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The Kallikrein-Kinin System

21

2. Acquired diseases
2.1 Sepsis

Although the pathophysiology of sepsis remains

largely unknown, experimental and clinical data point to
a role of kinins in sepsis. First, in vitro, different
bacterial strains responsible for sepsis interact with the
contact system of plasma leading to the release of BK.
In vivo, the factors of the contact system are consumed
in plasma of patients suffering from sepsis. Recently,
a significant increase of plasma BK was also measured
in plasma of patients suffering from 

S. aureus

 sepsis

while simultaneously treated with an ACEi (210). On the
other hand, the injection of LPS from 

E. Coli

 into the

dorsal skin of rats caused a dose-dependent increase in
vascular permeability and this increase caused by LPS
was attenuated by pretreatment with the B2R antagonist
HOE 140 (211). These observations could open a new
area for the clinical application of B2R antagonists.
However, and until now, the behavior of B1R agonists
(des-Arg

9

-BK and des-Arg

10

-KD) in septic patients and

the effect of sepsis on the kinin forming capacity of
fibrinolysis have not been documented.

Another therapeutic approach of sepsis could be the

use of the pentapeptide Arg-Pro-Pro-Gly-Phe, the stable
metabolite of BK (212). In fact, recent studies have
shown that this pentapeptide is able to increase signifi-
cantly the survival of rats treated with LPS (213) and to
inhibit the thrombin-induced platelet aggregation (214).

2.2 Anaphylactoid and severe hypotensive reactions

Contact system activation is also responsible for the

anaphylactoid reaction (AR) in hemodialysis and severe
hypotensive reaction (SHR) during blood product
transfusion in patients simultaneously treated with an
ACEi (see below) (215, 216).

3. Antithrombolytic treatment and kinins

A complex and dual relationship exists between the

fibrinolysis and the kinin system.

The application of recombinant technology has

allowed large-scale production of t-PA and its use
for thrombolytic therapy. Wide use of recombinant t-PA
(rt-PA) for thrombolysis in patients with myocardial
infarction stimulated intensive studies of structure func-
tion relationships of t-PA and development of mutant
forms of t-PA with improved pharmacokinetic pro-
perties, that is, with prolonged lifetime in circulation
and increased resistance to inhibitors and to cleavage by
plasmin (46). However, by stimulating the plasmin
formation, thrombolytic drugs not only dissolve the
clot but also activate factor XII, the complement cascade
and the kinin system (20, 217). In vitro, rt-PA at a thera-
peutic concentration generates significant quantities of

BK and des-Arg

9

-BK from human plasma, and this

kinin-forming activity depends on the activation of
plasmin which hydrolyzes HK, independently of the
activation of factor XII and PKK (47).

In addition, BK stimulates the exocytosis of t-PA

from the vascular endothelium. Studies using the B2R
antagonist HOE 140 indicate that ACEi also increase
endogenous t-PA release through a B2R-dependent
pathway (218). This potentiating effect of ACEi on
fibrinolysis constitutes a new aspect of the cardio-
protection by ACEi.

II- The Metabolism of Kinins

Until now, among the kinin metabolizing enzymes,

only ACE has been a clinically exploited pharmaco-
logical target. However, the effectiveness of this class of
metallopeptidase inhibitors has opened research in the
development for more potent inhibitors that inhibit two
metallopeptidases, namely ACE and NEP.

1. ACE: pathophysiology

The broad spectrum of substrates for ACE and its

wide distribution throughout the body indicates that
this enzyme, in addition to an important role in cardio-
vascular homeostasis, may be involved in additional
physiologic processes such as neovascularization,
fertilization, atherosclerosis, kidney and lung fibrosis,
myocardial hypertrophy, inflammation, and wound
healing (66). The deleterious effects of ACE on the
cardiovascular system were initially thought to be a
consequence of the formation of Ang II, which initiates
a cascade of events involving increased free radical
production and vascular smooth muscle cell prolifera-
tion (79). However, as BK is much more readily hydro-
lyzed by ACE than Ang I, the hydrolysis of BK may also
contribute to this phenomenon (219).

