(Hypertension. 2001;37:121.)
© 2001 American Heart Association, Inc.
Scientific Contributions |
Effect of Chronic Blockade of the Kallikrein-Kinin System on the Development of Hypertension in Rats
From the Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, Mich.
Correspondence to Oscar A. Carretero, MD, Education and Research, Suite 7123, Henry Ford Hospital, Hypertension and Vascular Research Division, 2799 W Grand Blvd, Detroit, MI 48202-2689. E-mail ocarretero{at}hfhs.org
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Abstract |
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Abstract—The kallikrein-kininogen-kinin system is an important vasodilator and vasodepressor component of the cardiovascular system. Acting mainly through B2 receptors, kinins may counterbalance the pressor effect of angiotensin II, salt, and mineralocorticoids plus salt. Using rats lacking the bradykinin precursors low- and high-molecular-weight kininogen or a B2 kinin receptor antagonist (icatibant), we investigated whether absence or blockade of the kallikrein-kinin system alters blood pressure (BP) in rats given (1) chronic infusion of Ang II, (2) a normal or high salt diet, or (3) chronic administration of deoxycorticosterone acetate (DOCA) plus salt. We confirmed the genotype and phenotype of Brown Norway Katholiek rats (BNK) and found that they had a G-to-A point mutation on the kininogen gene compared with Brown Norway (BN) and Sprague-Dawley (SD) rats, very low levels of high-molecular-weight kininogen (17±3 ng/mL) compared with BN and SD (1814±253 and 2397±302 ng/mL, respectively; P<0.01), and plasma low-molecular-weight kininogen concentrations below detectable limits compared with 1773±74 and 1781±140 ng/mL for BN and SD, respectively. Basal BP was the same in BNK and BN. Chronic infusion of icatibant did not alter BP in BN or Wistar rats. At doses that blocked the acute effect of bradykinin, icatibant did not potentiate the pressor effect of a chronic subpressor or pressor dose of angiotensin II in male and female Wistar rats nor that of a high salt diet (2%) plus unilateral nephrectomy in male Wistar rats. Moreover, blockade of the kallikrein-kininogen-kinin system in either BN rats given a very high dose of icatibant or kinin-deficient rats (BNK) did not potentiate the pressor effect of angiotensin II (nonpressor dose) or a high salt (3% NaCl) diet given for 2 weeks. Established DOCA-salt hypertension was not exaggerated in rats treated with icatibant but was partially attenuated by ramipril (1.5 mg · kg-1 · d-1 for 7 days; P<0.002). This antihypertensive effect was abolished by icatibant (P<0.002, ramipril versus ramipril plus icatibant). These results suggest that endogenous kinins do not participate in the maintenance of normal blood pressure or antagonize the development of hypertension induced by chronic infusion of angiotensin II, a high salt diet, or DOCA-salt. However, kinins appear to play an important role in the antihypertensive effect of angiotensin-converting enzyme inhibitors in DOCA-salt hypertension.
Key Words: kinins • receptors, B2 • angiotensin II • sodium • deoxycorticosterone acetate • hypertension, experimental • rats, Brown Norway Katholiek
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Introduction |
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Three types of kininogen are found in rat plasma: a low-molecular-weight form (LMWK, 9% to 14% of total plasma kininogen), a high-molecular-weight form (HMWK, 12% to 21%), and T-kininogen (65% to 77%).1 HMWK and LMWK are natural substrates of kallikrein, whereas T-kininogen is resistant to kallikrein but releases T-kinin by interacting with trypsin. Kinins are endogenous paracrine-autocrine vasodilators; acting by means of B2 receptors, they mediate the synthesis and release of prostaglandins and nitric oxide from the endothelium.2 This vasodilator effect has been extensively documented and reportedly plays an important role in the acute effect of angiotensin-converting enzyme inhibitors (ACEi),3 4 5 although its role in the chronic effects of ACEi remains controversial.6 7 8
Majima et al9 10 11 published a series of reports that indicated that Brown Norway Katholiek rats (BNK), which are deficient in kininogen, became hypertensive earlier than normal Brown Norway rats (BN) when subjected to both subpressor and pressor doses of angiotensin II (Ang II), a high salt diet, or deoxycorticosterone acetate (DOCA) plus salt. These authors also reported that normal rats given a subpressor dose of Ang II or a high salt diet became hypertensive when treated with either a kallikrein inhibitor (aprotinin) or a potent B2 kinin receptor antagonist (icatibant, administered subcutaneously). Using a different approach, Madeddu et al12 13 14 reported that chronic blockade of B2 receptors with icatibant (administered intraperitoneally) made rats hypertensive when chronically infused with either a pressor or nonpressor dose of Ang II or DOCA-salt. Both Majima et al and Madeddu et al concluded that defective kinin generation could contribute to arterial hypertension by fostering vasoconstrictor tone and sodium retention. In addition, Maddedu et al14 15 claimed that female rats were better models in which to study the cardiovascular effects of endogenous kinins because their renal kallikrein-kinin system was more active.
