Download Design, Synthesis, and Actions of a Novel Chimeric Natriuretic Peptide

Journal of the American College of Cardiology
© 2008 by the American College of Cardiology Foundation
Published by Elsevier Inc.
Vol. 52, No. 1, 2008
ISSN 0735-1097/08/$34.00
doi:10.1016/j.jacc.2008.02.077
PRECLINICAL RESEARCH
Design, Synthesis, and Actions
of a Novel Chimeric Natriuretic Peptide: CD-NP
Ondrej Lisy, MD, PHD,* Brenda K. Huntley,* Daniel J. McCormick, PHD,† Paul A. Kurlansky, MD,‡
John C. Burnett, JR, MD*
Rochester, Minnesota; and Miami, Florida
Objectives
Our aim was to design, synthesize and test in vivo and in vitro a new chimeric peptide that would combine the
beneficial properties of 2 distinct natriuretic peptides with a biological profile that goes beyond native peptides.
Background
Studies have established the beneficial vascular and antiproliferative properties of C-type natriuretic peptide
(CNP). While lacking renal actions, CNP is less hypotensive than the cardiac peptides atrial natriuretic peptide
and B-type natriuretic peptide but unloads the heart due to venodilation. Dendroaspis natriuretic peptide is a
potent natriuretic and diuretic peptide that is markedly hypotensive and functions via a separate guanylyl cyclase receptor compared with CNP.
Methods
Here we engineered a novel chimeric peptide CD-NP that represents the fusion of the 22-amino acid peptide
CNP together with the 15-amino acid linear C-terminus of Dendroaspis natriuretic peptide. We also determined
in vitro in cardiac fibroblasts cyclic guanosine monophosphate-activating and antiproliferative properties of
CD-NP.
Results
Our studies demonstrate in vivo that CD-NP is natriuretic and diuretic, glomerular filtration rate enhancing, cardiac unloading, and renin inhibiting. CD-NP also demonstrates less hypotensive properties when compared with
B-type natriuretic peptide. In addition, CD-NP in vitro activates cyclic guanosine monophosphate and inhibits cardiac fibroblast proliferation.
Conclusions
The current findings advance an innovative design strategy in natriuretic peptide drug discovery and development to create therapeutic peptides with favorable properties that may be preferable to those associated with
native natriuretic peptides. (J Am Coll Cardiol 2008;52:60–8) © 2008 by the American College of Cardiology
Foundation
An emerging innovative therapeutic strategy is the engineering of proteins, which fuses structural elements unique
for 1 peptide with active structures from separate peptides to
create chimeras, which possess attractive therapeutic propSee page 69
erties (1). Such an approach with natriuretic peptides is
advanced in the current study and involves the selection and
incorporation of isolated structural determinants from 2
native peptides that result in a unique peptide with a specific
From the *Cardiorenal Research Laboratory, Division of Cardiovascular Diseases,
and †Laboratory of Expression Proteomics and Protein Chemistry, Mayo Clinic and
Mayo Clinic College of Medicine, Rochester, Minnesota; and the ‡Miami Heart
Research Institute, Miami, Florida. This study was supported by Mayo Foundation
and grants from the Miami Heart Research Institute and National Institutes of
Health (RO1HL36634, PO1HL76611, and R01HL8373). CD-NP has been licensed by the Mayo Clinic to Nile Therapeutics and Dr. Burnett is Chair of its
Scientific Advisory Board.
Manuscript received December 27, 2007; revised manuscript received February 19,
2008, accepted February 26, 2008.
activity profile that could be more useful than their natural
counterparts as therapeutic agents in cardiorenal disease.
C-type natriuretic peptide (CNP) is a 22-amino acid
(AA) peptide that shares structural homology with other
members of the natriuretic peptide family, which includes
the cardiac hormones atrial natriuretic peptide (ANP)
and B-type natriuretic peptide (BNP), all of which
function via well-characterized particulate guanylyl cyclase receptors (i.e., NPR-A for ANP and BNP; NPR-B
for CNP) and the second messenger cyclic 3=5= guanosine
monophosphate (cGMP) (2– 4). While possessing structural similarity, CNP is genetically distinct from ANP
and BNP. Also unlike ANP or BNP, CNP lacks a
COOH-terminus (C-terminus) AA extension, which
may explain, in part, its lack of natriuretic properties
(5,6). Since its discovery in 1990, we have learned that
CNP is principally an endothelial cell-derived peptide
(7–13). In isolated venous and arterial rings, CNP
activates NPR-B receptors in veins while ANP and BNP
bind to NPR-A receptors in both arteries and veins,
Lisy et al.
