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(Circulation. 1995;91:2036-2042.)
© 1995 American Heart Association, Inc.

Articles

The Role of Neutral Endopeptidase in Dogs With Evolving Congestive Heart Failure

Kenneth B. Margulies, MD; Paul L. Barclay, PhD; John C. Burnett, Jr, MD

From the Section of Cardiology (K.B.M.), Temple University School of Medicine, Philadelphia, Pa; Pfizer Central Research (P.L.B.), Sandwich, England; and the Department of Internal Medicine (J.C.B.), Mayo Clinic and Foundation, Rochester, Minn.

Correspondence to Kenneth B. Margulies, MD, Assistant Professor of Medicine, Room 318, OMS Bldg, Temple University School of Medicine, 3401 N Broad St, Philadelphia, PA 19140.

	Abstract

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Background Recent studies suggest that neurohumoral mechanisms including decreased renal responses to increases in atrial natriuretic factor (ANF) play a central role in the progression from asymptomatic cardiac dysfunction to advanced congestive heart failure (CHF) with sodium retention, vasoconstriction, and reduced exercise tolerance. Recognizing that neutral endopeptidase 24.11 degrades ANF and may be enhanced in CHF, we hypothesized that chronic neutral endopeptidase inhibition (NEP-I) would potentiate renal responses to exogenous ANF and alter the temporal evolution of sodium retention in evolving CHF by potentiation of increased endogenous ANF.

Methods and Results We studied 13 conscious dogs with evolving CHF produced by rapid ventricular pacing at 250 beats per minute. Six of these dogs received NEP-I with candoxatril, 10 mg/kg PO BID, throughout evolving CHF. Responses to exogenous ANF, 10 µg/kg IV bolus, were assessed at baseline and after 6 days of CHF. Daily metabolic studies during evolving CHF with chronic NEP-I showed increased sodium excretion and renal cGMP generation consistent with enhanced renal activity of endogenous ANF compared with untreated controls. In addition, renal natriuretic and cGMP responses to exogenous ANF were intact in CHF with chronic NEP-I in contrast to markedly attenuated renal responses to exogenous ANF in untreated CHF. Despite enhanced ANF responsiveness and improved sodium balance in evolving CHF, a moderate degree of sodium retention was observed during chronic NEP-I in evolving CHF.

Conclusions Enzymatic degradation by neutral endopeptidase limits local renal responses to increases in endogenous and exogenous ANF in CHF independent of changes in systemic hemodynamics or augmented plasma concentrations of ANF. The moderate sodium retention observed during evolving CHF despite chronic NEP-I probably reflects the antinatriuretic effects of hemodynamic and humoral factors independent of ANF activity.

Key Words: heart failure • natriuretic peptides • sodium • atrial natriuretic factor

	Introduction

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Atrial natriuretic factor (ANF) is an endogenous peptide hormone that participates in the regulation of body fluid homeostasis by enhancing sodium excretion, inhibiting the renin-angiotensin-aldosterone system (RAAS) and decreasing venous return. These actions of ANF are mediated by increases in intracellular cGMP after ANF binding to its guanylate cyclase–linked cell surface receptors.^{1 2} However, despite marked increases in circulating ANF, congestive heart failure (CHF) is characterized by progressive sodium retention, RAAS activation, and vasoconstriction in the absence of increases in renal cGMP generation, a marker for ANF action.³ In fact, decreased renal responsiveness to exogenous ANF has emerged as a well-recognized feature of advanced CHF in humans⁴ and animal models.^{3 5} Interestingly, those factors previously shown to contribute to sodium retention in advanced CHF, including reduced renal perfusion pressure,⁶ increased renal sympathetic nerve activity,⁷ and RAAS activation,⁸ have more recently been implicated in the reduced renal sensitivity to ANF in CHF.^{9 10 11}

Neutral endopeptidase 24.11 (NEP) is a membrane-bound metalloenzyme that cleaves endogenous peptides at the amino side of hydrophobic residues. This ectoenzyme is localized principally within the kidney but also is found within the lung, gut, adrenal, brain, heart, and peripheral vasculature.^{12 13} The vasoactive peptide substrates of NEP include ANF and related peptides, bradykinin, endothelin, angiotensins, and substance P.^{14 15} Recent studies indicate that enzymatic degradation by NEP limits the in vivo renal responses to exogenous ANF under normal conditions.^{16 17 18} In addition, pharmacological NEP inhibition (NEP-I) enhances sodium excretion in humans and animals with CHF.^{5 19 20 21} Indeed, Cavero et al⁵ reported that NEP-I resulted in increased sodium, cGMP, and ANF excretion in dogs with advanced CHF resistant to exogenous ANF. However, it is unknown whether chronic NEP-I during evolving heart failure alters the sodium retention characteristic of the transition from compensated to decompensated cardiac dysfunction. Therefore, the present studies were designed to test the hypothesis that chronic NEP-I alters the temporal evolution of sodium retention and neurohumoral activation in evolving experimental CHF and potentiates renal responses to exogenous ANF.