ACEi act mainly by (1) abolishing the conversion of Ang I to Ang II, a potent vasopressor agent, and (2) inhibiting the breakdown of bradykinin into inactive fragments.16 17 Locally produced kinins appear to play an important role in the cardioprotective effect of ACEi in heart failure induced by myocardial infarction18 and preconditioning in a rat model of ischemia-reperfusion.19 These protective effects were absent in the presence of a B2 kinin receptor antagonist (icatibant).
Given the relevance of the findings of Majima et al9 10 11 and Madeddu et al,12 13 14 we attempted to confirm or refute them by testing the hypothesis that kinins play a key role in the balance between vasodilators and vasoconstrictors needed to maintain normal blood pressure (BP). For this, we compared BP in BNK versus BN rats and in BN or Wistar rats given a B2 kinin receptor antagonist versus controls. We also carefully examined changes in BP in BNK rats given either a high salt diet or chronic subpressor doses of Ang II using conventional tail-cuff plethysmography or telemetry and studied whether blockade of B2 kinin receptors with icatibant increases the pressor effect of chronic treatment with (1) a subpressor or pressor dose of Ang II, (2) a high salt diet, or (3) DOCA-salt. Finally, we tested whether the antihypertensive effect of ACEi in rats given DOCA-salt is mediated by kinins.
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Methods |
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Animals
Five-week-old male BN and BNK (obtained from Kitasato University, Kanagawa, Japan, and maintained and bred in Henry Ford Hospital animal care facilities) and 8- to 9-week-old female and male Wistar and male Sprague-Dawley rats (SD; Charles River, Wilmington, Mass) were used for these experiments. Rats were housed in an air-conditioned room with a 12-hour light/dark cycle and received standard rat chow (0.4% sodium) and tap water ad libitum. BN and BNK were given standard 0.3% NaCl rat chow obtained from NMF Oriental Yeast Co (Tokyo, Japan) and tap water. This study was approved by the Henry Ford Hospital Care of Experimental Animals Committee.
Surgical Procedures
All surgical procedures were performed under aseptic conditions
in rats anesthetized with either pentobarbital (50 mg/kg IP)
for uninephrectomy and catheterization or ether to
implant osmotic minipumps. The left kidney was removed by means of a
flank incision, which was then sutured and covered with a mixture of
collodion and iodine (20:1). Rats were allowed to recover in a warm
cage.
Blood Pressure
Systolic BP (SBP) was determined using a tail cuff.
After one week of training and adjustment to the new environment, SBP
was measured twice a week before starting treatment (baseline) as well
as for 2 to 3 weeks afterward. At the end of the study, mean BP (MBP)
was measured via the femoral artery in awake rats as described
previously.8 To determine whether blockade of
B2 receptors was successful, we injected
bradykinin intravenously or intra-arterially
into anesthetized rats.
Infusion of Ang II, Icatibant, or Vehicle
Infusions were performed by implanting
intraperitoneal or subcutaneous osmotic minipumps
(Alzet 2001 for 7 days or Alzet 2002 for 14 days) as indicated below.
Pumps were replaced when necessary.
Telemetry of BP
The telemetric device (Data Sciences International) was
implanted surgically in anesthetized rats in accord with the
manufacturer’s specifications and institutional procedures. Rats were
allowed to recover for 1 week in a quiet room. Baseline BPs were
measured for 1 week with a Data Science acquisition program, which
allows us to collect BP readings every 10 seconds for 24 hours. After
implanting osmotic minipumps filled with Ang II or vehicle, BP was
recorded for 2 more weeks. BP readings were averaged and plotted
against 4 different time frames: 6 AM to 12 PM,
12 to 6 PM, 6 PM to 12 AM, and 12
to 6 AM.
Phenotype and Genotype of BNK
We investigated whether the genotype and
phenotype that characterize BNK rats are conserved in these
rats as described below.
Measurement of Plasma Kininogen
To confirm that BNK are deficient in plasma kininogen, we
measured plasma kininogen from BN (n=10), SD (n=10), and BNK
rats (n=17). Rats given a normal diet were anesthetized with
pentobarbital sodium (50 mg/kg IP) and blood withdrawn through the
abdominal aorta with a heparin-soaked syringe. Plasma HMWK and LMWK
were measured by a modification of the method of Majima et
al.10 For HMWK, plasma prekallikrein
was primed through activation of coagulation factor XII by incubating
plasma with glass powder at 37°C for 30 minutes; kininase
inhibitors o-phenanthroline (6 mmol/L) and
Na2EDTA (60 mmol/L) were also added to the
reaction. HMWK was converted to kinins by plasma kallikrein, and
released bradykinin was measured by
radioimmunoassay.20 For LMWK, an additional plasma
sample was incubated with glass powder in the absence of kininase
inhibitors. Thus, kinins converted from HMWK by plasma
kallikrein were destroyed by kininases in plasma.10
Peptidases, kallikrein inhibitors, and plasma
prekallikrein were destroyed by acidification (pH 2):
for this, HCl (1 mol/L) was added for 30 minutes at 37°C. Plasma was
incubated with purified rat urinary kallikrein after pH was adjusted to
7.8 with 0.1 N NaOH to release kinins from LMWK. Kinins released from
kininogen were assayed by radioimmunoassay.20 Kininogen
levels were expressed as nanograms of kinin generated per milliliter of
plasma.
