05), indicating that cold exposure or treatments
with viral complexes did not affect the animals⬘ growth.
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synthesized by Integrated DNA Technologies (Coralville, IA), and
cloned into a AAV serotype-2 (AAV-2; Startagen, La Jolla, CA)
under the control of a human RNA polymerase II U6 promoter
(AAV.MRshRNA) (40). Scrambled shRNA was cloned into AAV-2
vector and used as a control viral construct (AAV.ControlshRNA).
Scrambled shRNA has been proved, by BD Clontech (Palo Alto, CA),
not to match with any other known gene sequences. The constructs
were packaged with pHelper and pAAV-RC to produce recombinant
vectors. Titers were determined by real-time PCR as described by
Rohr et al. (23).
Animals. This study was carried out according to the guidelines of
the National Institutes of Health on the Care and Use of Laboratory
Animals. This project was approved by the Institutional Animal Care
and Use Committee. Male albino mice of 8 wk of age were used in the
experiment. All mice were housed individually in wire-mesh cages
and were provided with Purina laboratory chow (No. 5001) and tap
water ad libitum throughout the experiment.
Animal study protocol. Six groups of albino mice (6 mice/group)
were used. The animals were handled daily to minimize handling
stress. The animals did not appear stressed during BP measurement.
Resting systolic BP was measured at room temperature (RT) from the
tail of each unanesthetized mouse using the tail-cuff method. The
tail-cuff procedure is a common method used by our laboratory (27,
30 –33) and others (17, 43) to delineate CIH. It has been confirmed by
the intra-arterial cannulation that the noninvasive tail-cuff method is
effective and reliable in monitoring systolic BP in animals exposed to
cold (5, 29).
During a 2-wk control period, BP, body weight (BW), food intake,
water intake, and urine output were measured. After the control
period, three groups were moved into to a climate-controlled walk-in
chamber (6.7 ⫾ 2°C), whereas the other three groups were kept in an
identical chamber maintained at RT (25 ⫾ 2°C, warm). In each
temperature condition, one group received a bolus injection of
AAV.MRshRNA (0.35 ⫻ 1011 particles/mouse, 0.5 ml iv via tail
vein), one group received AAV.ControlshRNA (0.35 ⫻ 1011 particles/mouse, 0.5 ml iv), and the last group received phosphate-buffered
saline (PBS; 0.5 ml iv) 48 h before exposure to cold. BP was
measured 24 h after the injection to ensure that the gene delivery did
not affect animal-resting BP at RT. BP and BW were measured at
least once a week throughout the experiment. Food intake, water
intake, and urine output were measured during weeks 3 and 5 of
exposure to cold. Urinary Na⫹ was measured by flame photometry as
described in previous studies from our laboratory (32, 40).
At day 32 after exposure to cold, all animals were deeply anesthetized with pentobarbital sodium (120 mg/kg ip) for blood collection,
followed by transcardiac perfusion with PBS. The heart and kidneys
were removed and weighed. The left kidney, the apical region of the
heart, and half of the brain were fixed in 4% paraformaldehyde in PBS
for immunohistochemical (IHC) and histological analyses. The rest of
the tissues were snap frozen and saved for Western blot analysis of
MR and AAV protein expression.
Measurements of plasma norepinephrine, plasma renin activity,
and plasma aldosterone. The plasma norepinephrine (NE) level was
measured using HPLC with an electrochemical detector as described
in previous studies from our laboratory (27, 32). Plasma renin activity
(PRA) and plasma aldosterone levels were measured using radioimmunoassay as described previously (32, 41).
Western blot analysis of MR and AAV capsid protein expression.
Tissues were homogenized in lysis buffer (50 mM Tris, 150 mM
NaCl, 1% sodium dodecyl sulfate, 1% sodium deoxycholic acid, 1
mM phenylmethylsulfonyl fluoride, 1 mM ethylene diamine tetracetic,
and 1% Triton X-100) containing a protease inhibitor complex and
centrifuged 5 min at 10,000 g. Supernatants were collected and
immediately mixed with an equal volume of electrophoresis loading
buffer for Western blot analysis of MR and AAV capsid protein
expression.