ACE2 has been implicated in cardiovascular patho-

logy, and the generation of ACE2-knockout mice
revealed that the enzyme is an essential regulator of
heart function (67, 220).

2. ACE inhibitors (ACEi)

The inhibitors of ACE have emerged as a first-line

therapy for a range of cardiovascular and renal diseases,
including hypertension, congestive heart failure, myo-
cardial infarction, and diabetic nephropathy. The first
clinically used ACEi was captopril (

K

i

=

1700 pM).

Numerous other more potent dipeptide and tripeptide
inhibitors, which bind to both catalytic sites in somatic
ACE have been synthesized subsequently for clinical
use in humans (61).

The inhibition of ACE activity is reported to improve

ME Moreau et al

22

endothelial function and to stimulate vascular remodel-
ing, as well as attenuate the progression of arterio-
sclerosis and the occurrence of cardiovascular events in
humans (221, 222). The identification of ACE as a
signalling molecule that can be activated by the binding
of ACEi may account for some of the beneficial effects
of this class of compounds on the cardiovascular system.

Since their discovery, a series of large, multicentre

clinical randomized trials have definitively established
the very important role of ACEi in cardiovascular
medicine (223).

2.1 Multicenter clinical randomized trials

Numerous clinical trials have established the role

of ACEi in ischemic heart disease, particularly after
acute myocardial infarction (MI). Early trials (SAVE,
TRACE, AIRE) focused on patients with left ventricular
dysfunction (224 – 226). More recent trials (HOPE,
PROGRESS, EUROPA) have demonstrated a benefit
of ACEi in preventing cardiovascular events even in
patients with normal ejection fraction.

The HOPE study, an international randomized trial,

showed that ramipril (

K

i

=

7 pM) was beneficial in a

broad range of patients without evidence of left ventri-
cular systolic dysfunction or heart failure who are at
high risk for cardiovascular events (222). The EUROPA
study extended the findings of HOPE to a group of
lower risk patients with coronary artery disease
frequently seen in clinical practice. PROGRESS demon-
strated that lowering blood pressure with an ACEi and
diuretic reduced strokes and cardiovascular events in
stroke patients. While some of the effect of ACEi may be
attributable to blood pressure lowering, the mortality
benefit is unequivocal. In addition, ACEi prevents the
progression of renal injury in diabetes (227) and a
number of diseases and reduces death due to MI in
diabetic population (228). Taken together these studies
present strong evidence that patients with evidence of
stable coronary heart disease, vascular disease, and

/

or

diabetes (plus one further risk factor), regardless of left
ventricular function, should be treated with an ACEi.
The challenge now is to translate this evidence-based

therapeutic management into cardiological practice (229).

The mechanism by which ACEi reduce cardio-

vascular mortality is the subject of intense investigation.
ACEi have been shown to improve endothelial function,
a marker of future cardiovascular events. While some
investigators have suggested that ACEi with higher
tissue affinity for somatic ACE may have a greater
impact on cardiovascular mortality, no comparative
trials have been conducted.

2.2 Role of BK in the cardiovascular effects of ACEi

The evidence for a contribution of kinins (mainly

BK) in the cardiovascular effects of ACEi is essentially
of pharmacological nature. These results have been
extensively reviewed elsewhere (230). In various experi-
mental models, in vitro, ex vivo, and in vivo, ACEi
mimic and potentiate the pharmacological effects of
BK, which can be suppressed by a B2R antagonism,
mainly by HOE 140 (Icatibant). These observations
have been made using cell models, where an ACEi
stimulates the NO and prostacyclin (PGI

2

) production

triggered by BK. In vivo, Icatibant has been shown to
suppress the antihypertensive, antihypertrophic, and
antiproliferative effects of ACEi to a variable extent,
depending on the experimental model. The effect on the
antihypertensive effects of ACEi is more controversial
and depends on the experimental model. In hypertensive
subjects, a short-term hypotensive effect of captopril
was abolished at least in part by Icatibant (231). Several
but not all studies indicate that BK contributes to the
effects of chronic ACEi in patients with congestive heart
failure.

