From the Clinica Medica and Terapia Medica, Medical School (P.M.), and
the Department of Animal Biology, Section of Pharmacology (A.F.M., B.S.),
University of Sassari, Sassari, Italy; the National Laboratory of the National
Institute of Biostructures and Biosystems (P.M., L.G., C.E.), Osilo, Italy;
the Department of Nephrology, Max Delbruck Center (W.G., A.L.), Berlin Buch,
Germany; and the Department of Vascular Pathology, Istituto Dermopatico
dell'Immacolata (C.E.), Rome, Italy.
Correspondence to Paolo Madeddu, MD, Clinica Medica, University of Sassari, Viale S. Pietro 8, 07100 Sassari, Italy. E-mail madeddu{at}ssmain.uniss.it
The Goldblatt hypertensive rat model was the first animal model shown
to have a reduction in the urinary excretion and the vascular content
of tissue kallikrein.9 10 11 12 In particular, the
two-kidney, one clip (2K1C) hypertension model is characterized, early
after clipping, by activation of the RAS in the ischemic kidney
and by suppression of renin release in the contralateral one, whereas
kallikrein activity is reduced principally in the compromised kidney,
with a normal or mildly decreased level of expression in the
contralateral one.11 The reason or reasons for
changes in system activity in opposite directions are not yet
explained. Nevertheless, the differential pattern of expression of the
2 systems, leading to unopposed vasoconstriction, could
represent an important pathogenetic factor in renovascular
hypertension. This theory has been further elaborated by Emond et
al13 in their study determining renal
B2-receptor density in 2K1C rats. They reported
increased levels of expression of the B2 receptor
in both kidneys, particularly in the nonclipped one, which also showed
augmented BK-stimulated prostaglandin
E2 release. In a recent review,
Margolius8 hypothesized that "these increases
could signify an augmented kinin-induced production of
eicosanoids and nitric oxide in response to the higher perfusion
pressure, that the nonclipped kidney faces." These changes could also
contribute to the augmented renal blood flow and sodium excretory
ability typical of the nonclipped kidney, aiming to prevent
hypervolemia and excessive increase in blood pressure. If this were the
case, abrogation of kinin function should worsen the development of
renovascular hypertension
Disruption of genes that control hormones or receptors related to the
regulation of arterial blood pressure has been used
recently to assess the role of the targeted mechanism in the
pathogenesis of certain forms of
hypertension.6 14 15 16 17 18 19 The "knockout" mouse
model B2-/-, in which the gene
encoding for the B2 receptor has been targeted
for disruption, appears to be an ideal tool to address the
hypothesis that kinins exert a protective role in renovascular
hypertension. Therefore, we evaluated whether 2K1C hypertension
develops more severely in
B2-/- than in wild-type and
heterozygous mice. In addition, we determined whether chronic blockade
of the BK B2 receptor by Icatibant also worsens
the development of renovascular hypertension.
Animals (male, 28 to 35 g body wt [BW]) were housed at a
constant room temperature (24±1°C) and humidity (60±3%) with a
12-hour light/dark cycle. During experiments, mice had free access to
chow (Mucedola, Settimo Milanese) and tap water. All procedures
complied with the standards for the care and use of animal subjects as
stated in Guide for the Care and Use of Laboratory Animals
(Institute of Laboratory Animal Resources, National Academy of
Sciences, Bethesda, Md) and were approved by the local animal care and
use committee.
D-Arg,[Hyp3,Thi5,D-Tic7,Oic8]-BK
(Icatibant) and
2-{N-propyl-N-[2'(1H-tetrazol-5yl,biphenyl-4-yl)methyl]amino}pyridine-3-carboxylic
acid (A-81988) were generous gifts from Hoechst AG (Frankfurt, Germany)
and Abbott Laboratories (Abbott Park, Ill), respectively;
2,2,2-tribromoethanol and tert-amyl alcohol were purchased from
Sigma-Aldrich.