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MR AND COLD-INDUCED HYPERTENSION
cold group than in the PBS-warm group, indicating that
MRshRNA failed to decrease heart weight to the warm control
level. MRshRNA did not affect heart weight significantly in
the mice maintained at RT (warm).
Cold exposure significantly increased kidney weight in the
ControlshRNA- and PBS-treated groups (Fig. 3B). MRshRNA
significantly decreased the cold-induced increase in kidney
weight but failed to decrease it to the warm control level.
MRshRNA did not significantly affect kidney weight in the
mice maintained at RT (warm).
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Fig. 1. Effects of adeno-associated virus (AAV) delivery of short-hairpin
small-interference RNA for mineralocorticoid receptor (MRshRNA) on coldinduced elevation of blood pressure (A) and body weight (means ⫾ SE; B).
Intravenous injections of AAV carrying MRshRNA (AAV.MRshRNA), AAV
carrying a scrambled sequence (AAV.ControlshRNA), or phosphate-buffered
saline (PBS) were carried out 2 days before exposure to cold. ⫹⫹⫹P ⬍ 0.001
compared with the ControlshRNA-cold group; n ⫽ 6 animals/group.
Food intake, water intake, urine output, and urinary sodium
output. Food intake, water intake, and urine output did not
differ among the six groups during the control period and
among all the warm-kept groups throughout the experiment
(P ⬎ 0.05). Although cold exposure did not affect BW, it
significantly increased food intake, water intake, and urine
output in all three groups compared with their corresponding
warm-kept groups (P ⬍ 0.01) during weeks 3 and 5 (Fig. 2, A–C).
No significant difference was found in food intake, water
intake, and urine output among the three groups in either
temperature condition. Cold exposure increased urinary sodium output significantly (data not shown). However, no significant difference was found in the urinary sodium output
between the MRshRNA- and ControlshRNA- or PBS-treated
groups in either temperature environment, indicating that
MRshRNA did not affect urinary sodium output.
Effects of AAV delivery of MRshRNA on cold-induced cardiac and renal hypertrophy. Cold exposure increased heart
weight to approximately the same extent in the ControlshRNAand PBS-treated groups (Fig. 3A). MRshRNA significantly
decreased the cold-induced increase in heart weight compared
with the ControlshRNA- or the PBS-cold groups. Nevertheless,
the heart weight was significantly greater in the MRshRNAAJP-Heart Circ Physiol • VOL
Fig. 2. Food intake (A), water intake (B), and urine output (means ⫾ SE; C)
in cold-exposed and room temperature-maintained rats. *P ⬍ 0.05 and **P ⬍
0.01 compared with the ControlshRNA-warm group. These were measured
during the control period (⫺1.0) and during week 3 and 5 of exposure to cold
(n ⫽ 6 animals). BW, body weight.
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MR AND COLD-INDUCED HYPERTENSION
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Fig. 3. Effects of AAV delivery of MRshRNA on heart weight (A) and kidney
weight (means ⫾ SE; B). These were measured when animals were euthanized
at 32 days after exposure to cold. ***P ⬍ 0.001 compared with the ControlshRNA-warm group; ⫹P ⬍ 0.05, ⫹⫹P ⬍ 0.01, and ⫹⫹⫹P ⬍ 0.001
compared with the ControlshRNA-cold group; n ⫽ 6 animals.
Effects of AAV delivery of MRshRNA on plasma NE, PRA,
and plasma aldosterone. The plasma NE concentration, PRA,
and plasma aldosterone concentration were significantly increased
in the ControlshRNA- and PBS-cold groups (Fig. 4, A–C), indicating that cold exposure activated the sympathetic nervous system (SNS) and the RAAS. AAV delivery of MRshRNA significantly decreased the plasma NE concentration, PRA, and plasma
aldosterone concentration of cold-exposed rats to the control
levels. MRshRNA did not affect these parameters in rats kept at
RT (warm).
