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Vol. 293, Issue 3, 989-995, June 2000
University of Illinois, Urbana-Champaign, Urbana, Illinois
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Neutral endopeptidase 24.11 (NEP) inhibitors are known to have vascular, diuretic, and natriuretic effects that may be helpful in the treatment of congestive heart failure (CHF). Most NEP inhibitors may act principally through intrarenal mechanisms, which are not completely understood. The purpose of this study was to determine the principal renal effects of the NEP inhibitor ecadotril in dogs with progressive CHF induced by rapid ventricular pacing. Renal function was measured before, during, and after acute i.v. infusion of normal saline in a total of six dogs during normal cardiac function, early left ventricular dysfunction, and overt CHF. During overt CHF, each dog was treated with either ecadotril or placebo orally for 1 week. Parameters measured included glomerular filtration rate, renal blood flow, urine output, sodium clearance, sodium fractional excretion, and proximal and distal sodium reabsorption. Ecadotril treatment resulted in increased urine output, sodium clearance, and renal sodium excretion relative to placebo-treated controls. The principal intrarenal effect of ecadotril was decreased distal renal tubular sodium reabsorption. Both glomerular filtration rate and renal blood flow declined during overt CHF and were unaffected by ecadotril treatment. The results of this study are consistent with the principal action of ecadotril occurring by way of intrarenal events as opposed to changes in renal hemodynamics. The principal effect of ecadotril on distal tubular sodium reabsorption suggests that inhibition of NEP activity in the proximal renal tubules may allow increased binding of filtered atrial natriuretic peptide to natriuretic peptide receptor sites in the distal renal tubules and collecting ducts.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Inhibitors
of neutral endopeptidase 24.11 (NEP; EC 3.4.24.11) are being considered
in the treatment of congestive heart failure (CHF) either as single
therapeutic agents or in combination therapy (Campbell et al., 1998
;
Lisy et al., 1998). NEP is active against several neuropeptides,
including natriuretic peptides, angiotensins, and bradykinins (Richards
et al., 1992; Yamamoto et al., 1992; Margulies and Burnett, 1993;
Roques et al., 1993; Lisy et al., 1998). In addition to cardiovascular
effects, inhibitors of NEP activity cause diuresis and natriuresis
(Margulies et al., 1990). At this time, the mechanism by which NEP
inhibition induces these effects during CHF is only partially
understood. Gaining such an understanding should be helpful in
determining the most effective uses of NEP inhibitors as single
therapeutic agents or in combination therapy.
Although NEP inhibitors can augment the activity of many natriuretic
peptides and enzymes, potentiation of endogenous atrial natriuretic
peptide (ANP) activity has been postulated for some time as the
principal target of such therapy (Borgeson et al., 1998; Cavero et al.,
1990; Margulies et al., 1990). Inhibition of NEP is thought to decrease
the degradation of atrial natriuretic peptide (ANP) and thereby augment
its activity through increased natriuretic peptide receptor binding,
primarily by way of the membrane guanylyl cyclase-A/natriuretic peptide
receptor-A (NPR-A). However, increased plasma ANP concentrations that
could result in increased delivery of ANP to the nephron are an
inconsistent finding with NEP inhibition (Cavero et al., 1990;
Willenbrock et al., 1996). This suggests that the most important
effects of NEP inhibition in the induction of natriuresis may be the
effects of NEP inhibition within the kidney itself. Renal NEP activity is highest on the luminal membrane in the brush border of proximal renal tubule epithelial cells (Berg et al., 1988; Olins et al., 1987;
Shima et al., 1988). In contrast, NPR-A is prevalent not only in the
proximal regions of the nephron but also within distal tubules and
inner medullary collecting ducts (Healy and Fanestil, 1986;
Grandclement and Morel, 1998). Hence, there are several potential
intrarenal sites of ANP-receptor interaction.