CD-NP: A Novel Chimeric Natriuretic Peptide
JACC Vol. 52, No. 1, 2008
July 1, 2008:60–8
which is consistent with the less hypotensive actions of
CNP as compared with ANP and BNP (14 –16).
In addition to preferential venodilating properties, studies
have also reported that CNP possesses more potent antiproliferative and collagen-suppressing properties in cardiac
fibroblasts (CFs) as compared with ANP and BNP (11).
Relevant to such fibro-inhibiting properties, studies have
shown that 14 days of continuous infusion of CNP in
rodents with acute myocardial infarction (AMI) markedly
attenuates ventricular dilation, cardiac fibrosis, and cardiomyocyte hypertrophy (17). The chronic infusion of CNP
was without any hypotensive actions.
In clear contrast to ANP and BNP, CNP lacks significant
natriuretic and diuretic actions when infused into humans.
This may explain its lack of utility in sodium- and waterretaining syndromes such as acute heart failure (AHF)
despite its attractive venodilating and antifibrotic properties
(15,16).
In 1992, Schweitz et al. (18) reported the in vitro
biological actions of a newly discovered peptide Dendroaspis
natriuretic peptide (DNP). We subsequently reported that
DNP, which was originally isolated from the green mamba,
was in vivo potently natriuretic and diuretic and possessed
cardiac unloading actions but with significant hypotensive
properties (19,20). In the original report of the vasoactive
actions of DNP, Schweitz et al. (18) concluded that DNP,
like ANP and BNP, functions via the NPR-A receptor as
saturation with ANP markedly attenuated the cGMPactivating actions of DNP in cultured human endothelial
cells. Indeed, the in vivo actions of DNP that include
natriuresis and hypotension are consistent with NPR-A
activation as such effects closely mimic the properties of
ANP and BNP but not CNP. Indeed, Singh et al. (21)
recently have reported that DNP has a higher affinity for the
NPR-A receptor in human myocardium as compared with
ANP and BNP.
A key structural feature of DNP is that it possesses the
longest C-terminus of the known natriuretic peptides consisting of 15 AA as compared with 5 AA for ANP, 6 AA for
BNP and none for CNP. Indeed, the long C-terminus of
DNP may render DNP highly resistant to degradation by
neutral endopeptidase (NEP) contributing to potent natriuretic and diuretic actions (22). Further, the lack of a
C-terminus for CNP may explain the observation that of
the 3 known endogenous natriuretic peptides CNP is the
most susceptible to NEP degradation, which could limit its
renal actions as NEP is expressed greatest in the kidney (23).
Based upon exploratory studies of the 15-AA C-terminus
of DNP, we hypothesized that fusion of the 15-AA
C-terminus of DNP into the C-terminus position of the
core 22-AA ring structure of CNP would result in a
synthetic chimeric peptide that in vivo would possess the
cardiac unloading actions of CNP with minimal hypotensive properties together with the additional renal effects of
natriuresis and diuresis. We also hypothesized that this
chimera called CD-NP would retain properties of CNP in
vitro in activating cGMP in CFs
and inhibiting cell proliferation.
Thus, we report the design, protein synthesis, and biological actions of a chimeric natriuretic
peptide with potentially beneficial efficacy and safety for the
treatment of cardiorenal diseases
states such as AHF and AMI.
Methods
61
Abbreviations
and Acronyms
AA ⴝ amino acid
AHF ⴝ acute heart failure
AMI ⴝ acute myocardial
infarction
ANP ⴝ atrial natriuretic
peptide
BNP ⴝ B-type natriuretic
peptide
BrdU ⴝ bromodeoxyuridine
CF ⴝ cardiac fibroblast
We synthesized 2 peptides based
upon the known sequence of DNP
cGMP ⴝ cyclic 3=5=
guanosine monophosphate
and CNP. First we synthesized the
CNP ⴝ C-type natriuretic
linear 15-AA C-terminus sepeptide
quence (PSLRDPRPNAPSTSA)
DNP ⴝ Dendroaspis
of DNP (Fig. 1). We call this
natriuretic peptide
peptide “C-terminus.” Secondly,
GFR ⴝ glomerular filtration
we designed and synthesized a chirate
mera of CNP and the C-terminus
NEP ⴝ neutral
of DNP (GLSKGCFGLKLendopeptidase
DRIGSMSGLGCPSLRDPRPNMP ⴝ
NAPSTSA), which is a 37-AA
1-methyl-2-pyrrolidinone
peptide that we call “CD-NP.”
