(Hypertension. 1995;25:1178-1184.)
© 1995 American Heart Association, Inc.
Articles |
Pulmonary and Renal Neutral Endopeptidase EC 3.4.24.11 in Rats With Experimental Heart Failure
From the Hypertension-Endocrine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health (Z.A.A., E.G., H.R.K.); Department of Microbiology, University of Maryland (S.K.), College Park, Md; and Centro di Fisiologia Clinica e Ipertensione, Ospedale Maggiore, Università di Milano (Italy) (F.P.).
Correspondence to Zaid A. Abassi, PhD, Hypertension-Endocrine Branch, National Heart, Lung, and Blood Institute, Bldg 10, Room 8C103, Bethesda, MD 20892-1754.
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Abstract Congestive heart failure is characterized by avid sodium retention and a blunted renal response to exogenous and endogenous atrial natriuretic peptide. Inhibition of neutral endopeptidase EC 3.4.24.11, the main enzyme that degrades natriuretic peptides, produces a natriuretic response in different models of congestive heart failure. This raises the possibility that an increase in either the expression or activity of neutral endopeptidase is responsible for these phenomena. In the present study, we examined (1) the renal effects of SQ-28,603, a neutral endopeptidase inhibitor, in rats with moderate and severe congestive heart failure induced by an aortocaval fistula compared with sham controls, and (2) neutral endopeptidase expression and activity in the lungs and kidneys of these rats. Infusion of SQ-28,603 (40 mg/kg IV) induced a significant natriuretic response in normal rats and rats with moderate congestive heart failure. This response was blunted in rats with severe congestive heart failure. Surprisingly, renal neutral endopeptidase mRNA levels, assessed by quantitative reverse transcriptase–polymerase chain reaction; protein levels, assessed by Western blotting; and activity, assessed by gelatin gels, were comparable in all groups. Pulmonary neutral endopeptidase mRNA levels decreased by 45% in rats with severe congestive heart failure but not in rats with mild congestive heart failure. In addition, pulmonary neutral endopeptidase immunoreactivity levels and activity were significantly decreased in congestive heart failure in correlation with the severity of the disorder. We conclude that an increase in neutral endopeptidase expression or activity does not occur in rats with congestive heart failure, and therefore, an upregulation of neutral endopeptidase is unlikely to account for the blunted natriuretic response to the atrial natriuretic peptide and the positive sodium balance in congestive heart failure.
Key Words: membrane metallo-endopeptidase • heart failure, congestive • kidney • lung • natriuresis
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Introduction |
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Chronic severe congestive heart failure (CHF) is characterized by salt retention and edema formation. Several factors have been suggested to contribute to the avid sodium and water reabsorption in this disease state. These include a decrease in renal perfusion pressure and activation of neural and hormonal systems such as the renin-angiotensin-aldosterone system (RAAS), catecholamines, endothelin, and vasopressin.1 2 3 4 5 On the other hand, plasma levels of atrial natriuretic peptide (ANP) and other related peptides are also elevated in humans3 6 7 and experimental animals8 9 with CHF. However, the efficiency of ANP as a natriuretic agent is extremely attenuated in advanced stages of this syndrome.8 9 10 11 Previously it was shown that rats with an aortocaval fistula (ACF), an experimental model of CHF, either develop progressive sodium retention (decompensated CHF) or compensate and maintain normal sodium balance (compensated CHF).8 12 13 Interestingly, plasma ANP levels were found to be equally elevated in both groups.8 ANP infusion into rats with compensated CHF induced diuresis and natriuresis, whereas rats with decompensated CHF showed a markedly blunted natriuretic response to ANP infusion, even at high doses.8 13 The mechanisms underlying the renal resistance to ANP are not fully known. Activation of the sodium-retaining systems mentioned above8 14 15 16 17 and downregulation of ANP receptors18 19 have been found to play major roles in the development of this phenomenon. Inhibition of the RAAS, either by angiotensin-converting enzyme inhibition8 14 or by blockade of angiotensin II type 1 receptors,16 partially restored the natriuretic response to endogenous and exogenous ANP.