The NEP inhibitor ecadotril
{N-(S)-[2-[(acetylthio)methyl]-1-oxo-3-phenyl
propyl]-glycine benzylester; sinorphan} has been shown to be
effective in the treatment of CHF (Varin et al., 1991; Wegner et al.,
1996; Kimura et al., 1998). The active metabolite of ecadotril is
S-thiorphan, which is known to potentiate the natriuretic
activity of exogenous ANP (Trapani et al., 1989). However, the
principal intrarenal effects of ecadotril during CHF have not been
determined. The purpose of this study was to determine how renal
function is altered by ecadotril during CHF by comparing the relative
contribution of various aspects of renal function on sodium excretion
by the kidneys. For this purpose, a model of progressive left
ventricular dysfunction, induced by rapid ventricular pacing in
combination with acute sodium volume expansion was used to accentuate
any effects on renal sodium handling that might be caused by ecadotril treatment.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals. Six conditioned, male mixed-breed dogs weighing ~25 kg each were housed at the University of Illinois at Urbana-Champaign Laboratory Animal Care Facility for at least 1 week before beginning the study. Each dog was screened for physical abnormalities that could interfere with the study by a complete physical examination, echocardiogram, and electrocardiogram. All dogs were fed a fixed sodium diet (58 mEq/day; Hills ID dog food) and evaluated throughout the study for advanced signs of heart failure and general malaise (e.g., anorexia, reluctance to move) with the contingency plan that dogs showing such signs would be removed from the study. After evaluation, each dog was anesthetized and a permanent transvenous ventricular pacemaker was implanted. A permanent arterial catheter also was placed into a femoral artery of each dog. The patency of the arterial line was maintained by biweekly heparin flushes. Each dog was allowed to recover for a minimum of 14 days before ventricular pacing was initiated.
Ventricular Pacing.
Progressive ventricular dysfunction was
induced by rapid ventricular pacing (Luchner et al., 1996, 1998;
Jougasaki et al., 1997). After a 14-day postimplantation recovery
period (termed study day 0), each dog's heart rate was set at 180 beats per minute (bpm) for 10 days. This rate and duration of
tachycardia cause early left ventricular dysfunction, characterized by
decreased cardiac function and increased plasma ANP and norepinephrine
concentrations; however, there are no measurable effects on renal
function, the renin/angiotensin/aldosterone system, or sodium excretion
(Redfield et al., 1993). In this study, all dogs were physically
asymptomatic for signs of CHF at this pacing rate; however, early left
ventricular dysfunction was confirmed by decreases in mean values for
ejection fraction, cardiac output, mean arterial pressure, and
increased atrial pressures over the prepacing values.
).
Dogs were randomly assigned to either the ecadotril treatment group
that received ~5 mg/kg ecadotril once a day orally or to the placebo
group that was given placebo capsules once a day orally. Treatment
began at the onset of the 220-bpm pacing period (day 25) and was
continued for 7 days.
Renal Function Studies.
Renal function was assessed in each
dog on day 0 (the onset of ventricular pacing), day 24 (early
ventricular dysfunction), and day 32 (7 days of treatment with either
ecadotril or placebo during overt CHF). On the afternoon before
assessing renal function, each dog was given 300 to 600 mg of lithium
carbonate orally. Lithium reabsorption by the kidney occurs principally
in the proximal renal tubules, and in parallel with sodium and water,
thereby allowing the measurement of lithium clearance to reflect
proximal tubule sodium clearance (Thomsen et al., 1981; Burnett et al., 1986; Skott, 1994; Lisy et al., 1998). Food, but not water, was withheld overnight. On the day of each study, the dogs were lightly sedated with acepromazine and buprenorphine and a flow-directed balloon
tip pulmonary artery catheter was placed via an external jugular vein
for measurement of cardiac output and filling pressures. Indwelling
i.v. catheters were placed in a saphenous vein for infusion of inulin
and para-aminohippurate (PAH) and in a cephalic vein for
saline infusion. A urinary catheter was placed for the collection of
urine samples.