Determination of a G-to-A Point Mutation on the Kininogen
Gene
The kinin deficiency in BNK rats is reportedly the result of a
G-to-A point mutation at kininogen nucleotide position
487.21 To confirm that our BNK rat colony had this point
mutation, we amplified 164-bp fragments containing kininogen
nucleotide position 487 by polymerase chain reaction, with
genomic DNA from BN, SD, and BNK rats (n=3 each) as
polymerase chain reaction templates. These fragments were then run on
an automated ABI DNA sequencer (model 373A).
Protocol 1
In protocol 1, BP response to chronically infused Ang II in BNK
rats (kinin deficient) or normal rats chronically treated with a
B2 kinin receptor antagonist
(icatibant) was tested.
Protocol 1a
To determine whether BNK rats (kinin deficient) are sensitive to
a subpressor dose of Ang II and examine diurnal changes in BP, we used
telemetry, which is less stressful and is well suited for long-term
drug studies. Rats weighing 100 to 135 g were subdivided into 2
groups that received either vehicle (n=4) or Ang II (30 µg/d SC;
n=5). After measuring basal MBP, osmotic minipumps filled with either
vehicle or Ang II were implanted and MBP was recorded for 2 more
weeks.
Protocol 1b
BN rats weighing 135 to 155 g were studied, because this
strain was used in the study of Majima et al.11 Rats were
subdivided into 2 groups that received either Ang II plus vehicle (n=6)
or Ang II plus icatibant (n=3). SBP was measured twice during the first
week (baseline), and then an infusion of Ang II (20 µg/d SC) was
started with osmotic minipumps and continued until the end of the
study. On day 7, a very high dose of icatibant (5 mg ·
kg-1 ·
d-1 IP) was infused and
continued until the end of the study. SBP was measured twice a week
until day 14. MBP was measured in awake rats on days 16 and
17.
Protocol 1c
Because Maddedu et al14 15 reported that the renal
kallikrein-kinin system is more active in female than male Wistar rats,
we compared the effect of a subpressor dose of Ang II (20 µg/d) on
SBP and MBP during blockade of bradykinin B2
receptors. Basal SBP was measured in untreated rats, which were then
subdivided into 2 treatment groups: (1) Ang II plus vehicle (n=5 males
and n=7 females), and (2) Ang II plus icatibant (500 µg ·
kg-1 ·
d-1; n=6 males and n=7
females). After basal SBP was measured, osmotic minipumps containing
Ang II combined with either vehicle or icatibant were implanted
intraperitoneally and SBP was measured twice a week
for 2 weeks following the protocol of Maddedu et al.14 On
day 14, MBP was measured directly in awake rats, after which a single
dose of bradykinin (100 ng IA) was injected into anesthetized
rats to confirm B2 kinin receptor blockade.
Protocol 1d
Male Wistar rats weighing 175 to 215 g were used in this
study as in the study of Madeddu et al.14 SBP was measured
twice during the first week (baseline), and then a subpressor to
pressor dose of Ang II (20, 40, 80, or 160 µg/d IP) combined with
vehicle or icatibant (500 µg ·
kg-1 ·
d-1 IP) was infused by
minipump and continued until the end of the study (n=4 to 5 for each).
SBP was measured twice a week until day 14. MBP was measured in awake
rats on days 16 and 17.
Protocol 2
This protocol measured the effect of a high salt diet on BP in
BNK (kinin deficient) and normal rats chronically treated with a
B2 kinin receptor antagonist
(icatibant).
Protocol 2a
Male BNK rats (120 to 180 g) were subdivided into 2 groups:
(1) those that received a 3% NaCl diet (n=7) and (2) those that
received standard 0.3% NaCl rat chow (n=8) for 3 weeks. SBP, 24-hour
urinary volume (UV) and sodium excretion (UNa+V),
and body weight were measured in each rat before the diet was started
and twice a week thereafter. At the end of the experiment, MBP was
measured in conscious rats.
Protocol 2b
Male BN rats (120 to 175 g) were divided into 2 groups that
received either (1) icatibant 5 mg ·
kg-1 ·
d-1 (n=11) or (2) vehicle
(n=12). Powdered rat chow containing 3% NaCl was given for 3 weeks
until the end of the protocol. One week after the high salt diet was
begun, osmotic minipumps containing icatibant or vehicle were implanted
subcutaneously. SBP, 24-hour UV and UNa+V, and
body weight were measured in each rat before the diet was begun and
twice a week thereafter. At the end of the experiment, MBP was measured
in conscious rats, after which the rats were anesthetized and
bradykinin injected through the femoral vein.
Protocol 2c
Male Wistar rats (180 to 210 g) were divided into 2 groups
that received either (1) icatibant 500 µg ·
kg-1 ·
d-1 (n=6) or (2) vehicle
(n=6). Rats were anesthetized and uninephrectomy performed.