	Methods

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Experimental Protocol
Experiments were performed on six male mongrel dogs weighing between 18 and 23 kg that were fed normal dog chow (Lab Canine Diet #5006, Purina Mills, Inc) and allowed free access to tap water. All studies conformed to the guiding principles of the American Physiological Society. Experimental CHF was produced by 8 days of rapid ventricular pacing. This experimental model consistently produces a state of low-output cardiac failure with a constellation of cardiovascular, renal, and neurohumoral abnormalities characteristic of chronic primary myocardial failure.^{3 4}

Programmable cardiac pacemakers (model 8426, Medtronic) were implanted before acute interventions. Under pentobarbital anesthesia (30 mg/kg) and via a left thoracotomy and pericardiectomy, the heart was exposed and a screw-in epicardial pacemaker lead was implanted into the right ventricle. The pacemaker lead was connected to a pulse generator implanted subcutaneously in the chest. The pericardium was sutured closed, and the parietal pleura and skin were closed in layers. The dogs were allowed to recover over a 3-day period, during which they received prophylactic antibiotic treatment with clindamycin and Combiotic (Pfizer, Inc). Four to six days after pacemaker implantation, after an overnight fast, dogs were briefly anesthetized with sodium pentothal (15 mg/kg) to allow percutaneous placement of a flow-directed, balloon-tipped pulmonary artery catheter (model 93A-131-7F, American Edwards Laboratories, AHS del Caribe, Inc) via an external jugular vein. At the same time, a second balloon-tipped catheter was inserted into the urinary bladder. An intravenous infusion of 0.9% saline was initiated, and the animals were placed in a minimally restraining sling and allowed to regain consciousness and equilibrate over 90 to 120 minutes.

At the conclusion of this equilibration period, the acute experimental protocol was performed with the animals in the conscious state. The bladder was emptied before and at the end of all urinary clearances. In addition, at the midpoint of these and subsequent clearance periods, cardiac hemodynamics were measured and a 20-mL blood sample was withdrawn from the jugular vein. Two 30-minute baseline urinary clearances were performed. After baseline clearances, synthetic ANF, 10 µg/kg, was administered intravenously over 1 minute. The atrial peptide used in these experiments was 28–amino acid -human ANF (Peninsula Laboratories, Inc). A 30-minute urinary clearance was performed immediately after the ANF administration. The pulmonary artery and urinary catheters then were removed, and the dogs were returned to metabolic cages.

One day after exogenous ANF administration, the first of seven consecutive 24-hour urinary clearances was performed; all clearances were performed with the animals in the conscious state. The dogs' bladders were emptied with a urinary catheter at the beginning and end of each of these clearance periods. The urine collection devices located beneath the metabolic cages were designed to assure prompt freezing of voided urine until the time of analysis. Blood samples were obtained from a foreleg vein at the conclusion of each of these 24-hour clearance periods. After the first 24-hour clearance was completed, dogs were divided into two experimental groups: In group 1 animals (n=7), the pacemaker was programmed to 250 beats per minute, and pacing was continued for the remainder of the experimental protocol. In group 2 (n=6), pacing was initiated 30 minutes after a 24 mg/kg loading dose of candoxatril (UK 79,300, Pfizer Central Research) to achieve systemic NEP-I. Candoxatril is the 5-indanyl ester pro-drug of candoxatrilat (UK 73,967, (s)-cis-4-([2-carboxy-3-(2-methoxyethoxy)-propyl]-1-cyclopentane carbonylamino)-1-cyclohexane carboxylic acid), a potent competitive and selective inhibitor of neutral endopeptidase.²² Pacing continued in group 2 during ongoing oral administration of candoxatril at a dose of 10 mg/kg twice daily. This dosing regimen was designed to maintain plasma drug levels above 70 ng/mL, the IC₉₅ for NEP, throughout the experimental period. In both groups 1 and 2, a series of six additional 24-hour urinary clearances were performed during the evolution of experimental CHF.