PFRNa ⴝ proximal
These 2 peptides were synthesized
fractional sodium
using solid phase methods by the
reabsorption
Mayo Protein Core Facility on an
PRA ⴝ plasma renin
ABI 431A Peptide Synthesizer
activity
(PE Biosystems, Foster City, California) on a pre-loaded Wang
resin with N-Fmoc-L-amino acids (SynPep, Dublin, California). Coupling of each AA to the resin-linked peptide was
performed in 1-methyl-2-pyrrolidinone (NMP) for 40 min.
Each Fmoc AA was activated in a solution of
1-hydroxybenzotriazole in NMP for 30 min. Deprotection of
the Fmoc protecting group was performed with 20% piperidine in NMP for 20 min before coupling of the next activated
AA. Subsequently, peptides were deprotected and removed
from the resin by treatment with a mixture of 82.5% trifluoroacetic acid/5% water/5% thioanisole/2.5% ethanedithiol/5%
phenol for 2 h at room temperature. Each peptide was washed
by precipitation in 3 ⫻ 50 ml of cold methyl t-butyl ether and
purified by reverse phase high performance liquid chromatography on a Jupiter C18 column (Phenomenex, Torrance,
California) in 0.1% trifluoroacetic acid/water with a
gradient of 10% to 70% B in 50 min.
The identity of each peptide was verified by electrospray
ionization mass analysis on a Perkin/Elmer Sciex API 165
Mass Spectrometer (PE Biosystems). Disulfide bridges in
CD-NP were formed by overnight air oxidation in a 50 mM
ammonium bicarbonate pH 8.5 buffer.
Integrative in vivo biological actions of C-terminus and
CD-NP in normal canines. Experiments were performed
in 5 separate groups of normal anesthetized dogs. All studies
conformed to the guidelines of the American Physiological
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Figure 1
Lisy et al.
CD-NP: A Novel Chimeric Natriuretic Peptide
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AA Sequence and Structure of a CNP and Novel Chimeric Natriuretic Peptide C-Terminus of DNP and CD-NP
C-type natriuretic peptide (CNP) is a 22-amino acid (AA) endothelial-cell-derived natriuretic peptide, C-terminus of
Dendroaspis natriuretic peptide (DNP) is a 15-AA chimeric natriuretic peptide, and CD-NP is a 37-AA-designed chimeric natriuretic peptide.
Society and were approved by the Mayo Clinic Animal Care
and Use Committee.
On the evening before the experiments, 300 mg of
lithium carbonate was administered orally for the assessment of segmental renal tubular function and then dogs
were fasted overnight. On the day of the acute experiment,
all dogs were anesthetized with pentobarbital sodium given
intravenously (30 mg/kg). Dogs were mechanically ventilated (Harvard respirator, Harvard Apparatus, Millis, Massachusetts) with 4 l/min of supplemental oxygen. A left
lateral flank incision was made, and the left kidney was
exposed. The ureter was cannulated for timed urine collection, and a calibrated electromagnetic flow probe was placed
around the left renal artery and connected to a flowmeter
(model FM 5010, Carolina Medical, King, North Carolina)
for monitoring of renal blood flow. Finally, the right
femoral vein was cannulated with 2 polyethylene catheters
(PE-240), 1 for infusion of inulin and the other for the
infusion of C-terminus. The right femoral artery was
cannulated for direct arterial blood pressure measurement
and arterial blood sampling. For determination of cardiac
filling pressures in those groups that received CD-NP or
BNP and measurement of cardiac output, a Swan-Ganz
catheter (Edwards, Mountain View, California) was inserted into the right internal jugular vein.
After completion of the surgical preparation, a priming
dose of inulin (ICN Biomedicals, Cleveland, Ohio) was
injected, followed by a constant infusion of 1 ml/min. The
dogs were allowed to equilibrate for 60 min without
intervention. After an equilibration period of 60 min, a
30-min baseline clearance (Baseline) was performed. This
was followed by a 15-min lead-in period during which
C-terminus infusion at 42 ng/kg/min in the first group (n ⫽
6) was begun intravenously after which the second 30-min
clearance (C-terminus) period was performed. A time
control with intravenous saline at 1 ml/min (n ⫽ 6) was
performed to serve as a control for the CD-NP group.