). Briefly, 25 µl of urine was incubated at room temperature for at least 15 min in
225 µl of a type III jack bean urease (Sigma Chemical Co., St. Louis,
MO) solution prepared by dissolving 5,000 to 10,000 U of urease in 100 ml of 0.05 M potassium phosphate, pH 7.0. After the incubation, 250 µl of the urease-treated urine, or 250 µl of plasma, was mixed with
2.5 ml of a protein precipitation solution consisting of 129 g of
p-toluene sulfonic acid and 57 g of dichloroacetic acid
in 1 liter of deionized H2O adjusted to a pH of
1.4 ± 0.03. After 10 min, each sample was centrifuged at
2500g for ~5 min and 1.0 ml of supernatant fluid mixed
with 1.0 ml of a solution containing 5.0 g of
p-dimethylaminobenzaldehyde in 300 ml of 95% ethanol and
200 ml of deionized H2O. The absorbance of each
solution was measured at 450 nm and the PAH concentration was
determined from a standard curve.
Plasma and urine inulin concentrations were determined based on a
previously described procedure (Davidson and Sackner, 1963). Briefly,
250 µl of urine diluted up to 1:100 with deionized
H2O and plasma was mixed with 2.25 ml of 0.44 N
trichloroacetic acid to precipitate proteins. The solutions were
allowed to stand at room temperature for at least 30 min and then
centrifuged at 2500g for 5 min. After centrifugation, 500 µl of the supernatant fluid was layered over 3 ml of ice-cold
anthrone solution consisting of 500 mg of anthrone, 500 ml of
concentrated H2SO4, and 130 ml of deionized H2O. Each sample was thoroughly
vortexed and placed on ice for 1 min and then incubated at 37°C for
50 min. The absorbance at 620 nm was read and the inulin concentrations
determined from a standard curve.
Data Analysis.
The following formulae were used for the
calculation of renal function parameters: glomerular filtration rate
(GFR) = (urine inulin concentration × urine flow) 
plasma inulin concentration; renal blood flow (RBF) = [(urine PAH
concentration × urine flow plasma PAH concentration) (1 
PCV)] body weight; lithium clearance
(LiCl) = (urine lithium concentration × urine flow) plasma lithium concentration; sodium clearance
(NaCl) = (urine sodium concentration × urine flow) plasma sodium concentration; fractional excretion
of sodium (FENa) = [(urine sodium
concentration × plasma inulin concentration) (plasma
sodium concentration × urine inulin concentration)] × 100%;
proximal fractional sodium reabsorption
(PFNa) = [1 (LiCl GFR)] × 100%; and distal fractional sodium reabsorption (DFNa) = [(LiCl NaCl) LiCl] × 100%.
). The level of
significant differences of all analyses was set at P 
.05.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The main signs of overt CHF observed included reduced exercise
tolerance, ascites, and pulmonary congestion. Symptoms tended to be
most severe in the placebo-treated dogs. The GFR significantly decreased (P < .0001) with the development of overt
heart failure, decreasing from an overall mean value of 6.2 ± 0.59 ml/kg/min on day 0 and 7.1 ± 0.70 ml/kg/min on day 24 to an
overall mean of 3.7 ± 0.39 ml/kg/min on day 32 of the study (Fig.
1). Ecadotril administration had no
significant effect on GFR compared with dogs receiving placebo. RBF
(Fig. 2) showed comparable effects to
GFR, with a significant decrease in RBF (P < .0001)
with overt CHF from 25.6 ± 1.58 ml/kg/min on day 0 and 23.5 ± 1.86 ml/kg/min on day 24 to 13.6 ± 0.45 ml/kg/min on day 32. As with GFR, a difference in RBF between dogs treated with ecadotril
and dogs treated with placebo during overt CHF was not observed.