The contribution of endogenous kinins in these

pharmacological effects is more controversial, and it is
still not clear, at the present time, which part is due to
the B1R and B2R agonists in the pharmacological
effects. In fact, depending on the animal model, ACEi
potentiate either the concentration of BK or des-Arg

9

-

BK. These apparent discrepanties could be the result
of varying importance of other metallopeptidases
responsible of the metabolism of kinins (72).

3. Vasopeptidase inhibitors (VPi)

The VPis possess the ability to inhibit simultaneously

two membrane-bound zinc metalloproteases, ACE and
NEP, with similar nanomolar inhibitory constants (232).
Omapatrilat was the first of this new class of drugs.
This dual inhibitor has been evaluated clinically for
the treatment of hypertension, heart failure, and renal
disease.

3.1 Multicenter clinical randomized trials

Preclinical (233) and early clinical studies conducted

with omapatrilat were very promising. Indeed, omapatri-
lat appeared to be a potent antihypertensive agent with
favorable effects on cardiac function in heart failure
patients (234, 235). In contrast to these early studies,
the large clinical trials were more disappointing.

The results of the Omapatrilat Cardiovascular Treat-

ment Versus Enalapril (OCTAVE) trial suggested that
use of a more efficacious monotherapy such as
omapatrilat can result in greater blood pressure reduc-
tions, but at the price of an AE rate more than threefold

The Kallikrein-Kinin System

23

higher than that of an ACEi in the overall population
(see below) (236).

In The Omapatrilat Versus Enalapril Randomized

Trial of Utility in Reducing Events (OVERTURE), the
patients with chronic heart failure and hypertension
treated with omapatrilat had a reduced morbidity and
mortality when compared with enalapril. AE was also
more common with omapatrilat (236 – 238).

3.2 Role of kinins in the cardiovascular effects of VPi

The protective effect of omapatrilat on BK degrada-

tion on both cardiomyocytes and endothelium, two
target sites for metallopeptidase inhibitors, has also been
compared to that of ACEi (239 – 241). These studies
have shown the complexity of the different metabolic
pathways, whose importance varies according to the
tissue but also the nature of the pathophysiological
background. As an example, Fig. 4 illustrates the
influence of insulin on the protective effect of an ACEi
and a VPi on the cardiac metabolism of BK in experi-
mental type I diabetes. Hence, BK may also be an
important mediator of this new class of drugs. The
effects of a single inhibitor of both ACE and NEP,
resulting in an indirect activation of the B2R, could be
responsible for additional beneficial therapeutic effects

of VPi such as omapatrilat (242, 243).

4. Kinins and side effects of metallopeptidase inhibitors

Despite their clinical effectiveness, ACEi can cause

chronic and acute side effects. The best known chronic
side effect is a non-productive cough. Although some
experimental and clinical evidences suggest a role of
BK in the pathophysiology of this effect, no definitive
arguments exist for such a role.

The nature of acute side effects of ACEi depends on

the clinical context. Angioedema has been reported in
hypertension and heart failure, but also in stroke patients
during fibrinolysis with rt-PA (244). AR occur in
patients dialysed with a negatively charged membrane
(119). Finally, SHR have been described during blood
product transfusion (216). In these cases, the majority of
the reactions occurred during transfusion of blood
components administered through a negatively charged
bedside leukocyte reduction filter. A similar reaction
also occurs during LDL and plasmapheresis (245).