Clipping
Blood Pressure Measurements
To measure mean blood pressure (MBP) directly, a polyethylene catheter
(a PE-10 soldered to a PE-50, Clay Adams) was inserted into the left
carotid artery and advanced into the thoracic aorta of
anesthetized mice. For drug injections, another catheter was
inserted into the left jugular vein. Catheters (filled with 5%
heparinized saline solution) were then tunneled under the skin and
exteriorized at the back of the neck. The following day, a Statham
transducer was connected to the carotid catheter, and
intra-arterial MBP of unanesthetized free-moving
mice was recorded continuously on Quartet polygraph (Ugo
Basile).
Experimental Protocols
SBP, HR, and BW were measured every week for 2 weeks (n=8 per group) or
4 weeks (B2-/-: 2K1C n=18,
Icatibant-treated 2K1C n=6, and sham-operated n=8;
B2+/-: 2K1C n=8, and
sham-operated n=8; B2+/+: 2K1C
n=16, Icatibant-treated 2K1C n=6, and sham-operated n=8). Standard ECG
was recorded in anesthetized mice on the occasion of
clipping, and it was repeated at the end of the experiment. On the last
day of the experimental period, animals were instrumented with carotid
and jugular catheters (as indicated above) and allowed to recover for 5
hours. MBP was then continuously measured before and for 30 minutes
after the acute intravenous injection of 10 µg A-81988, a
nonpeptidic antagonist of angiotensin type 1
(AT1) receptors. This dose of A-81988 is able to
block the vasopressor action of 10 pmol IV Ang II. Then mice received
an overdose of anesthesia, and the heart and both kidneys
were removed, cleaned, washed in saline, blotted, and weighed. Cardiac
weight index was calculated as heart wet weight (mg)/BW (g). The left
kidney weight to right kidney weight ratio was also calculated. Kidneys
were fixed in neutral formaldehyde, and hematoxylin-eosin–stained
sections were examined by conventional microscopy. At the end of the
experimental period, we observed a homogeneous distribution
of renal infarction (14% in average) among groups that underwent
clipping. This percentage is similar to that reported from another
group previously.21 Because functional exclusion
of the clipped kidney is generally not accompanied by the development
of hypertension, and because it was impossible to recognize the exact
time when infarction had occurred, we decided to exclude these animals
from the study. Therefore, group number (n; see above)
represents the animals that were actually considered for
statistical analysis.
Statistical Analysis
Under basal conditions, B2-/-
showed accelerated HR compared with
B2+/- and
B2+/+ mice (before sham
operation: 497±16 versus 443±11 and 420±14 bpm, P<0.05
for both comparisons; before clipping: 467±11 versus 431±11 and
419±10 bpm, P<0.05 for both comparisons). As shown in
Figure 1
ANOVA did not detect any difference among groups as far as BW gain over
time is concerned.
As shown in Figure 2
As shown in Figure 2
As shown in Figure 2
As shown in Figure 3
No change from basal ECG was detected in
B2+/+ at 4 weeks after clipping.
By contrast, as shown in Figure 4
Histological examination did not reveal any major
alteration in the kidneys of mice that underwent sham operation or
clipping, except in those showing renal infarction. Changes included
increased collagen content, glomerular sclerosis, tubular
atrophy, and monocyte infiltration of the interstitium. As stated in
"Methods," these animals were excluded from statistical
examination.
Successful development of 2K1C and 1K1C hypertension has been recently
reported by 2 independent groups.21 22 Using
0.12-mm clips (identical to those used by Wiesel et
al21 in C57BL/6 mice), we were able to show that
2K1C hypertension can be induced also in mice of J129 background.