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Urine output increased in response to sodium volume expansion during
normal cardiac function (day 0) and early CHF (day 24), averaging once
saline infusion was begun at 14.9 ± 5.12 and 16.8 ± 4.97 ml/kg/h, respectively (Fig. 3). In
contrast, urine output did not increase substantially with saline
infusion in the dogs with overt CHF (day 32) that were treated with
placebo, averaging 1.6 ± 0.41 ml/kg/h. This was significantly
different (P < .001) from urine output in dogs with
overt CHF that were treated with ecadotril. Urine output in the
ecadotril-treated dogs was comparable with that observed on days 0 and
24 of the study, averaging 14.2 ± 2.61 ml/kg/h after saline
infusion was begun.
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The effect of acute sodium volume expansion on days 0 and 24 of the
study was to increase NaCl (Fig.
4) and FENa (Fig.
5). However, by day 32, neither
NaCl nor FENa significantly
changed in response to acute sodium volume expansion in the dogs
receiving placebo. In contrast, in the ecadotril-treated group, both
NaCl and FENa increased
significantly (P < .0001) and to a comparable degree
to that observed in the two earlier studies.
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Acute sodium volume expansion also resulted in a significant
(P < .0001) and progressive decrease in
PFNa during normal cardiac function and both
phases of CHF (Fig. 6). The difference in
PFNa during normal cardiac function and during
overt CHF was not significant. In addition, PFNa
did not differ between the dogs treated with placebo and the dogs
treated with ecadotril. Although there was little change in
DFNa with sodium volume expansion in the
placebo-treated dogs, DFNa decreased
significantly (P = .001) during the first and second
studies and during overt CHF with ecadotril treatment (Fig.
7).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The principal intrarenal response to ecadotril observed in this
study was decreased distal tubular sodium reabsorption. Similar results
have been obtained in other studies, including those studies performed
with a competitive inhibitor of NEP (SQ 28,603) infused intrarenally to
anesthetized dogs with normal cardiac function (Margulies et al., 1990)
or to dogs with experimentally induced acute heart failure and
natriuretic peptide receptor inhibitors (HS-142-1; Stevens et al.,
1996). For the current study, it was evident that the major site of
effect on sodium reabsorption was after passage of filtrate through the
proximal renal tubule. The primary renal tubule-binding site of ANP is
guanylyl cyclase A/NPR-A. Although ANP receptor binding occurs in
glomerular and proximal renal tubular sites, ANP-binding sites have
been localized in high concentration to the inner medullary collecting
ducts (Healy and Fanestil, 1986; Koseki et al., 1986). In addition, ANP
has been found to inhibit tubular reabsorption primarily between the superficial late distal tubule and papillary collecting duct base and
the papillary collecting duct (Fried et al., 1988). However, infusion
of ANP results in not only distal renal tubule effects but also
proximal renal tubule effects, glomerular effects, and increased GFR
and RBF (Takeda et al., 1986; Ballermann and Brenner, 1987). Similar
effects are observed with other members of the ANP family of peptides,
including brain natriuretic peptide and urodilatin, when administered
systemically or by way of the renal blood system and in spite of the
fact that under physiologic conditions, urodilatin is probably more
involved in the regulation of electrolyte excretion than blood pressure
regulation (Forssmann et al., 1998; Meyer et al., 1998). Hence, it may
be possible that the intrarenal effect of NEP inhibition observed in
this study and in other studies does not represent potentiation of
normal physiologic pathways. Rather, the mechanism of induction of
natriuresis by ecadotril represents a supraphysiologic event that
allows access of filtered ANP to distal tubule sites that it normally
does not access.