Detection of AAV proteins and the effects of AAV.MRshRNA
on MR expression. The expression of AAV structural (capsid)
proteins VP1, VP2, and VP3 was used to assess gene delivery
and tissue transfection efficiency. VP1, VP2, and VP3 were
strongly expressed in hearts (Fig. 5A). A strong expression of
VP2 and VP3 was also found in the kidneys. VP2 was expressed in the hypothalamus. Thus the expression of at least
one of the AAV structural proteins was detected in the heart,
kidneys, and brain hypothalamus, indicating that AAV was
expressed in these tissues or cells.
Cold exposure significantly increased MR expression in the
heart, kidneys, and hypothalamus in ControlshRNA- and PBScold groups compared with their corresponding warm-kept
groups (Fig. 5, B–E). Thus MR expression was upregulated
by cold exposure. MRshRNA significantly decreased MR
protein expression in these tissues in cold-exposed rats. No
AJP-Heart Circ Physiol • VOL
Fig. 4. Effects of AAV delivery of MRshRNA on plasma concentration
(Conc) of norepinephrine (A), plasma renin activity (B), and plasma concentration of aldosterone (means ⫾ SE; C). ***P ⬍ 0.05 compared with the
ControlshRNA-warm group; ⫹⫹⫹P ⬍ 0.001 compared with the ControlshRNA-cold group; n ⫽ 6 animals.
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significant difference in MR expression was found between
the MRshRNA-cold and the PBS-warm groups (Fig. 5, C–E),
indicating that MRshRNA abolished the cold-induced increase
in MR expression.
IHC analysis of MR expression. The inhibitory effect of
MRshRNA on MR expression in the heart, kidneys, and
hypothalamus of cold-exposed mice was further examined by
IHC. Cold exposure increased MR immunostaining in the
hearts and kidneys in ControlshRNA- and PBS-treated mice
(Fig. 6). Strong MR expression was found in the nuclei of
cardiac myocytes and renal tubule epithelial cells of both
ControlshRNA- and PBS-cold groups. In the renal epithelial
cells, MR staining was also found in the cytoplasm. MRshRNA
greatly decreased MR staining in the hearts and kidneys of
cold-exposed mice (Fig. 6). Cold exposure increased interstitial
fibrotic tissue in the hearts of ControlshRNA- and PBS-treated
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MR AND COLD-INDUCED HYPERTENSION
mice (Fig. 6). MRshRNA greatly decreased cardiac fibrosis in
cold-exposed mice. For simplicity, the IHC photomicrographs
for MRshRNA- and ControlshRNA-warm mice were not
shown because their MR staining was not altered compared
with PBS-warm mice.
Cold exposure significantly increased the number of MRpositive neurons in the hypothalamus as determined by the
increased percentage of MR-positive neurons in ControlshRNA- and PBS-cold groups (Fig. 7, A and B). MRshRNA
significantly decreased the number of MR-positive neurons in
the hypothalamus in the cold-exposed group compared with the
ControlshRNA- and PBS-cold groups. MRshRNA decreased
the number of MR-positive neurons in the cold-exposed group
to that of the PBS-warm group (MRshRNA cold vs. PBS
warm, P ⬎ 0.05; Fig. 7B), indicating that AAV delivery of
MRshRNA abolished the cold-induced upregulation of hypothalamic MR.
DISCUSSION
The most exciting finding of this study is that the RNAi
inhibition of MR prevented the cold-induced elevation of BP.
AJP-Heart Circ Physiol • VOL
It is noted that one single dose of AAV.MRshRNA controlled
CIH for up to 32 days (length of the study). The RNAi
inhibition of MR also attenuated cardiac and renal hypertrophy. The MR was effectively inhibited by MRshRNA, as
evidenced by the fact that the cold-induced increase in the MR
was prevented in rats treated with AAV.MRshRNA. The prolonged antihypertensive effect of AAV.MRshRNA was probably due to the long-lasting AAV vector, which is safe,
nonpathogenic, noninflammatory, and extremely stable (19,
20). No signs of inflammation were seen during an autopsy,
and no viral effect was observed in the data analysis. Indeed,
AAV.shRNA did not have a toxic effect on animal growth and
BP regulation because AAV.ControlshRNA did not affect BW
and BP in either temperature condition. A recent study from
our laboratory indicated that RNAi inhibition of MR stopped
the progression of hypertension in rats with established CIH
although the BP was not lowered to normotensive level (40).