After 6 days of pacing and an overnight fast, dogs in each group were again briefly anesthetized with sodium pentothal (7 to 10 mg/kg) for placement of pulmonary artery and bladder catheters. The acute experimental protocol was performed with the animals in the conscious state in a manner identical to that described above for the baseline phase. Dogs in group 2 received a 10 mg/kg dose of candoxatril 2 to 4 hours before baseline clearances for these acute interventions. At the conclusion of the protocol, catheters were removed, and the dogs were returned to their cages. Because one of the group 2 animals suffered a cardiac arrest during pulmonary artery catheter placement after initiation of CHF, data from this animal are not included in Table 2 (ANF responses before and after initiation of CHF).

View this table: [in this window] [in a new window]   Table 2. Responses to Exogenous ANF Before and After CHF in the Presence and Absence of Chronic NEP-I

Analysis
Cardiac hemodynamic parameters measured during each 30-minute clearance period included right atrial pressure, pulmonary capillary wedge pressure, and cardiac output. Cardiac output was determined by thermodilution (cardiac output model 9510-A computer, American Edwards Laboratories), measured in quadruplicate, and averaged during each clearance period. All voided urine was collected on ice, measured using a graduated cylinder, and aliquots were made for measurement of sodium, creatinine, and cGMP. Urine samples for sodium and creatinine determination were refrigerated until analysis. Urine samples for cGMP determination were heated to 90°C and kept at -20°C until analysis. Net renal generation of cGMP was determined using the formula: Net renal generation of cGMP=(urinary cGMPxurine flow rate)-(plasma cGMPxcreatinine clearance).

Venous blood for sodium and creatinine determination was collected in heparinized tubes, placed on ice, and centrifuged at 2500 rpm at 4°C. After centrifugation, plasma was decanted and refrigerated until it was analyzed. Plasma and urinary sodium concentrations were measured using ion-selective electrodes (Beckman Instruments). Glomerular filtration rate (GFR) was determined by creatinine clearance. Plasma and urine creatinine concentrations were measured by the Jaffé reaction (Beckman Instruments).

Venous blood samples for hormone and cGMP analysis were placed in EDTA tubes, immediately placed on ice, and centrifuged at 2500 rpm at 4°C. Extracted venous plasma levels of ANF were measured by radioimmunoassay as previously described.²³ Plasma renin activity was determined by radioimmunoassay using the method of Haber et al.²⁴ Plasma samples for cGMP were extracted with ethanol, and plasma and urinary cGMP were measured by radioimmunoassay using the method of Steiner.²⁵

All data are presented as mean±SEM. Comparisons between pre-CHF and subsequent 24-hour clearances were analyzed by repeated-measures ANOVA followed by Dunnett's t test when appropriate. Exogenous ANF responses in baseline CHF phases were compared by paired Student's t test. Comparisons between NEP-I–treated and untreated groups were analyzed by repeated-measures ANOVA and Fisher's protected least-squares difference test when appropriate. Statistical significance was defined as P<.05.

	Results

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Metabolic Studies in Evolving CHF
The daily hormonal and renal parameters during the evolution of CHF produced by rapid pacing in untreated animals (group 1) and animals with chronic NEP-I (group 2) are presented in Table 1. With the exception of reductions in plasma ANF and renal cGMP levels in group 2, the groups were not significantly different at baseline. In group 1, the first day of evolving CHF was characterized by marked increases in plasma ANF and cGMP and associated increases in absolute and fractional sodium excretion. Renal cGMP excretion was increased almost threefold during the first day of evolving CHF. As CHF continued to develop, despite progressive increases in plasma ANF, plasma cGMP levels tended to plateau, and increases in plasma renin activity emerged and reached statistical significance on the sixth day of evolving CHF. After the first day of CHF, significant and progressive renal sodium retention was evident. GFR, as reflected by creatinine clearance, remained stable, and progressive decreases in fractional sodium excretion confirmed that avid sodium retention occurred as a result of increased tubular sodium reabsorption. Renal cGMP generation decreased toward baseline levels after the first day of evolving CHF.