An additional mechanism that could theoretically contribute to the abnormality in ANP action involves an increase in the ANP degradation rate in CHF.20 This possibility is supported by numerous reports that inhibition of neutral endopeptidase 24.11 (NEP), a key enzyme in the degradation of ANP and other natriuretic peptides, potentiated the natriuretic and hemodynamic responses to ANP and increased the urinary excretion of cGMP and ANP.21 22 23 24 NEP is a ubiquitous enzyme, predominantly found in the kidneys and lungs.25 Besides ANP, NEP is involved in the metabolism of other vasoactive peptides such as enkephalins, bradykinin, and endothelins.25 26 27 Since these peptides have major functions in sodium homeostasis and circulatory regulation, changes in NEP activity may interfere with their biological actions in CHF. The pulmonary and renal expression and activity of NEP in CHF have not been thoroughly examined.
To elucidate the role of NEP in determining sodium balance in experimental heart failure, we carried out the present study with the following aims: (1) to examine the renal effects of SQ-28,603, a specific NEP inhibitor, before and after the induction of CHF by the creation of an ACF in rats, and (2) to investigate NEP expression and activity in lungs and kidneys of rats with compensated and decompensated CHF.
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Methods |
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In Vivo Protocol
Experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Studies were performed on male Sprague-Dawley rats weighing approximately 300 g (Harlan Sprague Dawley Inc, Indianapolis, Ind). The rats were kept in controlled-temperature rooms with a 6 AM to 6 PM light cycle and were fed standard rat chow containing 170 mmol/kg sodium (Agway). Tap water and rat chow were provided ad libitum. An ACF was created between the abdominal aorta and inferior vena cava according to the method of Stumpe et al.28 Briefly, on the day of surgery the rats were anesthetized with pentobarbital sodium (40 mg/kg), and the vena cava and aorta were exposed via a midline abdominal incision. A side-to-side (1.2 to 1.4 mm) surgical anastomosis was created between the two blood vessels distal to the origin of the renal arteries. Rats that underwent sham operation served as a control group. The rats were placed in individual metabolic cages for daily monitoring of urine output and electrolyte excretion. One week after the operation, rats with ACF were divided into two groups according to their daily urinary excretion of sodium (UNaV): rats with UNaV less than 200 µmol/24 h (decompensated) and rats with UNaV greater than 1400 µmol/24 h (compensated). Rats with UNaV greater than 200 µmol/24 h and less than 1400 µmol/24 h were not included in this study. Postmortem examination revealed that decompensated rats developed additional signs of CHF, ie, ascites, edema, enlarged liver, pleural effusions, and hypertrophy of the heart. In addition, these rats displayed a profound activation of the RAAS (see "Results") and severe dyspnea, whereas these signs were mild or nonexistent in compensated rats.
On the day of the experiment, rats were anesthetized with 100 mg/kg IP thiobutabarbital (Inactin, BYK-Gulden) and prepared for clearance studies. Rats were placed on a heated table, and a tracheostomy was performed. Polyethylene catheters (PE-50) were inserted into the right carotid artery for blood pressure monitoring (Blood Pressure Analyzer, Micro Med) and blood sample collection; into the jugular vein for infusions; and into the bladder for urine collections. A solution of 0.9% NaCl containing methoxy-[3H]inulin (New England Nuclear) at a concentration of 4 µCi/mL was infused at a rate equal to 1% of body weight per hour. Rats were given 60 minutes for equilibration. Baseline urine then was collected for 60 minutes, with a blood sample withdrawn at the midpoint of the period. Thereafter, control rats (n=9) and rats with compensated (n=5) and decompensated (n=5) CHF were injected with 40 mg/kg IV of the NEP inhibitor SQ-28,603 (Bristol-Myers Squibb) dissolved in 1.5 mL/kg of 1% NaHCO3, and four 30-minute clearance periods were obtained.