The present study clearly shows that AAV delivery of
MRshRNA may be a promising approach for the prevention of
hypertension due to the overactive MR although there are
many challenges before it can be used clinically.
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Fig. 5. Western blot analysis of AAV capsid protein expression and mineralocorticoid receptor (MR) protein expression. A: representative Western blot of AAV
capsid proteins VP1, VP2, and VP3 in heart, kidneys, and hypothalamus in the MRshRNA-cold group. B: representative Western blot of MR expression in heart,
kidneys, and hypothalamus. Relative MR expression was normalized with GAPDH in heart (C), kidneys (D), and hypothalamus (E). These were measured at
32 days after exposure to cold. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 compared with the ControlshRNA-warm group; ⫹⫹P ⬍ 0.01 and ⫹⫹⫹P ⬍ 0.01
compared with the ControlshRNA-cold group; n ⫽ 6 animals.
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MR AND COLD-INDUCED HYPERTENSION
The present finding also supports the hypothesis that the MR
plays a critical role in the initiation and development of CIH
because cold exposure upregulated the MR and the normalization of MR expression prevented the onset of CIH. Previous
studies from our laboratory have shown that the hyperactivity
of the SNS and the RAAS is essential for the development of
CIH (27, 30 –33). It has been confirmed that the SNS initiates
CIH via the activation of the RAAS (27, 32, 33). It is inter-
esting to note that the AAV delivery of MRshRNA prevented
the cold-induced increase in the plasma NE level, PRA, and
plasma aldosterone level. This finding is unexpected because it
was originally assumed that RNAi inhibition of the MR would
increase plasma aldosterone levels due to compensatory feedback. The decreased plasma aldosterone levels by gene delivery of MRshRNA were probably due to the inhibition of the
SNS in cold-exposed mice, which decreased the activity of the
Fig. 7. Immunohistochemical analysis of MR
protein expression in the hypothalamus. A: representative sections from mice in the MRshRNAcold, ControlshRNA-cold, PBS-cold, and PBSwarm groups. Arrows indicate MR-positive
neurons (brown staining; ⫻200 magnification).
B: the percentage of neurons expressing MR in
the hypothalamus in the 4 groups of mice designated (means ⫾ SE). ###P ⬍ 0.001 compared
with the PBS-warm group. MR expression level
was evaluated by calculating the percentage of
MR-positive neurons in 150 neurons in at least 3
different areas (n ⫽ 6 animals).
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Fig. 6. Immunohistochemical analysis of MR protein expression in heart and kidneys. Representative sections from mice in the MRshRNA-cold, ControlshRNAcold, PBS-cold, and PBS-warm groups are shown. Arrows indicate MR protein staining (brown staining; ⫻200 magnification). MR staining in MRshRNA- and
ControlshRNA-warm groups is not shown for conciseness.
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MR AND COLD-INDUCED HYPERTENSION
AJP-Heart Circ Physiol • VOL
It is noted that RNAi inhibition of MR prevented the
cold-induced elevation of BP but only partially attenuated
cardiac and renal hypertrophy. Previous studies from our
laboratory indicate that cold-induced cardiac and renal hypertrophy are independent of pressure overload because the attenuation or prevention of CIH does not attenuate cold-induced
increases in heart and kidney weights (27, 32, 33). Therefore,
the partial attenuation of hypertrophy may be attributed to the
direct inhibition of the MR rather than its preventive effect on
BP. Indeed, the MR has been reported to be related to cardiac
hypertrophy (14, 16, 25a). The MR may also be involved in
some forms of renal hypertrophy (2, 10, 21). Nevertheless, the
present study suggests that MR partially contributes to the
pathogenesis of cold-induced cardiac and renal hypertrophy.