View this table: [in this window] [in a new window]   Table 1. Hormonal and Renal Function During Evolving CHF in the Presence and Absence of Chronic NEP-I

In group 2, the first day of evolving CHF in the presence of systemic NEP-I was characterized by increases in plasma ANF and cGMP, maintenance of sodium excretion, and marked increases in renal cGMP generation, a pattern not different from untreated animals. As CHF evolved, increases in plasma ANF and plasma cGMP levels were sustained but not greater in magnitude than those observed in untreated controls. Plasma ANF levels were actually lower in the NEP-I–treated group on the fifth day of evolving CHF. However, as illustrated in Fig 1, chronic NEP-I significantly altered the pattern of sodium retention in evolving CHF. Despite reductions in creatinine clearance, chronic NEP-I resulted in a lesser degree of sodium retention and reduced tubular sodium reabsorption in association with maintained increases in renal cGMP generation. Body weight did not change significantly in either experimental group during the 6 days of evolving CHF.

View larger version (24K): [in this window] [in a new window]   Figure 1. Plots show changes from baseline for daily fractional excretion of sodium (FENa) and renal cGMP generation during evolving congestive heart failure (CHF). §P<.05 for comparisons between untreated CHF and CHF with neutral endopeptidase inhibition (NEP-I).

Acute Responses to Exogenous ANF
Table 2 presents cardiac hemodynamic, hormonal, and renal parameters before and after bolus administration of ANF in baseline and CHF phases of the protocol. With the exception of differences in baseline creatinine clearance, the two experimental groups were well matched in the baseline (pre-CHF) phase. In each group, responses to exogenous ANF included increases in plasma ANF and cGMP, with associated increases in GFR, diuresis, and natriuresis. Responses to exogenous ANF were similar between groups in the baseline phase except for lower yet significant peak increases in plasma cGMP and creatinine clearance in group 2. Of note, marked increases in renal cGMP generation accompanied the renal responses to exogenous ANF, underscoring the role of this parameter as a specific marker for renal ANF activity.

After 6 days of rapid pacing, hemodynamic evidence of evolving CHF was present in both experimental groups, including reduced cardiac output and increased cardiac filling pressures. In addition, increases in plasma ANF and plasma cGMP of similar magnitude were observed in NEP-I–treated and untreated CHF. However, despite these similarities, group 2 animals receiving chronic NEP-I had higher urine flow rates and a tendency toward less tubular sodium reabsorption than untreated CHF controls. In addition, although cardiac hemodynamics in CHF were similar in the two groups, the mean change in pulmonary capillary wedge pressure from pre-CHF to CHF was 15.2±0.5 mm Hg in group 1 and 11.6±1.5 mm Hg in group 2 (P=.036), and the mean change in right atrial pressure from pre-CHF to CHF was 6.7±1.0 mm Hg in group 1 and 2.8±1.6 mm Hg in group 2 (P=.059). As in previous studies, when exogenous ANF was administered, untreated CHF controls exhibited no significant cardiac hemodynamic or renal responses despite significant further increases in plasma ANF and plasma cGMP. In particular, attenuated natriuretic and GFR responses to exogenous ANF in untreated CHF were associated with no increase in renal cGMP generation (Fig 2).

View larger version (24K): [in this window] [in a new window]   Figure 2. Bar graphs show acute renal responses to exogenous atrial natriuretic factor (ANF) (10 µg/kg IV). All values represent absolute changes from pre-ANF baseline values. In each group, open bars indicate responses to ANF before initiation of congestive heart failure (CHF); shaded bars indicate responses after 6 days of evolving CHF. GFR indicates glomerular filtration rate; UNaV, absolute sodium excretion; and FENa, fractional sodium excretion. P<.05 for comparisons between baseline and CHF phase responses; §P<.05 for comparisons between untreated CHF and CHF with neutral endopeptidase inhibition.

In animals with CHF and chronic NEP-I, exogenous ANF produced significantly greater increases in urine flow, GFR, and both absolute and fractional sodium excretion compared with untreated CHF controls. Despite similar increments in plasma ANF, these enhanced responses were associated with marked increases in renal cGMP generation, suggesting local renal potentiation of the exogenous ANF by chronic NEP-I. Moreover, as illustrated by Fig 2, renal responses to exogenous ANF in CHF were normalized by chronic NEP-I in that they did not differ from pre-CHF responses to ANF in these animals. In contrast, cardiac hemodynamic responses to exogenous ANF were not altered by chronic NEP-I in CHF.