Consistent with other reports, the magnitude of blood pressure
increase was less than that observed in renovascular hypertensive
rats.21 22 Interestingly enough, genetically
hypertensive mice also tend to have lower blood pressure levels than
spontaneously hypertensive rats and resemble more closely hypertensive
rabbit models, which develop target organ damage even with modestly
elevated blood pressure.23 In fact, a mild
pressure overload is sufficient to increase cardiac mass in wild-type
mice with Goldblatt hypertension.21 A direct
effect of Ang II on the heart could have amplified the adaptive
hypertrophic response. This possibility appears to be likely in
consideration of the fact that Ang II is able to stimulate protein and
DNA synthesis in cardiac cells.24
Similar to other species, murine 2K1C hypertension is sustained by a
continuously activated RAS.21 The
contralateral kidney is able to maintain an efficient natriuresis, thus
preventing hypervolemia and further incremental rises of blood
pressure. Therefore, it is conceivable that any alteration of the
neurohumoral mechanisms that regulate the function of the nonclipped
kidney can accelerate the development of hypertension. Our
attention was focused on kinins because these potent vasodilators are
known to modulate water and electrolyte homeostasis by directly
affecting tubular function and renal hemodynamics and
by interacting with the RAS and other paracrine and endocrine
systems.25 Despite reduced levels of expression
of tissue kallikrein in the ischemic kidney of 2K1C
hypertensive rats, this defect is reduced or even absent in the
contralateral kidney in the early phases of
hypertension.9 10 11 These changes may only be
secondary to the increase in B2 receptor
expression that reportedly occurs, especially in the contralateral
kidney.13 It is tempting to interpret the main
finding of the present study, ie, the accelerated development of
2K1C hypertension in mice with chronic blockade or genetic disruption
of the B2 receptor, as a consequence of a
defective compensatory excretory response to increased perfusion
pressure by the untouched kidney, leading to total body fluid
expansion. Unfortunately, our attempts to obtain consistent
measurements of separate renal excretory function failed because of the
high variability of renal perfusion pressure in mice under the effects
of general anesthesia (P.M., unpublished observations,
1998).
In the mouse, resistance to vasoconstriction induced by acute renin
infusion has been reported, possibly due to low
angiotensinogen concentration, which represents a
rate-limiting factor for the enzymatic reaction catalyzed by
renin.26 No difference exists between
B2 receptor knockout mice and wild-type controls
with regard to plasma renin activity and to the tissue expression of
various components of the RAS, including angiotensinogen
and angiotensin-converting enzyme.5
This discounts the possibility that the accelerated development of
hypertension in B2-/- is due
to more favorable kinetic conditions for Ang II generation.
In contrast to rats, in which different levels of hypertension can be
obtained depending on the size of the clip opening, an all-or-nothing
blood pressure response to clipping is observed in mice, ie, sizes
>0.13 mm do not cause renal damage but are not able to induce
hypertension, whereas sizes of
In vitro studies have shown that Ang II stimulates NO
production in vascular endothelial cells by
enhancing the synthesis and release of BK.27 In
the early phase of 2K1C hypertension, when increased blood pressure is
sustained by elevated Ang II, endothelium-dependent
vasodilation is not reduced and may actually be increased. In fact, in
2K1C hypertensive rats, inhibition of NO synthase results in an
exaggerated increase in blood pressure and a decrease in contralateral
renal blood flow.28 This suggests that increased
NO release could exert a compensatory response on the vasoconstrictor
activity of activated RAS. The same vasodilator mechanism could
contribute to buffering of angiotensin-independent
vasoconstriction during chronic phases of 2K1C
hypertension.29 Deficiency in NO release, due to
the interrupted signaling by endogenous kinins, could have
exacerbated the development of renovascular hypertension in our mouse
model lacking the BK B2 receptor. Dysfunctional
endothelium-dependent vasodilation in these mice is
documented by the observation that they show a reduced blood pressure
response to chronic NO synthase inhibition and a rightward shift in the
dose-pressure response curve to
acetylcholine.5 30 In addition to NO, other
relaxant factors such as PGE2 and
endogenous cannabinoid derived from
arachidonate are known to mediate the vascular effects of
BK.31 32 Further studies are necessary to
determine whether deletion of the B2-receptor
gene worsens 2K1C hypertension by affecting the production of
PGE2 and/or endogenous cannabinoid.
Other unforeseen adaptation mechanisms taking place in animals that are
subjected to higher blood pressures during development might have also
contributed to the amplification of the pressor response to clipping in
B2-/- mice.