Glomerular filtration rate (GFR) was determined via inulin clearance calculated from the concentration of methoxy-[3H]inulin in 10-µL samples of urine and plasma as measured by a liquid scintillation counter (model LS 9000, Beckman Instruments) with the use of Hydrofluor (National Diagnostic Inc). The sodium concentration in samples of plasma and urine was measured by an ion-selective electrode (Synchron EL-ISE Electrolyte System, Beckman). Six to 8 days after creation of the ACF, a separate group of normal rats (n=15) and rats with compensated CHF (n=15) or rats with decompensated CHF (n=12) were decapitated for determination of plasma renin activity, plasma aldosterone concentration, and plasma ANP levels. Blood was collected in precooled tubes containing potassium EDTA and immediately centrifuged at 4°C. Plasma samples were stored at -70°C until analysis. Plasma renin activity was measured by radioimmunoassay for angiotensin I and expressed as nanograms angiotensin I formed per deciliter plasma generated during a 1-hour incubation period. Plasma aldosterone concentration was measured in unextracted samples with a commercially available radioimmunoassay kit (Endocrine Sciences). Plasma ANP levels were determined by Nichols Institutes (San Juan Capistrano, Calif) with the use of a specific commercial radioimmunoassay kit.
In Vitro Protocols
Control rats and rats with compensated and decompensated CHF
were anesthetized with an intraperitoneal injection
of pentobarbital sodium, and their kidneys and lungs were removed and
immediately placed in liquid nitrogen. RNA was extracted from the
tissues as described by Chomczynski and Sacchi29 with the
use of a commercial solution (RNAzol B, Tel-Test Inc) and was
quantified by spectrophotometry. Since we were unable to detect RNA
encoding NEP by standard Northern blotting with 20 µg total RNA on
each lane (most likely because of using total RNA instead of mRNA),
quantitative reverse transcription followed by quantitative polymerase
chain reaction (RT-PCR) was applied.
RT-PCR
NEP cDNA was synthesized from 2 µg total RNA with the use of a
specific primer: 5'-TGTGATTTCATGTCCGATGACC-3' (bases 1719-1740, see
Reference 3030 ; synthesized by Lofstrand Laboratories, Gaithersburg, Md).
Avian myeloblastosis virus reverse transcriptase (AMV-RT, 8 U per
reaction; Promega) was used for RT as well as the reaction mixture
recommended by the enzyme manufacturer in a volume of 20 µL. PCR was
then applied with the use of 2 µL of the resulting cDNA and the
GeneAmp kit (Cetus Perkin-Elmer), with the upstream primer
5'-CTATCCTGATGACATCATCTC-3' (bases 1422-1442, see Reference 3030 ) and the
downstream primer used for RT. Each PCR reaction mixture contained 200
µmol/L each of dATP, dGTP, and dTTP; 100 µmol/L "cold" dCTP;
and 0.8 µCi 32P-labeled dCTP (New England Nuclear).
Primers were chosen to span introns in order to distinguish by size PCR
products derived from cDNA from those derived from genomic DNA
contaminants.
In a preliminary study we found that the minimum number of PCR cycles necessary to obtain a visible product on an acrylamide gel was 21 and that the quantity of the product yielded after 21 PCR cycles is directly proportional to the amount of cDNA used. Therefore, after an initial denaturation step at 94°C for 3 minutes, cycles of annealing at 56°C for 1 minute, elongation at 72°C for 1 minute, and denaturation at 94°C for 1 minute were applied with the use of 10% of the cDNA described above. The expected size of the NEP PCR product is 322 bp (Cetus Perkin-Elmer). The RT-PCR product of the gene encoding ß-actin served as a quantity control. The ß-actin primers were upstream, 5'-GACTACCTCATGAAGATCCTGACC-3' (nucleotides encoding amino acids 210-217) and downstream, 5'-TGATCTTCATGGTGCTAGGAGCC-3' (nucleotides encoding amino acids 320-327).