During each clearance, renal hemodynamic and excretory
response was recorded. In the third group, CD-NP was
infused at 3 concentrations that were 10, 50, and 100
ng/kg/min each for 30 min with a 15-min lead-in period for
each dose. Groups 4 (n ⫽ 6) and 5 (n ⫽ 6) received an
equimolar concentration of 10 and 50 ng/kg/min of
CD-NP or BNP, respectively.
Plasma and urine electrolytes including lithium were
measured by flame-emission spectrophotometer (IL943,
Flame Photometer, Instrumentation Laboratory, Lexington, Massachusetts). Plasma and urine inulin concentrations
were measured by the anthrone method, and glomerular
filtration rate (GFR) was measured by the clearance of
inulin. The lithium clearance technique was employed to
estimate the proximal fractional sodium reabsorption
(PFRNa) and distal fractional sodium reabsorption. Proximal fractional sodium reabsorption was calculated by the
following formula: [1 ⫺ (lithium clearance/GFR)] ⫻ 100.
Distal fractional sodium reabsorption was calculated by the
formula: [(lithium clearance ⫺ sodium clearance)/lithium
clearance] ⫻ 100. Plasma and urinary cGMP and plasma
renin activity (PRA) were measured by commercially available radioimmunoassay as described previously.
In vitro studies on CD-NP cGMP generation and
inhibition of cell proliferation in human CFs. Studies
were performed in human CFs (ScienCell, San Diego,
California) as we have previously reported (24). Only cell
passages 1 through 4 were used for experiments. Cells were
exposed to CD-NP (10⫺11 to 10⫺6 M) for 15 min to
determine cGMP activation. The samples were assayed
using a competitive radioimmunoanalysis cGMP kit
(Perkin-Elmer, Boston, Massachusetts). Briefly, samples
and standards were incubated with anti-human cGMP
polyclonal antibody and I125-antigen for 18 h. Cyclic GMP
assay buffer was added to the samples, and they were
Lisy et al.
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centrifuged for 20 min at 2,500 rpm. The free fraction was
aspirated off, and the bound fraction was counted and
concentrations determined. Values are expressed as pmoles/
ml. There was no cross-reactivity with ANP, BNP, CNP,
and endothelin, and ⬍0.001% cross-reactivity with cyclic
adenosine monophosphate, GMP, guanosine diphosphate,
adenosine triphosphate, and guanosine triphosphate.
For CFs proliferation studies, 70% to 80% confluent cells
at passage 1 to 4 cells were treated for 24 h with 10⫺8 M
cardiotrophin-1 to induce cell proliferation. CD-NP at the
concentration of 10⫺8 M was added to cardiotrophin-1stimulated CFs to determine its effect on cell proliferation.
Untreated fibroblasts were processed as a control. Colormetric bromodeoxyuridine (BrdU) cell proliferation
enzyme-linked immunoadsorbent assay (Roche, Indianapolis, Indiana) was performed as directed. Briefly, CFs were
labeled with BrdU for 2 h in a CO2 37°C incubator.
Antibromodeoxyuridine was added and allowed to react for
90 min at room temperature. The anti-BrdU was removed,
and the CFs were washed 3 times with a washing solution.
Colormetric substrate solution was added, and color was
allowed to develop for 30 min. Absorbance at 370 nm was
measured on a SpectraMax spectrophotometer (Molecular
Devices, Sunnyvale, California).
Statistical analysis. Results of quantitative studies are expressed as mean ⫾ SE. Statistical comparisons within 1
group when comparing 2 clearances was performed using
Student t test, and when comparing multiple clearances,
repeated measures analysis of variance (ANOVA) followed
by post hoc Dunnett’s test. Statistical comparison between
groups was performed using 2-way ANOVA followed by
Bonferroni post-test. Statistical significance was accepted
for a p value ⬍0.05.
Results
In vivo actions of C-terminus of DNP and CD-NP. The
C-terminus of DNP that is lacking the core ring structure of
DNP was natriuretic and diuretic (Fig. 2). We localized this
natriuretic action to the proximal segment of the renal
nephron as we observed a decrease in PFRNa suggesting
that this part of the nephron contributes to the renal actions
of this small peptide (Table 1). We noted no change in
GFR, renal blood flow, or urinary cGMP excretion. Importantly, in a time control group that also received saline at 1
ml/min as in the C-terminus group, parameters of renal
function were unchanged (data not shown).