Powdered rat chow containing 2% NaCl was given for 3 weeks until the
end of the protocol. One week after the high salt diet was begun,
osmotic minipumps containing icatibant or vehicle were implanted
subcutaneously. SBP was measured before the diet was begun and twice a
week thereafter. At the end of the experiment, MBP was measured in
conscious rats.
Protocol 3
Protocol 3 measured the effect of chronic blockade of
B2 kinin receptors on maintenance
of hypertension and the antihypertensive effect of ACE inhibition in
DOCA-salt hypertensive rats.
Male SD weighing 225 to 250 g were given standard rat chow (0.28% NaCl) with free access to tap water and given 1 week to acclimate to the housing conditions. Basal SBP was measured by tail cuff, and then a silicone rubber sheet (No. 3110 RTV) containing DOCA (150 mg/kg) was implanted subcutaneously in uninephrectomized rats. Rats that received DOCA were given a solution of 1% NaCl and 0.2% KCl to drink. SBP was measured once a week for 4 weeks, the time needed to develop sustained hypertension. Osmotic minipumps filled with (1) vehicle (saline; n=7); (2) ramipril (1.5 mg · kg-1 · d-1; n=8); (3) icatibant (35 µg/d; n=7); or (4) ramipril plus icatibant (n=8) were implanted IP and the femoral artery catheterized as above. MBP was recorded every day for 1 week in conscious animals housed in a plastic restrainer. After MBP was measured, 100 ng bradykinin dissolved in 100 µL saline was injected intra-arterially into anesthetized rats and MBP recorded to determine whether blockade of B2 receptors was successful.
Materials
Porcine pancreatic kallikrein, o-phenanthroline,
NaCl, Na2EDTA, and DOCA were purchased from
Sigma. Ang II was obtained from Peptides International. Osmotic
minipumps were obtained from Alza, polyethylene catheters from
Clay-Adams, and silicone rubber from Dow Corning. Ramipril and
icatibant were generously donated by Hoechst (Cincinnati,
Ohio).
Data Analysis
Results are expressed as mean±SEM. Values were compared by
ANOVA for repeated measures, except for the UV,
UNa+V, and BP responses to bradykinin (in
BN and Wistar rats), which were analyzed by paired t
test. BP response to bradykinin in BNK rats given different levels of
salt in the diet was compared using a 2-sample t test.
Pairwise comparisons were made using Holm’s method22
to adjust for multiple testing to compare BP of the 4 groups listed in
protocol 3. P<0.05 was considered significant.
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Results |
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Kininogen Deficiency and Presence of a G-to-A Point Mutation on the Kininogen Gene in BNK Rats
Plasma HMWK in BNK was 17±3 ng/mL, 107-fold lower than BN (1814±253) and 140-fold lower than SD (2397±302; P<0.01). Plasma LMWK was below detectable limits in BNK (<5 ng/mL) compared with BN (1773±74) and SD (1781±140; P<0.01). We also confirmed by polymerase chain reaction and sequence analysis that BNK rats (but not BN or SD) have a G-to-A point mutation of nucleotide 487 on the amplified cDNA fragment, in the heavy chain of the kininogen gene.
Effect of Kininogen Deficiency or Chronic Blockade of
B2 Kinin Receptors on Basal BP
Comparison of SBP among all rats showed that BNK (kinin deficient)
had SBP almost identical to BN (124±4 versus 123±3 mm Hg;
n=15 and 23, respectively). Icatibant (500 µg ·
kg-1 ·
d-1 for 14 days) tended to
increase SBP in Wistar rats (123±3 mm Hg; n=5) compared with
controls (115±4 mm Hg; n=5), but did not reach significance.
Protocol 1
Protocol 1a
Telemetric BP in BNK rats given a subpressor dose of Ang II (30
µg/d) was not significantly different from rats given saline (Figure 1); BP was higher at night than during
the day in rats given Ang II or vehicle, which is to be expected since
rats are nocturnal animals. Although it did not reach significance, BP
was lower at week 2 compared with baseline or week 1 during the day in
both Ang II and vehicle groups.
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Protocol 1b
In BN, chronic infusion of a subpressor dose of Ang II (20 µg/d)
did not alter SBP or MBP either in the absence or presence of a very
high dose of icatibant (5 mg ·
kg-1 ·
d-1) (Figure 2).
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) or combined with a very high dose of icatibant (5 mg · kg-1 · d-1; •) on SBP and MBP in BN. The effect of Ang II was unaltered by chronic blockade of B2 receptors. Veh indicates vehicle; B2-ant, B2 antagonist. Values are mean±SEM.
Protocol 1c
SBP and MBP were measured during chronic infusion of a subpressor
dose of Ang II (20 µg/d) alone or combined with icatibant in Wistar
rats. Icatibant did not increase SBP or MBP in either females or males
(Figure 3); however, in the same rats
bradykinin (100 ng) given intra-arterially decreased BP by
15±2 mm Hg in females and 14±2 mm Hg in males. This
depressor effect was significantly lowered to 0±1
(P=0.0004) and 2±2 mm Hg (P=0.0001) by
chronic icatibant treatment.