The finding that heterozygosity did not confer the same blood pressure
phenotype after clipping was unexpected, since a condition of
partial deficiency has been shown to be sufficient to elicit increased
sensitivity to chronic Ang II infusion.5 However,
heterozygous mice share with
B2-/- mice the feature of a
tachycardic response during the first 2 weeks after clipping. The
reasons for such discrepancies remain unknown. Chronic infusion allows
one to evaluate the effects of Ang II as a blood-borne hormone, but it
might not reflect the paracrine actions of an excess of
endogenous peptide generated endogenously. In
addition, redundancy of the B2 receptor, known to
be a spare receptor, might compensate for heterozygosity, thus allowing
full response to kinins.13
A puzzling aspect of our results is that at 4 weeks after clipping, the
MBP of 2K1C B2+/+ mice given
Icatibant was higher compared with 2K1C
B2+/+ mice without concomitant
blockade of the BK B2 receptor, but the
difference in SBP was lost at the same time. This discrepancy could be
due to the different experimental conditions used to measure direct or
indirect blood pressure. In addition, the pattern of SBP might reflect
a progressive decrease in cardiac output with persistently elevated
total peripheral resistances.33
Interestingly, SBP was maintained at high levels in Icatibant-treated
2K1C knockout mice. Thus, the pattern observed in wild-type mice could
be related to the long-term interaction between the
antagonist and its receptor (ie, receptor upregulation)
rather than other unforeseen effects of Icatibant.
The mechanisms mediating the exaggerated HR responses of mice with
genetic or pharmacological manipulation of the BK
B2 receptor remain unknown. Previous studies
showed that BK can modulate the reflex response to increments in blood
pressure by interacting with other peptidergic
systems.34 35 The accelerated HR found in
B2-/- mice after clipping
could be the consequence of an unbalanced central effect of Ang II.
Recent reports have suggested a direct inhibitory action of
kinins on myocardial and vascular proliferation, instrumental for
cardiovascular protection of converting enzyme
inhibitors.36 We found that under
basal conditions, heart weight is increased in
B2-/- mice compared with
wild-type mice.5 In addition, analysis of
transverse chamber diameter showed that hypertrophy is
associated with dilation of the left ventricle, suggesting that the
heart of adult B2-/- mice
could have almost reached the limit for a compensatory hypertrophic
response (P. Madeddu and G. Olivetti, unpublished results,
1998). This could explain why in
B2-/- mice, despite a greater
pressure load, the increase in heart weight after clipping was similar
to that observed in B2+/+ mice.
By contrast, the potential for further hypertrophic adaptation in
wild-type mice is revealed by the finding that long-term blockade of
the BK B2 receptor brought heart weight to the
levels of knockout mice. Changes in ECG observed after clipping in
B2-/- mice probably reflect
augmented duration of ventricular depolarization,
consequent to heart enlargement. Collectively, our findings indirectly
favor the possibility that activation of the KKS can prevent target
organ damage in renovascular hypertension. The potential protective
role of kinins in cardiac function is documented by the observation
that adenovirus-mediated kallikrein gene delivery causes a reduction of
the left ventricular mass and cardiac myocyte size in 2K1C
hypertensive rats.37 38
In conclusion, our results indicate that kinins are part of a
homeostatic response that buffer the vasoconstrictor activity of Ang
II. Development of renin-dependent renovascular hypertension could be
accelerated by the concomitance of genetic defects relevant to this
counterbalancing mechanism.
Received April 6, 1998;
first decision April 27, 1998;
accepted May 21, 1998.
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© 1998 American Heart Association, Inc.