Figure 3 reports the in vivo responses to CD-NP infusion. All 3 doses of CD-NP decreased pulmonary capillary
wedge pressure while 2 doses decreased right atrial pressure.
These cardiac unloading responses were associated with
minimal decreases in mean arterial pressure even during the
infusion of a high dose of CD-NP. We also observed potent
natriuretic and diuretic responses with CD-NP in normal
canines (Fig. 4). Specifically, both medium and high dose
CD-NP increased urinary excretion of sodium and urine
Figure 2
63
Effect of C-Terminus of DNP
on UNaV and UV in Normal Dogs
Data are expressed as means ⫾ SE. C-terminus, intravenous infusion of 42
ng/kg/min of C-terminus, n ⫽ 6. *p ⬍ 0.05 versus baseline. DNP ⫽ Dendroaspis natriuretic peptide; UNaV ⫽ urinary excretion of sodium; UV ⫽ urine
flow.
flow (Fig. 4). Despite a slight decrease in mean arterial
pressure during the infusion of a high dose of CD-NP,
GFR increased. There was no change in renal blood flow
during CD-NP infusion (240 ⫾ 40 ml/min [baseline] to
245 ⫾ 27 ml/min [dose-10] to 248 ⫾ 25 ml/min [dose-50]
to 253 ⫾ 22 ml/min [dose-100] and to 221 ⫾ 32 ml/min
[recovery]). Natriuresis and diuresis involved a decrease in
proximal tubular reabsorption of sodium as PFRNa during
the infusion of medium dose of CD-NP was reduced. In
addition, a decrease in distal fractional sodium reabsorption
during the infusion of medium and high doses of CD-NP
was also observed, which indicates that CD-NP also involves activities in the terminal nephron (Fig. 5). These
renal parameters returned to baseline levels after discontinuation of the infusion of CD-NP. These actions were also
associated with suppression of PRA during the infusion of
low and medium doses of CD-NP (Table 2). Plasma renin
activity returned to baseline levels after discontinuation of
infusion. Medium and high doses of CD-NP increased
plasma and urinary cGMP (Table 2).
At a dose of 50 ng/kg/min, CD-NP significantly increased GFR in contrast to a lack of increase in GFR in the
group receiving an equimolar dose of BNP (Fig. 6). Impor-
64
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CD-NP: A Novel Chimeric Natriuretic Peptide
JACC Vol. 52, No. 1, 2008
July 1, 2008:60–8
Renal Hemodynamic
Response
to Infusionand
of C-Terminus
Excretory
Renal Hemodynamic and Excretory
Table 1
Response to Infusion of C-Terminus
Baseline
C-Terminus
GFR (ml/min)
34 ⫾ 6
44 ⫾ 6
RBF (ml/min)
213 ⫾ 19
198 ⫾ 27
PFRNa (%)
77.9 ⫾ 2.7
71.0 ⫾ 2.6*
DFRNa (%)
98.7 ⫾ 0.3
97.8 ⫾ 0.5
1,674 ⫾ 213
2,275 ⫾ 231
UcGMPV (pmol/min)
Data are expressed as means ⫾ SE. ⴱp ⬍ 0.05 versus baseline.
C-terminus ⫽ infusion of 42 ng/kg/min of C-terminus of Dendroaspis natriuretic peptide (n ⫽ 6);
DFRNa ⫽ distal fractional sodium reabsorption; GFR ⫽ glomerular filtration rate; PFRNa ⫽
proximal fractional sodium reabsorption; RBF ⫽ renal blood flow; UcGMPV ⫽ urinary cyclic 3=5=
guanosine monophosphate excretion.
ring structure of CNP with the 15 AA C-terminus of DNP,
is potently natriuretic and diuretic and GFR enhancing,
which is in contrast to the reported lack of significant renal
actions of CNP (5,15,16). The natriuretic action of CD-NP
was localized to both the proximal and distal nephrons.
Contributing to the natriuretic and diuretic action of
CD-NP was an increase in GFR. The increase in GFR,
therefore, must be mediated by a decrease in pre-glomerular
vascular resistance together with an increase in postglomerular vascular tone to increase glomerular capillary
hydrostatic pressure while there was no significant change
in renal blood flow. Alternatively, an increase in the
glomerular ultrafiltration coefficient (Kf) could contribute
tantly, this dose of CD-NP was associated with a less
hypotensive action than BNP.