Eight microliters of the PCR product was electrophoresed on a 4% to 20% Tris-glycine gel (Novex). The resulting gel was exposed to x-ray film for several hours until clear bands were visible. Negative controls for the PCR reaction included tubes lacking either template or AMV-RT. NEP and ß-actin mRNA were quantified by densitometric analysis (IMAGE 1.55, National Institutes of Health).
Determination of NEP Immunoreactivity Levels by Western Blot
Analysis
Membranes were prepared from kidneys and lungs of sham-operated
rats and rats with compensated and decompensated CHF as described by
Maeda et al.31 Briefly, rats were decapitated and their
kidneys and lungs removed, minced, and resuspended in 3 mL of 10 mmol/L
sodium phosphate buffer, pH 7.4, containing 1 mmol/L
MgCl2, 30 mmol/L NaCl, 0.02% sodium azide, 20 mg/L
bestatin, 20 mg/L leuopeptin, and 10 µg/L DNAse. Tissues then were
homogenized for 30 seconds with a polytron homogenizer (Brinkmann
Instruments) at a setting of 7.0. The homogenate was layered on an
equal volume of 41% (wt/vol) sucrose and centrifuged at
100 000g for 30 minutes with an ultracentrifuge (model
L5-50B, Beckman). The buffer/sucrose interface, which includes the
membranal preparation, was collected and washed twice with 10 mmol/L
Tris, pH 7.4, and resuspended in an appropriate volume of the same
buffer and stored at -70°C until used. Prestained molecular markers
(Bio-Rad) were used for determination of the molecular weight of the
immunoreactive products. Approximately 10 µL of membrane preparation
(80 µg protein) from each tissue of the different experimental groups
was treated with 20 µL sample buffer (10% sodium dodecyl sulfate
[SDS], 50% glycerol, 1.0 mol/L Tris, 0.1% bromphenol blue, and 1
mol/L dithiothreitol, pH 6.8) and placed in a boiling water bath for 5
minutes. Samples then were electrophoresed on SDS and polyacrylamide
gels (4% to 16%) and transferred electrophoretically to a
nitrocellulose membrane (100 V for 1 hour). The nitrocellulose membrane
was incubated with polyclonal NEP antibodies and visualized by
successive incubation with goat anti-rabbit IgG–alkaline phosphatase
conjugate and alkaline phosphatase substrate. A pure recombinant NEP
(Genentech) was used as a positive marker.
Determination of NEP Activity by Substrate Gel
Electrophoresis
Renal and pulmonary NEP activities were determined by use of a
polyacrylamide gel containing SDS and gelatin as copolymerized
substrates. This method is based on the fact that gelatin is a good
substrate for proteases; therefore, it is widely used for
characterization and determination of protease and
collagenase activities in bacteria and mammalian
tissues.32 Renal and pulmonary membrane preparations (10
µg) were mixed with an equal volume of sample buffer and loaded on
4% to 10% polyacrylamide gels containing 2% SDS copolymerized with
0.1% gelatin as substrate. After electrophoresis, gels were incubated
in 2.5% Triton X-100 for two successive 30-minute periods at room
temperature. Gels were removed and washed with substrate buffer (50
mmol/L Tris-HCl, 5 mmol/L CaCl2, and 0.02%
NaN3, pH 8.0) and incubated in the same buffer for 4
to 12 hours at 37°C. After incubation, gels were stained with 0.5%
Coomassie blue R-250 (Bio-Rad) solution containing 10% acetic acid,
30% isopropyl alcohol, and 60% water for 20 minutes. Gels were
destained in distilled water. Clear zones indicated proteolytic
activity of the enzyme. Recombinant NEP was used as a positive
control.