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Protocol 1d
The pressor effect of Ang II at doses of 20, 40, 80, or 160 µg/d
for 14 days was measured in male Wistar rats infused with vehicle or
icatibant (500 µg ·
kg-1 ·
d-1). Icatibant did not
increase SBP in rats chronically infused with subpressor or pressor
doses of Ang II (Figure 4).
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Protocol 2
Protocol 2a
A high salt diet did not increase SBP or MBP in BNK (kinin
deficient) (Figure 5A). In BNK given a
high salt diet (3% NaCl), UV and UNa+V increased
to 46.9±5.5 mL per 100 g body weight per 24 hours and
7.1±0.4 mmol per 100 g body weight per 24 hours,
respectively, compared with rats on a normal diet (UV, 10.9±0.7;
UNa+V, 0.8±0.06).
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Protocol 2b
BN given a high salt diet did not exhibit any significant changes
in SBP, either in the absence or presence of a very high dose of
icatibant (5.0 mg ·
kg-1 ·
d-1; Figures 5B and 5D). MBP was very similar in BN given high salt alone or
combined with icatibant (114±2 versus 113±3 mm Hg; n=10 in each
group). With sodium loading, BN excreted higher amounts of sodium
(UNa+V, 8.1±1.6 mmol per 100 g body
weight per 24 hours) and urine (UV, 50.5±3.9 mL per 100 g body
weight per 24 hours) compared with rats given normal sodium
(UNa+V, 0.58±0.1; UV, 8.4±0.5). Icatibant did
not affect UNa+V or UV in rats given a high salt
diet (UNa+V, 8.2±0.7; UV, 43.5±3.3). Exogenous
bradykinin (10 µg IV) lowered MBP in anesthetized BN given
high salt alone by 33±3 mm Hg (n=5), and its effect was almost
completely blocked in rats given high salt plus icatibant (3±1
mm Hg; n=9; P<0.01).
Protocol 2c
SBP was significantly increased in Wistar rats fed a high salt
diet (P<0.05). Icatibant did not increase the pressor
effect of high salt (Figures 5C and 5D). MBP was also
similar in rats given a high salt diet alone or combined with
icatibant.
Protocol 3
In DOCA-salt hypertensive rats treated with an ACE
inhibitor (ramipril 1.5 mg ·
kg-1 ·
d-1 for 7 days), MBP
decreased from 176±9 (n=7) to 158±7 mm Hg (n=8;
P=0.066). This effect was blocked by icatibant, which
resulted in an MBP of 184±7 mm Hg (n=5; P<0.05).
Blockade of B2 kinin receptors with icatibant (35
µg/d IP) did not aggravate the hypertension induced by DOCA-salt in
conscious rats (Figure 6) but
significantly attenuated the hypotensive effect of exogenous bradykinin
(100 ng IA) from 56±8 to 13±3 mm Hg (n=3 for each group;
P<0.01).
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), or ramipril plus icatibant (
) on DOCA-salt hypertension in SD. Icatibant did not alter MBP in rats treated with DOCA-salt, whereas ramipril decreased MBP. Icatibant blunted the mild hypotensive effect of ramipril. Values are mean±SEM. *P<0.01 and **P<0.002 vs control. 
P<0.01 and 
P< 0.005 vs ramipril plus icatibant. |
Discussion |
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This study was designed to test whether chronic blockade or absence of kinins (1) increases BP in animals fed a normal diet; (2) potentiates the hypertensinogenic effect of Ang II, high salt intake, or DOCA-salt (a model of low renin and high kinins), or (3) prevents the antihypertensive effect of ACE inhibitors in established DOCA-salt hypertension. Two different approaches were followed: (1) use of kininogen-deficient BNK rats and (2) chronic blockade of kinin B2 receptors with a kinin antagonist (icatibant). First, we confirmed the presence of a G-to-A point mutation at nucleotide 487, which causes a substitution of Thr for Ala163 in the heavy chain of HMWK and LMWK in BNK but not normal BN or SD rats21 and also verified that only traces of HMWK and undetectable levels of LMWK were found in BNK compared with normal levels in BN and SD.9 This point mutation is reportedly responsible for the defective secretion of both kininogens by the liver in BNK rats.21 23 24 Our results only partially confirm the findings by Yang et al,19 since we found that BNK rats merely had traces of HMWK and LMWK, whereas Yang et al observed very low HMWK and normal LMWK concentrations in plasma. This could be due to the source of glandular kallikrein used in the present study and the studies of Majima et al9 10 11 (purified urinary kallikrein) compared with the study of Yang et al, in which glandular kallikrein (porcine pancreas) was obtained from Sigma. Indeed, we confirmed that porcine pancreatic kallikrein from Sigma was still capable of releasing kinins from LMWK. It is not clear how commercial kallikreins release kinins in BNK, but they could act like trypsin, releasing kinin from T-kininogen.