CD-NP–mediated cGMP generation in CFs and inhibition of proliferation. Figure 7 reports findings from in
vitro studies in cultured human CFs. These studies demonstrate that CD-NP activates cGMP in a dose-dependent
manner (Fig. 7A). Further, when compared with CNP,
BNP, and DNP, the cGMP generated with CD-NP was
similar to CNP and significantly greater (p ⬍ 0.05) than
DNP and BNP at 10⫺6 M (data not shown). We then
sought to define if CD-NP possessed antiproliferative
actions. We utilized the pro-fibrotic and hypertrophic
cytokine cardiotrophin-1 that has been reported by others
and ourselves as a markedly robust activator of CFs and
which is activated in states such as AMI and heart failure
(25–27). CD-NP suppressed cell proliferation in human
CFs induced by cardiotrophin-1 as assessed by BrdU uptake
as a measure of DNA synthesis and cellular proliferation
(Fig. 7B).
Discussion
Here we report the design, protein synthesis, and in vivo
cardiorenal actions of a chimeric natriuretic peptide that is a
fusion of the core 22-AA ring structure of CNP together
with the 15-AA C-terminus of DNP. Unlike CNP, which
lacks significant renal actions, CD-NP is potently natriuretic and diuretic, GFR enhancing, and PRA inhibiting.
CD-NP reduces cardiac filling pressures and when compared with BNP is less hypotensive. In vitro studies in
cultured CFs establish that CD-NP, like CNP, activates
cGMP and suppresses CF proliferation induced by
cardiotrophin-1, which is known to be activated in heart
failure and myocardial infarction. This modular design
incorporating sequences of 2 native peptides thus emerges as
attractive from a therapeutic standpoint as many of the
current native natriuretic peptides for the treatment of AHF
and AMI such as ANP and BNP optimize 1 function such
as cardiac unloading while sacrificing another such as
maintenance of blood pressure, which is overcome by
CD-NP.
The current study also demonstrates that the chimeric
peptide CD-NP, which combines the full-length 22 AA
Figure 3
Effect of CD-NP on MAP,
RAP, and PCWP in Normal Dogs
Data are expressed as means ⫾ SE. n ⫽ 6. *p ⬍ 0.05 versus baseline.
CD-NP 10 ⫽ dose of CD-NP 10 ng/kg/min; CD-NP 50 ⫽ dose of CD-NP 50
ng/kg/min; CD-NP 100 ⫽ dose of CD-NP 100 ng/kg/min; MAP ⫽ mean arterial
pressure; PCWP ⫽ pulmonary capillary pressure; RAP ⫽ right atrial pressure.
Lisy et al.
CD-NP: A Novel Chimeric Natriuretic Peptide
JACC Vol. 52, No. 1, 2008
July 1, 2008:60–8
65
enhanced NPR-B activation by CD-NP that then could
mediate increases in GFR together with natriuresis and
diuresis. Of note, addition of AAs to the C-terminus of
ANP has been demonstrated to increase ANP stability by
increasing resistance to NEP degradation (33). This concept
would be consistent with reports that inhibition of NEP
during DNP administration potentiates the renal actions of
DNP, which is observed during administration of native
ANP or BNP together with NEP inhibition (22,34,35). It
is also possible that this linear peptide (i.e., C-terminus of
DNP) could retard the degradation of endogenous ANP or
BNP by competing with NEP and/or reduce binding of the
native natriuretic peptides to the natriuretic peptide clearance receptor (i.e., NPR-C) making more endogenous ANP
and BNP available for NPR-A binding in the kidney.
Finally, it is possible that the C-terminus of DNP also
altered the ability of CNP to activate NPR-A directly,
making CD-NP an NPR-B and NPR-A agonist. Clearly,
CD-NP is natriuretic, diuretic, and GFR enhancing with
less hypotensive actions but by mechanisms that will need to
Figure 4
Effect of CD-NP on UNaV,
UV, and GFR in Normal Dogs
Data are expressed as means ⫾ SE. n ⫽ 6. *p ⬍ 0.05 versus baseline.
GFR ⫽ glomerular filtration rate; other abbreviations as in Figures 2 and 3.
to the GFR increase. Importantly, we observed that the
renal hemodynamic and tubular actions of CD-NP were
associated with increases in plasma and urinary cGMP
concentrations consistent with particulate guanylyl cyclase receptor activation.