The incidence of these acute side effects has

traditionally been around 0.1% to 0.2%. However, this
incidence must be revised in light of the OCTAVE study
(236, 246). This study has shown an incidence of AE
with enalapril equal to 0.68% in Caucasian hypertensive
patients. This incidence was still higher in African
Americans (1.62%) and in smokers (0.81%), whereas
patients with diabetes had lower rates of AE. This
study showed an increased incidence of AE associated
with omapatrilat in Caucasians (2.17%), African
Americans (5.54%), and smokers (3.93%). These results
are important because they highlight 3 factors: the
presence of inflammation (smokers), the genetic aspect
(African Americans), and the nature of the metallo-
peptidase inhibitor that inhibits one or two peptidases,
specifically or non-specifically.

In fact, acute side effects of ACEi and, by extension,

of other metallopeptidase inhibitors (VPi) could result
in the meeting of at least 3 different factors. The first
one is the presence of a drug that inhibits specifically
ACE (or ACE plus NEP) but could also non-specifically
inhibit other enzymes involved in the metabolism of
kinins, such as APP or CPN. The second factor may be a
pathophysiological or physicochemical trigger for the
release of kinins. It has been identified as the negatively
charged surface in AR and as the negatively charged
filter in SHR. The triggering factor remains unknown in
AE but could be linked to the inflammatory status, as
suggested by the higher incidence in smokers. The third
factor could be intra- or inter-individual genetic or
environmental factors that effect non-ACE inhibitor
mediated pathways metabolizing vasoactive peptides
such as the kinins.

Fig. 4.

Influence of insulin on the cardiac metabolism of exogenous

BK in experimental type I diabetes. The half-life of exogenous BK
incubated with heart membranes from Sprague-Dawley control rats
(CTL), streptozotocin (STZ)-treated rats 8 weeks post-injection and
STZ-treated rats before and after (7 days) insulin administration
(+INS-1 week). Membranes were incubated with exogenous BK
without enzymatic inhibitor (shade) or in presence of enalaprilat
(grey) or omapatrilat (black). Values are means

±

S.E.M. *

P

<

0.05

versus without inhibitor; 

†

P

<

0.05 versus enalaprilat. (Reproduced

with permission from P. Leclair, MSc Thesis, p. 79, Université de
Montréal, 2000)

ME Moreau et al

24

We have investigated the metabolism of endogenous

kinins from the plasma of patients who developed AE
while being treated with an ACEi. While we found no
abnormality in the metabolism of BK, Caucasian
patients with a history of AE exhibited slower degrada-
tion of des-Arg

9

-BK, correlating with a decreased

activity of plasma APP (247, 248). This anomaly could
also be observed in patients who presented an AR (119)
or a SHR (245, 249). We hypothesized that the accumu-
lation of the B1R agonist could be responsible for
the symptoms that characterize AR and SHR, but our
observations cannot be related to the local inflammatory
reaction that characterizes AE.

As no inhibitor of APP is present in plasma (119), we

investigated the genetic aspect of AE. A recent report
provides evidence that variable APP activity in humans
is partially regulated by genetic factors. From data
ascertained from 8 Caucasian kindreds whose probant
suffered either an AE whilst being treated with an
ACEi, we estimated that 34% of the phenotypic varia-
tion results from genetic differences in the linkage
analysis. We described two sequence variations, a
nonsense mutation resulting in a truncated protein and a
potentially regulatory single nucleotide polymorphism
(SNP) that segregated with reduced plasma APP acti-
vity, suggesting an increased susceptibility to ACEi
associated acute side-effect (250). These findings do
not mean that this active metabolite, des-Arg

9

-BK, is

necessarily the only mediator of AE; a local release of
neurokinins or a decrease in DPP IV activity (substance
P-degrading enzyme) seems to be associated with this
potentially life-threatening side effect (251), particularly
in African Americans. The precise mechanism respon-
sible for ACEi-associated AE is not known.