We found that chronic blockade of the kallikrein-kinin system did not
increase SBP or MBP (1) under normal conditions, (2) in Wistar rats
(male or female) given a chronic subpressor or pressor dose of Ang II,
or (3) in BN or BNK given a chronic subpressor dose of Ang II. These
data are at odds with those of Majima et al,11 who found a
difference of >40 mm Hg in SBP between BN and BNK (kinin
deficient) after infusion of a subpressor dose of Ang II for 2 weeks,
and Maddedu et al,14 who found that chronic blockade of
the B2 kinin receptor increased the effect of
both pressor and subpressor doses of Ang II in female Wistar rats. At
present, we cannot explain these discrepancies. Similarly, chronic
blockade of the kallikrein-kinin system did not increase the pressor
effect of a high salt diet in Wistar, BN, or BNK rats. Interestingly,
we found that under normal conditions, Wistar rats had lower BP than
either normal BN or BNK (Figure 5); while the reason for this is
unknown, differences in strain and age could account for the variations
in BP. Although we did not study sodium balance (nor did Majima et
al10 ), we confirmed that BNK had increased UV (9-fold) and
UNa+V (5-fold) when given a high salt versus a
normal diet; similarly, BN treated with icatibant exhibited increased
UV (14-fold) and UNa+V (5-fold) on a high salt
versus a normal diet, which confirms that the rats received a high salt
diet. These data contrast with those previously reported by Majima et
al9 10 for reasons that remain unclear but agree with
those of Madeddu et al.12 Majima and
colleagues10 found that SBP was increased by 
40
mm Hg in BNK but not BN given a high salt diet, whereas Madeddu et
al12 reported that icatibant did not increase the pressor
effect of high salt in Wistar rats. Dr Majima was invited to our
laboratory to discuss the reasons for these discrepancies. He kindly
performed 2 different sets of experiments with us to try to confirm his
previous studies9 10 : (1) normal BN rats were given a
subcutaneous infusion of Ang II or high salt diet alone or combined
with icatibant (given intraperitoneally) and (2)
BNK rats were given a subcutaneous infusion of a nonpressor dose of Ang
II or a high salt diet. Although the experimental conditions (BNK rats,
powdered rat chow, acidified water, and Ang II) were identical to those
used in Japan, we were unable to reproduce any of their findings or
those of Maddedu et al.14 The subpressor effect of Ang II
was not increased by icatibant either
intraperitoneally or subcutaneously (N.-E.R. and
O.A.C., unpublished observations, 1999). Madeddu et
al14 showed that the BP response to chronic infusion of a
subpressor dose of Ang II (subcutaneous) was increased in the presence
of icatibant given intraperitoneally. We could not
explain these dramatic differences between our findings and those of
Majima et al or Maddedu et al. One could also question whether changes
in BP occur only at night, when the rats are active; however, this
possibility was ruled out because telemetry showed no difference in BP
between BNK infused with vehicle or a subpressor dose of Ang II either
during day or night.
Alfie et al25 found that mice genetically lacking the B2 kinin receptor gene had higher SBP and MBP only when given a diet very high in salt. This study was recently confirmed by Cervenka et al,26 who started in a mouse model a high salt diet before birth and continuing it in the pups for 4 months after birth. Both groups found that permanent inactivation of B2 receptors does not cause adult hypertension, which refutes the findings of Madeddu et al.27 B2 receptors are well established as the predominant if not the only means by which kinins may induce the release of endogenous vasodilators such as nitric oxide, hyperpolarizing factors, and prostaglandins. Therefore, eliminating B2 receptors from the body may contribute to the imbalance between vasoconstrictor agents and kinins, and this may have occurred in mice in which the B2 receptor gene was disrupted and a high salt diet was given longer than in BNK (6 to 8 versus 2 to 3 weeks). In contrast to these transgenic mice, BNK were still able to produce active kinins through release of T-kinin following the action of trypsin-like enzymes on T-kininogen,21 28 which, in turn, may act on the B2 receptor. Findings of Alfie et al25 and Cervenka et al26 contrast with those of the present study, since that we found that even a very high dose of icatibant, which completely blocked B2 kinin receptors, did not increase the pressor effect of a high salt diet or chronic infusion of Ang II. This discrepancy may be due to 3 fundamental differences: (1) our protocol was designed for just 2 to 3 weeks follow-up after a subpressor dose of Ang II or high salt diet was begun, whereas Alfie and colleagues did not observe increased BP until 6 to 8 weeks after a high salt diet was started and did not notice any changes in BP after 3 to 4 weeks (M.E. Alfie, O.A. Carretero, unpublished observations, 1996); (2) B2 receptors were absent in the transgenic mice since the fetal stage, whereas BN rats were treated with the B2 kinin receptor antagonist for only a short time; and (3) icatibant may not be as efficient as animals that lack B2 receptors, because icatibant may be degraded before it reaches the lumen of the distal nephron. We infused radiolabeled icatibant in rats and found that it was completely degraded in the urine (O.A.C, unpublished observations, 1999). Thus, whether the kallikrein-kinin system is physiologically linked to regulation of sodium balance (and, hence, BP regulation) remains unclear.