Scientific Contributions
Renovascular Hypertension in Bradykinin B2-Receptor Knockout Mice
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Abstract—We evaluated whether
kinins exert a protective action against the development of two-kidney,
one clip (2K1C) hypertension, a model characterized by an
activated renin-angiotensin system in the
ischemic kidney and increased expression of the bradykinin (BK)
B2 receptor in the contralateral kidney. BK
B2-receptor knockout (B2-/-),
wild-type (B2+/+), and heterozygous
(B2+/-) mice underwent clipping of the left
renal artery, with the other kidney remaining untouched. Basal
systolic blood pressure (SBP, via tail-cuff plethysmography)
was higher in B2-/- mice than in
B2+/- or B2+/+ mice
(121±2 versus 113±2 and 109±1 mm Hg; P<0.05
for both comparisons). SBP did not change from basal values after sham
operation, but it increased in mice that underwent clipping. The
increase in SBP was greater in 2K1C B2-/-
mice than in B2+/- or
B2+/+ mice (28±2 versus 14±2 and 14±2
mm Hg, respectively, at 2 weeks; P<0.05 for both
comparisons). Blockade of the BK B2 receptor by Icatibant
enhanced the pressure response to clipping in
B2+/+ mice (29±2 mm Hg at 2 weeks).
Intra-arterial mean blood pressure (MBP) was higher in 2K1C
than in respective sham-operated mice, with the MBP difference being
higher in B2-/- mice (32 and 38 mm Hg,
at 2 and 4 weeks, respectively), and higher in
B2+/+ mice given Icatibant (30 and 32
mm Hg) than in B2+/+ mice without Icatibant
(17 and 18 mm Hg). At 4 weeks, acute injection of an
angiotensin type 1 receptor antagonist
normalized the MBP of 2K1C hypertensive mice. A tachycardic response
was observed 1 week after clipping in B2-/-
and B2+/- mice, but this effect was delayed
in B2+/+ mice. However, the HR response to
clipping in B2+/+ mice was enhanced by
Icatibant. Within each strain, heart weight to body weight ratio was
greater in 2K1C hypertensive mice than in sham-operated control animals
(B2-/-: 5.7±0.1 versus 5.2±0.1;
B2+/+: 5.1±0.1 versus 4.5±0.1;
P<0.01 for both comparisons). The clipped kidney weight
to nonclipped kidney weight ratio was consistently reduced in
mice with 2K1C hypertension. Our results indicate that kinins acting on
the BK B2 receptor exert a protective action against
excessive blood pressure elevation during early phases of 2K1C
hypertension.
Key Words: angiotensin • hypertension, renovascular • mice, kinin B2-receptor, knockout • hypertrophy, myocardial • kallikrein-kinin system
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
The hypothesis that arterial hypertension is
the consequence of an imbalance between vasoconstrictor and vasodilator
mechanisms is now widely accepted. Recently, evidence that a defective
activity of the vasodilator and natriuretic
kallikrein-kinin system (KKS) is implicated in the pathogenesis of
various forms of hypertension has been provided by a series of
epidemiological surveys,1
gene-polymorphism-cosegregation
analyses,2 and studies on animal models
with long-term blockade or disruption of the bradykinin (BK)
B2 receptor.3 4 5 6 The
elevated blood pressure of these models is reportedly caused by blunted
basal activation of the nitric oxide (NO) pathway that leads to the
amplification of vasoconstrictor activity.5 In
particular, the slow pressor response to angiotensin II
(Ang II) is exaggerated in animals with pharmacological or genetical
deficiency of the KKS, thus indicating that endogenous
kinins could buffer the long-term pressure effect of an excess of Ang
II.3 5 7 Together, these results reinforce the
notion that the KKS and renin- angiotensin system (RAS)
are functionally interrelated in the regulation of blood pressure and
total body fluid volume homeostasis.8
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
The knockout strain was developed by gene targeting and
homologous recombination on a J129 Sv genetic
background.20 Injection of 129Sv/Ev embryonic
cells, carrying the targeted mutation, into C57Bl/6J blastocysts
produced highly chimerical mice. They were mated with 129Sv/Sl mice (a
substrain closely related to 129Sv/Ev), and only the offspring (F1)
that were heterozygous for the knockout (thus having both sets of
chromosomes of 129Sv origin) were used for subsequent mating to
homozygous (F2) mice. This strategy has important advantages over the
practice of breeding the chimerical animals to C57Bl6/6J, which results
in a hybrid (129SvxC57Bl/6J). Studies with F2 mice derived from these
hybrids may suffer from the problem that the observed phenotype
is influenced by genes linked to the mutation.