Statistical Analysis
Statistical significance was assessed by one-way ANOVA for
repeated measurements within groups followed by Fisher's test. Two-way
ANOVA was used for comparisons between different experimental groups,
followed by unpaired Student's t test for comparison of
each period between these groups. A value of P<.05 was
considered statistically significant.
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Results |
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In Vivo Protocol
Plasma ANP levels were significantly and equally elevated in rats with ACF compared with sham-operated rats (32±2 pg/mL in controls, 54±4 pg/mL in compensated, and 56±4 pg/mL in decompensated) (Fig 1a). Plasma renin activity was significantly higher in rats with decompensated CHF (895±173 ng/dL per hour) compared with sham controls (394±67 ng/dL per hour) and compensated rats with ACF (573±111 ng/dL per hour) (Fig 1b). Similarly, plasma aldosterone concentration was significantly elevated in decompensated rats (143±60 ng/dL) compared with sham controls (33±4 ng/dL) and compensated rats (21±6 ng/dL) (Fig 1c).
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Fig 2 shows data on renal responses to NEP inhibition. In control rats, injection of the NEP inhibitor induced significant diuresis and natriuresis throughout the experiment. Urine flow rate increased from a baseline of 12±2 µL/min to a maximum of 99±16 µL/min (P<.0001) after 90 minutes. UNaV and fractional sodium excretion (FENa) increased from 1.22±0.35 µmol/min and 0.32±0.09% to a maximum of 9.4±1.8 µmol/min (P<.0003) and 3.0±0.67% (P<.0001), respectively. These effects were associated with only minor changes in GFR. Administration of the same dose of NEP inhibitor to rats with compensated CHF resulted in a significant increase in urine output (from 14±7 µL/min to a maximum of 49±9 µL/min, P<.0012) and sodium excretion (FENa increased from 0.15±0.05% to a maximum of 1.96±0.33%, P<.0001). However, the absolute increases in these parameters were slightly attenuated compared with controls. In contrast, NEP inhibition did not significantly alter urine flow rate in rats with severe CHF (from 14±8 µL/min to a maximum of 25±12 µL/min, P=NS) or UNaV and FENa throughout the study (UNaV increased from 0.18±0.06 µmol/min to a maximum of 0.56±0.39 µmol/min, P=NS; FENa, from 0.08±0.03% to a maximum of 0.38±0.22%, P=NS). Renal responses to NEP inhibition were independent of changes in GFR (Fig 2b). While NEP inhibition significantly decreased mean arterial pressure in both normal and compensated rats with CHF to the same extent, it induced slight and not significant reductions in decompensated rats (Table).
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In Vitro Protocols
Quantitative RT-PCR
PCR amplification detected a single band of the expected length
for NEP. The amounts of this product, compared with the actin quantity
control, were similar in kidneys of control rats (NEP/ß-actin: 1.05)
and rats with either compensated (NEP/ß-actin: 1.14) or decompensated
(NEP/ß-actin: 0.84) CHF. In contrast, the amounts of the PCR product
were lower in lungs of decompensated rats (NEP/ß-actin: 0.17)
compared with either compensated rats (NEP/ß-actin: 0.29) or control
rats (NEP/ß-actin: 0.31) (Fig 3). Thus, renal NEP mRNA
does not increase as a result of CHF.
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Western Blot Analysis
A polyclonal antibody to rat NEP was used for determination of NEP
immunoreactivity in membranal preparations extracted from lungs and
kidneys of the different experimental groups. A major band of 95 000
D, comigrating with pure recombinant NEP, was detected. In agreement
with the PCR findings, the intensity of this band in renal tissue was
similar in sham controls and compensated rats and slightly less in
decompensated rats. Amounts of immunoreactive NEP in lungs of
decompensated rats were significantly less than in control and
compensated rats (Fig 4).