We found that ACE inhibitors have a mild antihypertensive effect on DOCA-salt hypertension, which was blocked by a B2 kinin receptor antagonist. Icatibant alone did not increase BP further in rats with established DOCA-salt hypertension, in contrast to the results of Madeddu et al.12 13 They found that chronic blockade of B2 kinin receptors not only facilitated the development of hypertension, but also resulted in exaggerated established hypertension. On the other hand, Majima et al9 and Katori et al29 found that blockade of the kallikrein-kinin system only facilitated development of DOCA-salt hypertension, which we could not confirm in mice that lacked B2 kinin receptors.30 Our results indicate that blocking the B2 receptor with icatibant does not increase MBP in DOCA-salt hypertension but does blunt the hypotensive effect of ACEi, which confirms Carretero’s findings.31 This may indicate that kinins play a role in the antihypertensive effect of ACEi in DOCA-salt hypertension, in which kallikrein-kinin and ACE activity are reportedly increased.32 However, the role of kinins in the long-term antihypertensive effect of ACE inhibitors in other models of hypertension remains controversial.7 8 30 33
We conclude that kinins do not oppose the pressor effect of a chronic infusion of Ang II or a high salt diet; thus, results of the present study fail to confirm the data of Majima et al9 10 11 or Maddedu et al14 15 for reasons that remain unclear. Although kinins did not regulate BP, they did mediate the beneficial effect of ACEi in DOCA-salt hypertensive rats.
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Acknowledgments |
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We wish to express our deep appreciation to Dr Masataka Majima for trying to help us to resolve the discrepancies between our data and theirs. This study was supported by NIH grant HL-28982.
Received September 16, 1999; first decision October 13, 1999; accepted July 27, 2000.
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1. Suzuki H, Bouhnik J, Alhenc-Gelas F, Corvol P, Ménard J. Direct radioimmunoassay for rat high molecular weight kininogen: measurement of immunoreactive high molecular weight kininogen in normal and deficient plasma. Agents Actions Suppl.. 1987;22:277–287.[Medline] [Order article via Infotrieve]
2. Carretero OA, Scicli AG. Local hormonal factors (intracrine, autocrine, and paracrine) in hypertension. Hypertension. 1991;18(suppl I):I-58–I-69.
3.
Benetos A, Gavras I, Gavras H. Hypertensive effect of
a bradykinin antagonist in normotensive rats.
Hypertension. 1986;8:1089–1092.
4. Carbonell LF, Carretero OA, Stewart JM, Scicli AG. Effect of a kinin antagonist on the acute antihypertensive activity of enalaprilat in severe hypertension. Hypertension. 1988;11:239–243.[Abstract]
5.
Danckwardt L, Shimizu I, Bönner G, Rettig R,
Unger T. Converting enzyme inhibition in kinin-deficient Brown Norway
rats. Hypertension. 1990;16:429–435.
6. Bao G, Gohlke P, Unger T. Role of bradykinin in chronic antihypertensive actions of ramipril in different hypertension models. J Cardiovasc Pharmacol. 1992;20(suppl 9):S96–S99.
7. Linz W, Schölkens BA. A specific B2-bradykinin receptor antagonist HOE 140 abolishes the antihypertrophic effect of ramipril. Br J Pharmacol. 1992;105:771–772.[Medline] [Order article via Infotrieve]
8.
Rhaleb N-E, Yang X-P, Scicli AG, Carretero OA. Role of
kinins and nitric oxide in the antihypertrophic effect of ramipril.
Hypertension. 1994;23:865–868.
9.
Majima M, Katori M, Hanazuka M, Mizogami S, Nakano T,
Nakao Y, Mikami R, Uryu H, Okamura R, Mohsin SSJ, Oh-Ishi S.
Suppression of rat deoxycorticosterone-salt hypertension by
kallikrein-kinin system. Hypertension. 1991;17:806–813.
10.
Majima M, Yoshida O, Mihara H, Muto T, Mizogami S,
Kuribayashi Y, Katori M, Oh-Ishi S. High sensitivity to salt in
kininogen-deficient Brown Norway Katholiek rats.
Hypertension. 1993;22:705–714.
11.
Majima M, Mizogami S, Kuribayashi Y, Katori M, Oh-Ishi
S. Hypertension induced by a nonpressor dose of angiotensin
II in kininogen-deficient rats. Hypertension. 1994;24:111–119.
12.
Madeddu P, Parpaglia PP, Demontis MP, Varoni MV,
Fattaccio MC, Tonolo G, Troffa C, Glorioso N. Bradykinin
B2-receptor blockade facilitates
deoxycorticosterone-salt hypertension. Hypertension. 1993;21:980–984.
13. Madeddu P, Anania V, Parpaglia PP, Demontis MP, Varoni MV, Fattaccio MC, Glorioso N. Chronic kinin receptor blockade induces hypertension in deoxycorticosterone-treated rats. Br J Pharmacol. 1993;108:651–657.[Medline] [Order article via Infotrieve]
14.
Madeddu P, Parpaglia PP, Demontis MP, Varoni MV,
Fattaccio MC, Glorioso N. Chronic inhibition of bradykinin
B2-receptors enhances the slow vasopressor
response to angiotensin II. Hypertension. 1994;23:646–652.