In the past, we and others have speculated that under
physiologic condition renally synthesized CNP acts in an
autocrine/paracrine fashion to activate NPR-B receptors,
which are expressed in the kidney, resulting in natriuretic
and diuretic actions (28 –32). This would be in contrast to
intravenously infused CNP, which lacks significant renal
actions due to rapid degradation by renal NEP. It is
tempting to speculate that the addition of the C-terminus of
DNP protects CNP from degradation by NEP resulting in
Figure 5
Effect of CD-NP on Proximal and Distal
Fractional Sodium Reabsorption in Normal Dogs
Data are expressed as means ⫾ SE. n ⫽ 6. *p ⬍ 0.05 versus baseline.
DFRNa ⫽ distal fractional sodium reabsorption; PFRNa ⫽ proximal fractional
sodium reabsorption; other abbreviations as in Figure 3.
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Neurohumoral Response to Infusion of CD-NP in Normal Dogs
Table 2
Neurohumoral Response to Infusion of CD-NP in Normal Dogs
PcGMP (pmol/ml)
UcGMPV (pmol/min)
PRA (ng/ml/hr)
Baseline
CD-NP 10
CD-NP 50
CD-NP 100
Recovery
9.4 ⫾ 1.2
14.6 ⫾ 1.6
30.3 ⫾ 2.1*
50.1 ⫾ 2.1*
32.7 ⫾ 6.7*
2,133 ⫾ 234
4,566 ⫾ 621*
9,999 ⫾ 1,590*
6,661 ⫾ 734*
6.3 ⫾ 1.1
9.0 ⫾ 1.8
1,648 ⫾ 151
8.2 ⫾ 0.8
5.3 ⫾ 1.1*
3.9 ⫾ 0.7*
Data are expressed as means ⫾ SE. ⴱp ⬍ 0.05 versus baseline.
CD-NP 10 ⫽ infusion of 10 ng/kg/min of CD-NP; CD-NP 50 ⫽ infusion of 50 ng/kg/min of CD-NP; CD-NP 100 ⫽ infusion of 100 ng/kg/min (n ⫽ 8 except n ⫽ 6 for high dose); PcGMP ⫽ plasma cyclic
3=5= guanosine monophosphate; PRA ⫽ plasma renin activity; UcGMPV ⫽ urinary cyclic 3=5= guanosine monophosphate excretion.
be addressed in future studies. It should be noted that at the
lower doses of CD-NP, PRA was suppressed.
CD-NP unloaded the normal heart with reductions in
both right atrial pressure and pulmonary capillary wedge
pressure. At the doses employed in the normal dogs, cardiac
unloading occurred with no or only modest changes in
arterial pressure. These are consistent with the conclusion
that the reduction in cardiac filling pressures may involve
reductions in cardiac pre-load via venodilation as occurs
with CNP (14). Thus, CD-NP may address the need of a
cardiac unloading natriuretic peptide with renal-enhancing
actions with less hypotension than ANP or BNP, thus
better maintaining renal function, which was the goal of our
engineered chimeric peptide.
An important property of CNP and the natriuretic
peptides is antifibrotic actions in the heart and other organs,
which involve inhibition of fibroblast proliferation and
suppression of collagen synthesis. Importantly, Horio et al.
(11) demonstrated that CNP was more antifibrotic in
cultured human CFs as compared with ANP or BNP. Such
fibro-inhibiting actions have clinical relevance as demonstrated by studies of Soeki et al. (17) in which chronic CNP
infusion in a rodent model of AMI for 14 days suppressed
Figure 7
Figure 6
Effect of CD-NP and Equimolar Doses
of Human BNP in 2 Groups of Normal Dogs
Data are expressed as means ⫾ SE. n ⫽ 6 in each group. *p ⬍ 0.05 versus
baseline; †p ⬍ 0.05 between groups. Solid bars ⫽ CD-NP; open bars ⫽ B-type
natriuretic peptide (BNP). Abbreviations as in Figures 3 and 4.
Effect of CD-NP on cGMP Generation and
Antiproliferative Actions of CD-NP in Human CFs
(A) Cyclic 3=5= guanosine monophosphate (cGMP) generation in human cardiac
fibroblasts (CFs). Data are expressed as means ⫾ SE. *p ⬍ 0.05 versus no
treatment; **p ⬍ 0.05 versus CP-NP 10⫺11M; ⫹p ⬍ 0.05 versus CD-NP 10⫺8
M. (B) Antiproliferative actions of CD-NP in human CFs. Bar graph reports colormetric bromodeoxyuridine (BrdU) uptake in optical density units as measured
by colorimetry. Data are expressed as means ⫾ standard error. *p ⬍ 0.05 versus control. Cardiotrophin-1 ⫽ proliferation of fibroblasts by cardiotrophin-1;
Cardiotrophin-1 ⫹ CD-NP ⫽ CD-NP added to cardiotrophin-1 stimulated fibroblasts; Control ⫽ untreated human cardiac fibroblasts.