Thus the mechanisms in addition to the MR may be involved
in cold-induced cardiac and renal hypertrophy. The hypothesis
that cold-induced cardiac and renal hypertrophy is largely
mediated by thyroid hormones needs to be tested because cold
exposure increases the secretion of thyroid hormones that are
required for nonshivering thermogenesis.
In conclusion, cold exposure upregulated MR expression,
which played a critical role in the initiation and development of
CIH. AAV delivery of MRshRNA may serve as a new approach for the prevention of CIH.
GRANTS
This study was supported by National Heart, Lung, and Blood Institute
Grant R01-HL-077490 (to Z. Sun).
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RAAS. The plasma aldosterone level did not increase in
MRshRNA-cold mice probably because that MR expression
was decreased only to the control level, which may not elicit a
compensatory feedback mechanism. It is interesting to note
that MR expression was upregulated against the background of
an increased plasma aldosterone level (PBS- and MRshRNAcold groups), suggesting that the physiological regulation of
the aldosterone-MR relationship disappeared in cold (stress)
conditions. The brief anesthesia procedure did not affect the
plasma aldosterone level because the plasma aldosterone levels
(⬇49 pg/ml) of the control mice were similar to the levels
obtained when the mice were decapitated without anesthetic
(⬇50 pg/ml) (42). Thus the prevention of CIH by the RNAi
inhibition of MR may be attributed to its inhibitory effect on
the SNS and the RAAS. The suppression of the SNS by
MRshRNA might be due to the inhibition of the central MR,
which was upregulated by cold exposure. This hypothesis,
however, needs to be tested by a blockade of the central MR.
Indeed, MR protein expression and the number of the MRpositive neurons in the hypothalamus were decreased by the
AAV delivery of MRshRNA. The present finding suggests, for
the first time, that the upregulation of the central MR may be
responsible for the cold-induced increase in the SNS activity.
The brain MR may be involved in the modulation and regulation of the SNS activity and BP (8, 22). We convincingly
demonstrated that the AAV carried MRshRNA into the hypothalamus, as evidenced by the expression of AAV capsid
protein VP2 in the hypothalamus and by a decrease in MR
expression in neurons in the hypothalamus in the MRshRNAtreated mice. Thus the recombinant AAV.shRNA can cross the
blood-brain barrier. The AAV may get into the hypothalamus
via the circumventricular organ (18).
Our previous studies have established that cold exposure
increases food intake, water intake, urine output, and urinary
sodium output (32, 34). It is interesting to note that coldexposed rats did not gain additional BW although they consumed more food, probably owing to the increased metabolic
rate and heat production to maintain body temperature. Although MRshRNA inhibited the renal MR, it did not affect the
cold-induced increases in food intake, water intake, urine
output, and urinary sodium output measured during weeks 3
and 5 after exposure to cold. A recent report indicated that
inhibition of the renal MR transiently increased Na⫹ excretion
within 24 h of treatment in both normotensive and hypertensive
rats, but the natriuretic effect disappeared and the sodium
balance was reestablished within 5 days (15). Therefore, compensatory mechanisms for conserving Na⫹ may be activated
during the chronic inhibition of the renal MR, e.g., the maintenance of ␣-epithelial Na⫹ channel content (15). In contrast,
AAV.MRshRNA increased urinary Na⫹ excretion in rats with
established CIH at 3 wk after gene delivery (40). In that study,
urinary Na⫹ excretion was measured at 8 wk after exposure to
cold. Thus long-term exposure to cold seems to impair the
renal compensatory reabsorption of Na⫹ although the mechanism remains to be determined.
Nevertheless, the limitation of this study is that it cannot
determine the relative importance of the brain MR and kidney
MR in the development of CIH. Further studies are needed to
test the effect of RNAi inhibition of the central MR on CIH by
an intracerebrovascular delivery of AAV.MRshRNA.
MR AND COLD-INDUCED HYPERTENSION
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