B2-/- mice were a generous
gift of Dr Fred Hess (Merck Laboratories, West Point, Pa).
B2+/+ (129Sv/Ev strain) were
purchased from Jackson Laboratories (Bar Harbor, Maine). Heterozygous
mice were obtained by breeding pairs of
B2-/- and
B2+/+ mice.
Mice were anesthetized with 2,2,2-tribromoethanol
(88 mmol/100 g BW, IP) dissolved in tert-amyl alcohol. The left
kidney was exposed through a small flank incision, and the renal artery
was individualized over a short segment by blunt dissection. A U-shaped
stainless steel clip (3x2x1 mm with a 2-mm-long cleft and
0.12-mm opening width, Exidel SA) was placed around the renal artery
close to the aorta, according to the procedure described by Wiesel et
al.21 Then the kidney was gently pushed back into
the retroperitoneal cavity, and the muscle layer and the skin incision
were sutured. Sham-operated mice underwent the same surgical procedure
except without clipping.
Measurements of systolic blood pressure (SBP) and heart
rate (HR) were performed in unanesthetized mice by the
tail-cuff plethysmography method. They were warmed for 10 minutes at
37°C in a thermostatically controlled heating cabinet for better
detection of tail artery pulse. With the mouse kept gently wrapped in a
cotton hand towel, the tail was passed through a miniaturized cuff (1
cm long, 0.8 cm internal diameter) and a tail-cuff sensor that was
connected to an amplifier (Recorder 8002, Ugo Basile). The
amplified pulse was recorded during automatic inflation and
deflation of the cuff. SBP was defined as the cuff inflation pressure
at which the waveform becomes indistinguishable from baseline noise. As
an inclusion criterion, we required that at least 10 of 12 measurements
were successful. Final SBP value was obtained by averaging 10 to 12
successful readings. Experimental measurements were performed between
10 AM and 2 PM by a single investigator, and
then they were judged by an independent investigator in a blind
fashion. HR was recorded automatically by a counter triggered by
the pulse wave.
Basal measurements of SBP, HR, and BW were performed on 3
occasions (3 days apart). Mice then were randomly assigned to control
or experimental groups that underwent sham operation or clipping of the
left renal artery, respectively. In an additional group of 2K1C
B2+/+ mice, we tested the
cardiovascular effects of the BK
B2-receptor antagonist Icatibant (50
nmol in 10 µL saline/100 g BW twice a day, SC). The dose and timing
of administration were chosen on the basis of preliminary experiments
in B2+/+ mice showing the
ability of Icatibant to antagonize the vasodepressor effect of BK but
not that of acetylcholine or prostaglandin
E2 (PGE2).
All data are expressed as mean±SEM.
Multivariate repeated-measures ANOVA was performed to
test for interaction between time and grouping factor.
Univariate ANOVA then was used among groups and over time.
Differences within and between groups were determined using paired or
unpaired Student's t tests, respectively, with the
Bonferroni multiple comparisons adjustment.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
Basal SBP levels were higher in
B2-/- than in
B2+/- or
B2+/+ mice before sham operation
(122±2 versus 112±2 and 113±1 mm Hg; P<0.05 for
both comparisons) or clipping (121±2 versus 113±2 and 109±1
mm Hg; P<0.05 for both comparisons). As shown in Figure 1
, SBP remained unchanged after sham
operation. After clipping, it increased more in
B2-/- than in
B2+/- or
B2+/+ mice (28±2 versus 14±2
and 14±2 mm Hg, respectively, at 2 weeks; P<0.05 for
both comparisons). Chronic blockade of the BK B2
receptor by Icatibant enhanced the pressure response to clipping in
B2+/+ mice (29±2 versus
14±2 mm Hg at 2 weeks in mice without Icatibant;
P<0.05), thus leading to the hypertensive blood pressure
levels of B2-/- mice.
View larger version (19K):
[in a new window]
Figure 1. Absolute changes in SBP (top) and HR (bottom)
after sham operation ( 
) or clipping of the left renal artery in mice
with (
) or without Icatibant treatment (
). Values are mean±SEM.