Lisy et al.
CD-NP: A Novel Chimeric Natriuretic Peptide
JACC Vol. 52, No. 1, 2008
July 1, 2008:60–8
ventricular remodeling and fibrosis with reductions in collagen III synthesis in the absence of reductions in blood
pressure. In our in vitro studies in human CFs, we observed
that CD-NP activated cGMP and possessed antiproliferative actions to cardiotropin-1, which is a potent pro-fibrotic
factor that is known to be activated in heart failure and AMI
(25–27). This action of CD-NP was demonstrated by
inhibition of cardiotropin-1 augmentation of BrdU uptake.
To further understand mechanisms by which CD-NP is
antifibrotic, future studies will investigate collagen turnover
in CFs in response to fibrogenic cytokines.
The natriuretic peptide BNP was approved in 2002 as a
new drug for the treatment of acute decompensated heart
failure based upon the randomized trial VMAC (The
Vasodilation in the Management of Acute CHF) which
demonstrated that, compared with nitroglycerin, BNP was
superior in reducing cardiac filling pressures and compared
with placebo was superior in improving dyspnea (36). With
continued use of BNP, concerns were raised regarding renal
function with a key study reporting a lack of action of BNP
on kidney function in human heart failure and even a
worsening of renal function (37,38). As the level of GFR is
a robust predictor of survival in heart failure, preservation of
renal function or even improvement of renal function is a
high priority (39,40). We have previously reported that
experimental reductions in renal perfusion pressure by
suprarenal aortic constriction abolished ANP-mediated increases in GFR and sodium excretion underscoring the
importance of maintaining renal perfusion pressure in heart
failure during infusion of natriuretic peptides (41). Indeed,
in recent human studies in heart failure and during cardiac
bypass surgery, low-dose BNP without hypotension has
been demonstrated (42– 44). Further, the chronic intermittent use of BNP in human heart failure was unassociated
with any worsening of renal function compared with placebo. While further studies in disease states are warranted,
CD-NP represents a novel therapeutic strategy, which
possesses properties of a peptide-like BNP and ANP but
with apparent less hypotension. The beneficial properties of
CD-NP will be tested in future studies in models of
experimentally induced heart failure.
Conclusions
In summary, we report the design, synthesis, and in vivo
cardiorenal actions of a chimeric natriuretic peptide
CD-NP that represents a fusion peptide of the full-length
22-AA structure of CNP together with the 15-AA
C-terminus of DNP. Specifically, CD-NP is natriuretic and
diuretic, GFR enhancing, renin inhibiting, and unloads the
normal heart, with less hypotensive properties when compared with BNP. In addition, CD-NP in vitro possesses
cGMP-activating and antiproliferative properties in CFs.
As CD-NP contains the native CNP peptide, which activates NPR-B, the therapeutic potential of CD-NP is made
highly relevant by recent reports that document the down-
67
regulation of the NPR-A receptor in the heart and kidney in
human and experimental heart failure (45,46). Our findings
are significant and demonstrate the successful fusion of
native CNP with an AA sequence taken from the
C-terminus of DNP producing a chimera that now possesses clinically relevant renal actions with minimal hypotensive actions and favorable cardiovascular and humoral
properties. These properties are especially attractive in acute
cardiorenal syndromes such as AHF and AMI. Further
studies in experimental models of cardiorenal disease and
ultimately in human disease states will answer the question
of whether this strategy will indeed yield new efficacious and
safe drugs.
Acknowledgments
The authors cordially acknowledge the expert help of
Sharon Sandberg and Denise Heublein with the assays and
Gail Harty with the animal studies.
Reprint requests and correspondence: Dr. Ondrej Lisy, Cardiorenal Research Laboratory, Guggenheim 9, Mayo Clinic College
of Medicine, 200 First Street SW, Rochester, Minnesota
55905. E-mail: [email protected].
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Key Words: peptide y kidney y blood pressure y cGMP y sodium
excretion y fibroblasts.