The prolonged and progressive method of induction of CHF used in this
study may have accentuated the distal effects observed due to
down-regulation of more proximal binding sites. With time, one effect
of CHF is elevation of circulating ANP concentrations (Wilkins et al.,
1990; Wilkins and Needleman, 1992). Prolonged exposure to increased
exogenous ANP concentrations results in a suppressed renal response due
to down-regulation of receptor numbers. With inhibition of NEP in the
proximal tubules, a larger percentage of the ANP filtered through the
glomerulus would gain access to receptor sites in the distal tubules
and collecting ducts. Compared with proximal tubule sites, these sites
are relatively isolated from systemic ANP and therefore their response
to ANP is anticipated to be less suppressed. It had been proposed that inhibition of NEP by thiorphan allows filtered ANP to gain access to
renal tubule sites that are normally inaccessible (Wilkins et al.,
1990). It was further proposed that NEP inhibition would allow filtered
ANP to reach normally inaccessible renal tubule sites where receptors
would not have been down-regulated due to their isolation from the high
circulating ANP levels. The findings of our study are consistent with
this hypothesis. It also has been shown that the concentrations of
ecadotril are relatively high in the kidneys for prolonged
periods.4 This and
regional differences in the intrarenal concentration of NEP 24.11 also
may have been a factor.
This study has shown that ecadotril administered orally for 1 week can effectively induce diuresis and natriuresis in dogs with progressive CHF caused by rapid ventricular pacing. The use of the progressive left ventricular dysfunction pacing model in conjunction with acute sodium volume expansion enhanced greatly our ability to detect the effects of ecadotril on renal function during overt CHF. The rapidity and degree of the response in the ecadotril-treated group to saline infusion was comparable with that observed during periods of normal cardiac function and early asymptomatic ventricular dysfunction. In contrast, ecadotril almost completely prevented pathophysiologic changes as a consequence of CHF induced by rapid pacing.
Our data also have shown that during CHF ecadotril administration is
not associated with alterations to either RBF or GFR. This finding is
similar to that of an earlier study that examined the effects of a
different NEP inhibitor (SQ 28,603) during CHF and acute volume
overload and that concluded that induction of natriuresis by NEP
inhibition under such conditions may be independent of alterations to
systemic or renal hemodynamics (Cavero et al., 1990).
In conclusion, the findings of this study demonstrate that the oral administration of ecadotril for 1 week during progressive CHF induced by rapid ventricular pacing results in a significant diuretic and natriuretic effect in response to acute sodium volume expansion. The effects of ecadotril occur by way of intrarenal events as opposed to changes in renal hemodynamics. The principal renal effect of ecadotril was on distal tubular sodium reabsorption. This is consistent with the hypothesis that the chain of events leading to increased natriuresis by the metabolite of ecadotril, S-thiorphan, occur through inhibition of NEP 24.11, diminished natriuretic peptide degradation, enhanced NPR-A receptor binding in the distal tubules and collecting ducts, and activation of NPR-A receptors.
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Acknowledgment |
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We thank the University of Illinois, College of Veterinary Medicine Toxicology Laboratory, for the plasma and urine lithium analyses.
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Footnotes |
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Accepted for publication February 7, 2000.
Received for publication November 17, 1999.
1 This study was supported by Bayer AG, Monheim, Germany.
2 Current address: University of California-Davis, Veterinary Medical Teaching Hospital, Davis, CA, 95616-8737.
3 Current address: Bayer AG, GB-TG/Pharmakologie, Monheim 6700, 51368 Leverkusen, Germany.
4 Steinke W and Schwarz T. [14-C]BAY y 7432: Distribution of the radioactivity in rats after single oral and i.v. administration (whole body autoradiography). PH-report 23165, April 11, 1994, Bayer AG.
Send reprint requests to: Philip F. Solter, University of Illinois, College of Veterinary Medicine, 1008 W. Hazelwood Dr., Urbana, IL 61802.
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Abbreviations |
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NEP, Neutral endopeptidase 24.11; CHF, congestive heart failure; ANP, atrial natriuretic peptide; NPR-A, natriuretic peptide receptor-A; bpm, beats per minute; PAH, para-aminohippurate; VE, volume expansion; PCV, packed cell volume; GFR, glomerular filtration rate; RBF, renal blood flow.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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