15. Madeddu P, Glorioso N, Maioli M, Demontis MP, Varoni MV, Anania V, Xiong W, Chai K, Chao J. Regulation of rat renal kallikrein expression by estrogen and progesterone. J Hypertens. 1991;9(suppl 6):S244–S245.
16.
Erdös EG. Angiotensin I converting
enzyme. Circ Res. 1975;36:247–255.
17.
Campbell DJ, Kladis A, Duncan A-M. Effects of
converting enzyme inhibitors on angiotensin and
bradykinin peptides. Hypertension. 1994;23:439–449.
18. Liu Y-H, Yang X-P, Sharov VG, Nass O, Sabbah HN, Peterson E, Carretero OA. Effects of angiotensin-converting enzyme inhibitors and angiotensin II type 1 receptor antagonists in rats with heart failure: role of kinins and angiotensin II type 2 receptors. J Clin Invest. 1997;99:1926–1935.[Medline] [Order article via Infotrieve]
19.
Yang X-P, Liu Y-H, Scicli GM, Webb CR, Carretero OA.
Role of kinins in the cardioprotective effect of preconditioning: study
of myocardial ischemia/reperfusion injury in
B2 kinin receptor knockout mice and
kininogen-deficient rats. Hypertension. 1997;30:735–740.
20. Allen TA, Wilke WL, Fettman MJ. Captopril and enalapril: angiotensin-converting enzyme inhibitors. J Am Vet Med Assoc. 1987;190:94–96.[Medline] [Order article via Infotrieve]
21.
Hayashi I, Hoshiko S, Makabe O, Oh-Ishi S. A point
mutation of alanine 163 to threonine is responsible for the defective
secretion of high molecular weight kininogen by the liver of Brown
Norway Katholiek rats. J Biol Chem. 1993;268:17219–17224.
22. Holm S. A simple sequentially rejective multiple test procedure. Scand J Stat. 1979;6:65–70.
23. Hayashi I, Maruhashi J, Oh-Ishi S. Functionally active high molecular weight-kininogen was found in the liver, but not in the plasma of Brown Norway Katholiek rat. Thromb Res. 1989;56:179–189.[Medline] [Order article via Infotrieve]
24. Lattion AL, Baussant T, Alhenc-Gelas F, Seidah NG, Corvol P, Soubrier F. The high-molecular-mass kininogen deficient rat expresses all kininogen mRNA species, but does not export the high-molecular-mass kininogen synthesized. FEBS Lett. 1988;239:59–64.[Medline] [Order article via Infotrieve]
25.
Alfie ME, Sigmon DH, Pomposiello SI, Carretero OA.
Effect of high salt intake in mutant mice lacking
bradykinin-B2 receptors. Hypertension. 1997;29:483–487.
26.
Cervenka L, Harrison-Bernard LM, Dipp S, Primrose G,
Imig JD, El-Dahr SS. Early onset salt-sensitive hypertension in
bradykinin B2 receptor null mice.
Hypertension. 1999;34:176–180.
27.
Madeddu P, Varoni MV, Palomba D, Emanueli C, Demontis
MP, Glorioso N, Dessi-Fulgheri P, Sarzani R, Anania V.
Cardiovascular phenotype of a mouse strain with
disruption of bradykinin B2-receptor gene.
Circulation. 1997;96:3570–3578.
28. Hayashi I, Oh-Ishi S, Kato H, Enjyoji K, Iwanaga S, Nakano T. Identification of T-kininogen in high and low molecular weight kininogens deficient rat (Brown Norway Katholiek strain). Thromb Res. 1985;39:313–321.[Medline] [Order article via Infotrieve]
29. Katori M, Majima M, Mohsin SSJ, Hanazuka M, Mizogami S, Oh-Ishi S. Essential role of kallikrein-kinin system in suppression of blood pressure rise during the developmental stage of hypertension induced by deoxycorticosterone acetate-salt in rats. Agents Actions. 1992;38:235–242.
30.
Rhaleb N-E, Peng H, Alfie M, Shesely EG, Carretero OA.
Effect of ACE inhibitor on DOCA-salt- and aortic
coarctation-induced hypertension in mice: do kinin
B2 receptors play a role?
Hypertension. 1999;33:329–334.
31. Carretero OA. High-mineralocorticoid conditions: kinins (paracrine hormones) in the regulation of renal function and blood pressure. In: Mornex R, Jaffiol C, Leclère J, eds. Progress in Endocrinology: The Proceedings of the Ninth International Congress in Endocrinology, Nice 1992. London, England: Parthenon; 1993:536–540.
32.
Nakagawa M, Nasjletti A. Plasma kinin
concentration in deoxycorticosterone-salt hypertension.
Hypertension. 1988;11:411–415.
33. Sugimoto K, Fujimura A. Role of bradykinin in the reduction of left ventricular hypertrophy induced by angiotensin-converting enzyme inhibitors in spontaneously hypertensive rats. Jpn J Pharmacol. 1998;76:431–434.[Medline] [Order article via Infotrieve]
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