*P<0.05 vs sham-operated mice; +P<0.05
vs 2K1C mice without Icatibant.
, no significant change in HR was observed after sham
operation. A prompt tachycardic response was detected 1 week after
clipping in B2-/- and
B2+/- mice, whereas
tachycardia was delayed in
B2+/+ mice. However, the HR
response to clipping in B2+/+ mice
was enhanced by Icatibant. , within each group
the MBP of 2K1C mice was higher than that of respective sham-operated
controls, with this difference being more pronounced in
B2-/- mice (32 and 38
mm Hg at 2 and 4 weeks, respectively) and in
B2+/+ mice given Icatibant (30
and 32 mm Hg) than in
B2+/+ mice without blockade of
the B2 receptor (17 and 18 mm Hg). The
pressure level observed in
B2+/- mice was similar to that
of B2+/+ mice.
View larger version (37K):
[in a new window]
Figure 2. MBP, heart weight to body weight ratio (HW/BW),
and left kidney to right kidney weight ratio (LKW/RKW) at 2 or 4 weeks
after sham operation (open bars) or clipping of the left renal artery
in mice with (solid bars) or without Icatibant (hatched bars). Values
are mean±SEM. *P<0.05 vs sham-operated mice;
+P<0.05 vs 2K1C mice without Icatibant. , clipping increased heart weight to BW ratio in
all groups. However, 2K1C B2+/+
mice given Icatibant showed a greater increase compared with mice of
the same strain without blockade of the BK B2
receptor. , left kidney weight to right kidney weight ratio
was diminished consistently in all groups with 2K1C
hypertension (averaging from 0.73 to 0.78). , acute
AT1 receptor blockade induced a more pronounced
MBP decrease in 2K1C mice than in sham-operated mice
(P<0.05). There was no significant difference in the MBP
decrease induced by AT1 receptor blockade in
B2-/- and
B2+/+ mice with 2K1C
hypertension.
View larger version (11K):
[in a new window]
Figure 3. Absolute changes in MBP induced by acute
intravenous injection of 10 µg A-81988, a nonpeptidic
antagonist of AT1 receptors, in
sham-operated ( , n=8 per group) or 2K1C mice (). Values are
mean±SEM. *P<0.05 vs sham-operated mice., both
the duration and the amplitude of QRS were increased in
B2-/- mice.
View larger version (20K):
[in a new window]
Figure 4. Typical ECG trace (50 mm/s, 20 mm/mV)
under basal condition and at the end of the experimental period in a
B2-/- mouse with 2K1C hypertension.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
We found that pharmacological blockade or genetic disruption of
the BK B2 receptor accelerates the development of
renovascular hypertension induced in mice by clipping of the left renal
artery and leaving the other kidney untouched. 
0.10 mm always result in
infarction of the clipped kidney.21 The rate of
occurrence of a very small white kidney, indicating loss of excretory
capacity, was consistently low in both
B2-/- and
B2+/+ mice, similar to that
reported previously by Wiesel et al,21 who used
the same solid stainless steel clip to cause hypertension in C57BL/6
mice. Renal infarction was not associated with hypertension, probably
because of the functional exclusion of the stenotic kidney. In
our study, successful development of renovascular hypertension led to
consistent reduction of clipped/nonclipped kidney weight ratio
in B2-/- and
B2+/+ mice. Four weeks after
clipping, plasma renin activity was similarly elevated in the 2 strains
(P.M., unpublished observations, 1998), and no significant difference
was detected in the MBP response to acute AT1
receptor blockade. Together, these results speak against the
possibility that in knockout mice, hypertension was worsened by more
severe constriction of the renal artery and/or by a greater degree of
activation of the RAS.
Acknowledgments
This work was supported in part by grants from the Minister of
Universities and Scientific Research (MURST, Area Scienze Mediche: Il
Cuore Insufficiente, No. F07C/1997) and the National Research Council
(CNR, No. 97.04476.CT04). In addition, the financial support of
Telethon-Italy (grant A.105) is gratefully acknowledged.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
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Ash KO, Kuida H, Williams RR. A gene for high kallikrein may protect
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