III- Kinin Receptors

1. Receptor polymorphisms and pathology

The potential role of kinin receptors in human

disease is derived from studies evaluating kinin
receptor polymorphisms, measurement of differences
in kinin receptor expression level, and detection in
differences in receptor functions. However, reports
concerning a correlation between SNP in kinin receptor
genes and complex pathologies should be carefully
interpreted. Usual analysis involves a statistical com-
parison between case and control populations. Neverthe-
less, this method does not always take in account the
influence of a nearby gene rather than the gene of
interest or the possibility of a true relationship between
a SNP with a disease which were not at first directly
correlated. The specific situation of the kinin receptor
locus is particularly problematic in this respect: as both

kinin receptor genes lay very close to each other in the
same locus, a genetic marker in one of them may
point out to a functional alteration of the other; there
is some experimental support for this idea (252).

Further pharmacological studies, appropriate family-based

designs, or case-control designs analyzing haplotypes
rather than single SNP (253) should also be used to
demonstrate the predicted interrelation.

Many polymorphisms of B2R and B1R have been

described (254). Some of them have been related to
pathophysiological events.

1.1 Polymorphism of B2Rs

Left ventricular hypertrophy and cardiomyopathy are

some of the adverse effects of hypertension. These
pathologies have been linked to kinin receptors (255).

1.1.1 +9 /

−

9, exon 1 polymorphism

A +9

/

−

9 exon 1 polymorphism of the B2R was

strongly associated with the left ventricular growth
response among normotensive white males undergoing a
ten-week physical training program. Individuals with the
lowest levels of B2R (+9

/

+9 genotype) and the highest

levels of ACE (DD genotype) had the greatest increase
in left ventricular mass (256). Subjects with B2R +9

/

+9

genotype showed less left ventricular mass regression
compared with other genotypes. B2R (+9

/

−

9) poly-

morphism was also shown to be significantly associated
with higher skeletal muscle metabolic efficiency and
with endurance in athletic performance (257).

The (

−

9

/

−

9) genotype of B2R exon 1 was also

examined for the contractile response of human umbili-
cal veins to stimulation with kinins (252). This poly-
morphism was associated with increased contractile
efficiency of the B1R agonist, suggesting that the B2R
gene may be in linkage disequilibrium with the B1R
gene.

Cardiovascular risk and increase in blood pressure

associated with hypertension are also influenced by the
presence of B2R (+9) allele, an effect not identified
in individuals homozygous for B2R (

−

9) (258). This

suggests a role of B2R in human coronary vascular
disease.

B2R (+9

/

−

9) polymorphism was significantly asso-

ciated with diabetic nephropathy (259).

The B2R (

−

9) allele has been associated with the

most symptomatic cases of C1INH deficiency (here-
ditary angioedema with angioedema crises) and thus is
proposed to modulate in a dominant manner the pheno-
type of the basic genetic defect of this disorder (130).
However, this polymorphism did not predict the
incidence of the most common side effect of ACEi,
the non-productive cough (260).

The Kallikrein-Kinin System

25

1.1.2 (C

−

58

→

T), promoter region polymorphism

Several studies have detected a significant association

between the B2R (C

−

58

→

T) polymorphism and hyper-

tension. A consistent finding that the C

−

58

 allele has

higher frequency in hypertensive individuals compared
to normotensive individuals has been reported in various
populations (261 – 263). The allele frequencies are
similar in Caucasians and Afro-American populations,
whereas the T allele is slightly more frequent in the
Japanese.

The genotypic and allelic frequencies of (C

−

58

→

T)

polymorphism of general hypertensive subject were
analyzed (264). The frequencies of the TT genotype
and the T allele of (C

−

58

→

T) are higher in the subjects

with cough than in subjects without cough. Moreover,
T

−

58

 was found to have a higher transcription rate than

that of C

−

58

. The high transcriptional activity of the

B2R promoter might be involved in the occurrence of
ACEi-related cough.