	Discussion

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The present studies confirm and extend previous investigations by demonstrating that the ectoenzyme NEP 24.11 is a major factor limiting renal responses to exogenous ANF in evolving CHF. In untreated evolving CHF, we observed progressive sodium retention, gradually increasing plasma renin activity, and decreasing renal cGMP generation despite progressive increases in plasma ANF, suggesting renal resistance to increasing endogenous ANF. Responses to exogenous ANF in established untreated CHF were characterized by markedly diminished glomerular, tubular, and cGMP responses compared with the pre-CHF baseline. In evolving CHF with chronic NEP-I, we observed reduced sodium retention and enhanced renal cGMP generation in the absence of altered circulating ANF levels consistent with local renal potentiation of endogenous ANF. In established CHF with chronic NEP-I, normalization of glomerular, tubular, and renal cGMP responses to exogenous ANF further supported the concept that the effects of chronic NEP-I on sodium balance during evolving CHF were a consequence of enhanced renal responsiveness to increases in endogenous ANF.

Previous studies have demonstrated that the emergence of sodium retention in evolving CHF is consistently associated with a preserved GFR, increased tubular sodium reabsorption, and renal resistance to the natriuretic action of ANF.^{3 26 27 28} The tubular segment primarily responsible for sodium retention in advanced CHF remains unclear, but studies have suggested enhanced distal nephron sodium reabsorption in advanced CHF.²⁹ In the present study, evolving CHF was characterized by initial maintenance of sodium excretion followed by progressive sodium retention. The marked decreases in fractional excretion of sodium and preserved GFR observed in untreated CHF indicate that increasing tubular sodium reabsorption was responsible for this progressive sodium retention in evolving CHF. Moreover, the possible role of endogenous ANF as a modulator of sodium excretion in evolving CHF is suggested by the close association between sodium excretion and renal cGMP generation during chronic studies and after exogenous ANF administration in untreated CHF. The parallel increases in sodium and renal cGMP excretion observed during evolving CHF with chronic NEP-I support the concept that ANF degradation by NEP may contribute to sodium retention in CHF via renal resistance to increases in endogenous ANF.

To further clarify whether chronic NEP-I alters attenuated renal responses to ANF, we administered a pharmacological dose of exogenous ANF before and after induction of CHF. In untreated CHF, our results confirm previous investigations by demonstrating that reduced renal glomerular and tubular responses to exogenous ANF in untreated CHF occur in association with reduced renal generation cGMP.³ In evolving CHF with chronic NEP-I, renal glomerular, diuretic, and natriuretic responses to exogenous ANF were significantly enhanced in association with marked increases in renal cGMP generation. The magnitude of responses to ANF in CHF with chronic NEP-I did not differ from the responses observed before the induction of experimental CHF, consistent with the concept that enzymatic degradation by NEP represents a major factor limiting renal responses to exogenous ANF in CHF.

The present studies support the concept of local renal potentiation of ANF action by chronic NEP-I in evolving CHF based on the observation that enhanced sodium excretion and renal cGMP generation occurred in the absence of significant changes in circulating ANF levels and systemic hemodynamics. Such local potentiation of ANF action by NEP-I in the present studies is consistent with previous studies from our laboratory and others.^{5 16} Potentiation of renal responses to ANF by chronic NEP-I does not exclude a role for other peptide modulators of renal function. Mechanisms through which NEP-I potentiates renal ANF action might also involve a contribution by other factors, notably kinins, which are degraded by NEP.³⁰ In humans with advanced CHF, Munzel et al²⁰ observed marked increases in urinary excretion of the prostacyclin metabolite 6-keto-PGF-1 during natriuretic responses to NEP-I, further implicating kinins as a cofactor for ANF potentiation by NEP-I. Therefore, the altered pattern of sodium excretion and responses to exogenous ANF produced by chronic NEP-I in the present studies may be a consequence of synergistic local potentiation of ANF, kinins, and prostaglandins occurring without increases in circulating ANF.

The lesser increases in pulmonary capillary wedge pressure and the tendency for lesser increases in right atrial pressure in CHF with chronic NEP-I may be related to improved sodium balance observed in these animals. In humans with established moderate to severe CHF, Elsner et al³¹ observed similar reductions in cardiac preload after 10 days of NEP-I with candoxatril. In addition, reductions in endogenous ANF secretion owing to lesser increments in right and left atrial pressures in CHF with chronic NEP-I may in part account for the absence of augmented plasma ANF levels in these animals. One might speculate that if reductions in cardiac preload and endogenous ANF secretion were of sufficient magnitude to reduce the natriuretic effects of NEP-I, such actions might serve as a potential feedback mechanism to prevent excessive volume depletion in CHF.