1.1.3 (C

181

→

T), exon 2 polymorphism

Presence of the nonsynonymous coding region B2R

exon 2 (C

181

→

T) was associated with increased

contractile potency in response to stimulation with
BK consistent with the enhanced in vitro potency of BK
at the Arg

14

→

Cys mutant receptor (252) and with

significantly lower systolic and diastolic blood pressure
(265).

1.2 Polymorphisms of B1Rs: (G

−

699

→

C) substitution

Cardiovascular risk and increase in blood pressure

associated with hypertension are influenced by the
presence of B1R (G

−

699

 substitution) allele, an effect

not identified in the presence of the homozygous B1R
(C

−

699

 substitution) allele (258). This suggests a role of

B1R in human coronary vascular disease.

The B1R polymorphism consisting of a single base

substitution (G

−

699

→

C) in a positive control region of

the promoter exhibits an altered frequency in patients
with end-stage renal failure (266). A deficit of the C
allele was observed among diseased patients and in
some etiologic groups (polycystic kidneys, pyelone-
phritis, and interstitial nephritis) compared with healthy
volunteers. This polymorphism may be a marker of
prognostic significance for the renal function in diseased
individuals. Profound alteration of allele frequencies
were found in patients with inflammatory bowel disease
such as ulcerative colitis or Crohn’s disease in a small
case-control study (267). Thus, the B1R promoter gene
does not have a specific etiologic influence but rather
modifies a downstream inflammatory pathway common
to these disorders.

2. Kinin receptors as pharmacological targets

Roles for the kallikrein-kinin system in inflammation

have been investigated and reviewed extensively (268).
As a great number of disease states such as chronic
inflammatory pain, edema, asthma, and sepsis have their
basis in the inflammatory response, the development of
novel antagonist drugs targeted at the B1R and B2R
provides a novel therapeutic opportunity. The clinical
development of these drugs is at an early stage, with few
human clinical studies reported until now and mainly
based on peptide compounds (269 – 272). These charged
and rather large molecules may not show the full
therapeutic effect of kinin receptor blockade due to
limitations in oral bioavailability, distribution, and
stability. The potential therapeutic applications of
kinin receptor ligands (not always antagonists) include
cardiovascular and renal disorders, inflammation, pain,
diabetes, asthma, and perhaps cancer.

2.1 Cardiovascular and renal diseases

BK is known for its multiple effects on the cardio-

vascular system and particularly by its vasodilatation
and plasma extravasation properties (273), leading to an
inflammatory response. Vasodilatation is normally
mediated by B2R (274), but under inflammatory condi-
tions, B1R up-regulation mediates kinin-induced vaso-
dilatation and hypotension (275, 276). BK-related
peptides act as vasodilators through endothelial cells
from which secondary mediators are released to affect
the vascular smooth muscle. In humans, cardiovascular
actions of kinins are mainly correlated to preformed
B2R stimulation (leading to NO and PGI

2

 formation)

and the contribution of the B1R is not detectable
(130). NO is derived from 

L

-arginine by eNOS. NO

diffuses from the endothelium to the smooth muscle
where it activates guanylate cyclase. NO-independent
ion channels are also suspected to mediate endothelial-
dependent vasorelaxation. Prostacyclin is also released
by kinins from the endothelial cells, probably via the
cytosolic Ca

2+

-sensitive isoforms of PLA

2

 and stimulates

cAMP production in smooth muscle cells (277). These
physiological effects of kinins are potentially useful to
treat hypertension and ischemic disorders and to main-
tain renal function (as the kallikrein-kinin system plays a
role in handling salt excess).

The local generation of kinins or the inhibition of

their degradation and the resulting B2R stimulation
could be of interest to reduce blood pressure or to
promote cardioprotective effects (231, 278, 279).

On the other hand, B1R activation has been shown

to exert a protective effect after cardiac ischemia in
mice (280). The peptidase-resistant B1R agonist Sar-[

D

-

Phe

8

]des-Arg

9

-BK has been used to further stimulate