The present studies also suggest, for the first time, that NEP may contribute to the progression of CHF in that chronic inhibition of this enzyme system throughout the early evolution of CHF produced significant alterations in renal function and sodium balance. Previous studies of NEP-I in CHF have used either acute NEP-I at various stages of CHF^{5 20 32 33 34 35 36} or chronic NEP-I in established CHF.^{21 31} By initiating NEP-I concurrently with rapid pacing, the present studies support a putative role for the NEP enzyme system in the early adaptations to cardiac dysfunction. From this perspective, NEP emerges as a potent modulator of renal responses to endogenous peptides during the hemodynamic stress associated with evolving CHF, with a net effect of enhancing renal tubular sodium reabsorption while maintaining glomerular filtration.

Although chronic NEP-I clearly potentiates renal responses to exogenous ANF and attenuates sodium retention in evolving CHF, a moderate degree of sodium retention nevertheless occurred despite continuous chronic NEP-I. This residual degree of sodium retention probably is a result of other abnormalities occurring in early CHF including decreases in renal perfusion pressure,⁶ increased renal sympathetic nerve activity,⁷ and RAAS activation,⁸ which have been shown to contribute to sodium retention in CHF. In fact, chronic NEP-I did not prevent increases in renin during evolving CHF. Based on recent studies in which angiotensin antagonism with chronic angiotensin-converting enzyme inhibition markedly augmented renal responses to acute NEP-I in CHF,³³ the local or systemic RAAS may limit the effects of chronic NEP-I and contribute to the residual degree of sodium retention that occurs despite augmented responses to endogenous and exogenous ANF. If this hypothesis is true, chronic inhibition of both angiotensin-converting enzyme and NEP might be an attractive means of shifting the neurohumoral balance from vasoconstrictor-antinatriuretic hormones to vasodilator-natriuretic hormones in evolving CHF.

Several potential limitations of the present studies should be noted. First, although candoxatril is a specific inhibitor of NEP 24.11, the enzyme itself hydrolyzes several vasoactive peptides other than ANF including brain natriuretic peptide, kinins, angiotensins, endothelin, and substance P. Thus, despite clear potentiation of cGMP excretion and renal responses to exogenous ANF by chronic NEP-I, reduced metabolism of peptides other than ANF may have contributed to the changes in sodium balance produced by chronic NEP-I in these studies. In addition, it is also possible that differences in ANF and renal cGMP excretion at baseline may have affected our results, although the magnitude of these baseline differences was small compared with subsequent increments in these parameters after the initiation of CHF. Despite an equilibration period and its brief hemodynamic actions, the use of brief sodium pentothal anesthesia for pulmonary artery catheterization before acute studies with subjects in the conscious state could affect vasoactive peptides. However, this agent was used similarly in all animals studied to minimize the potential for confounding results. The potential for bias in an unblinded physiological study should be noted, but such effects should be minimized by the use of objective metabolic, hemodynamic, and hormonal outcome measures with a high degree of reproducibility rather than more subjective clinical signs.

Conclusions
These studies extend previous investigations into the factors contributing to sodium retention in evolving CHF by demonstrating that NEP limits local renal responses to increases in endogenous and exogenous ANF in CHF independent of changes in systemic hemodynamics or augmented plasma concentrations of ANF. In addition, these studies suggest that NEP is a modulator of renal glomerular and tubular adaptations in this pathophysiological state. While providing insights into the role of NEP as a modulator of vasoactive peptides in CHF, these studies also suggest that NEP-I may be a promising therapeutic strategy to favorably influence the balance between vasodilator-natriuretic systems and vasoconstrictor-antinatriuretic systems to alter the progression of CHF in humans.

	Acknowledgments

 
This research was supported in part by a Grant-in-Aid from the Minnesota Affiliate of the American Heart Association. Additional support was provided by Pfizer Central Research, Sandwich, Kent, England. The authors gratefully acknowledge the expert technical assistance of Denise M. Heublein and Larry L. Aarhus.

	Footnotes

 
Dr Barclay is employed in the Department of Cardiovascular Biology at Pfizer Research. Candoxatril, the neutral endopeptidase inhibitor used in these studies, is made by Pfizer Research. Dr Burnett's laboratory received funding from Pfizer Central Research to support the investigator-initiated studies described in this article.

Received July 20, 1994; revision received September 26, 1994; accepted October 14, 1994.

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