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Activity Gel
Incubation of the recombinant NEP with gel copolymerized with
gelatin resulted in gelatin degradation in the zone corresponding to 95
kD. Incubation of renal and pulmonary membranal preparations from the
different experimental groups created proteolytic zones at the same
level as that produced by recombinant NEP. When equal amounts of
protein were loaded onto each lane of the activity gel, no differences
were observed between NEP activity in the renal tissues of sham
controls and the two rat subgroups with CHF. Consistent with the
findings mentioned above, NEP activity in lungs of rats with
decompensated CHF was significantly less than that obtained in those of
control and compensated rats. Interestingly, pulmonary NEP activity in
compensated rats was slightly less than it was in sham controls but
greater than the activity in the lungs of decompensated rats (Fig 5).
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Taken together, the results show that NEP mRNA, immunoreactive NEP, and NEP activity do not differ in kidneys from the different rat groups, whereas rats with severe CHF exhibit decreased pulmonary levels of NEP.
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Discussion |
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The present study demonstrates that NEP inhibition in normal rats and rats with compensated CHF induced significant diuresis and natriuresis, whereas the renal response to NEP inhibition was blunted in rats with decompensated CHF. NEP mRNA, immunoreactivity, and activity were similar in renal tissues of control rats and rats with CHF. In contrast, NEP expression, concentration, and activity were lower in lungs of rats with decompensated CHF compared with either compensated rats or sham-operated controls. These findings suggest that NEP inhibition would be more beneficial in mild than in severe CHF. Renal NEP activity was not affected by CHF, whereas pulmonary NEP activity was downregulated in decompensated CHF, possibly as a mechanism to preserve ANP, a valuable circulatory factor in this condition.
The involvement of NEP in the metabolism of ANP and other natriuretic peptides has been established.25 33 34 This enzyme, most abundant in the brush border of the proximal nephron and in the lungs,25 33 cleaves other biologically active peptides, including enkephalins and bradykinin.25 26 27 These NEP substrates, especially ANP, are beneficial in the regulation of renal function and sodium homeostasis.35 Therefore, modulation of the enzyme responsible for their degradation, ie, NEP, was pursued as a therapeutic strategy in cardiorenal disease states characterized by a disorder of sodium balance. Consistent with this assumption, NEP inhibition with specific inhibitors induced natriuresis in both physiological36 37 and pathophysiological21 22 23 24 25 conditions, including CHF. However, the efficacy of NEP inhibition as a natriuretic agent, as well as NEP expression and activity in renal and pulmonary tissues, was not determined in CHF of different severities. The present study shows that SQ-28,603 was a more effective natriuretic agent in rats with compensated CHF than in decompensated rats. Similarly, Northridge et al22 demonstrated that intravenous infusion of UK 69,578 (another specific NEP inhibitor) into patients with mild cardiac failure induced a significant increase in plasma ANP levels in association with natriuresis and diuresis. Wilkins et al23 also reported that infusion of the NEP inhibitor thiorphan into rats with ACF induced a natriuretic response (UNaV increased approximately fourfold) that exceeded that seen in normal rats. The authors did not specify the severity of the heart failure of these rats, but the renal response in their study is similar to that in our rats with compensated CHF. Margulies et al38 showed that when SQ-28,603 was given intravenously to dogs with severe CHF, it induced only a slight and insignificant increase in UNaV. This pattern of natriuretic response after NEP inhibition is similar to that observed after ANP infusion, in which ANP induces significant natriuresis in mild but not in severe CHF.8 13 This suggests that NEP inhibition acts through an ANP-mediated mechanism. The assumption is supported by findings that plasma ANP levels increase after NEP inhibition25 33 and that administration of polyclonal antibodies to ANP abolish the NEP inhibitor natriuretic response.39 However, such an interpretation is not consistent with the known attenuated renal response to ANP in heart failure.8 9 10 11 Since even a high dose of ANP would not induce a large natriuretic response in CHF, it is unclear how increasing the endogenous level of ANP by NEP inhibition would be beneficial. It could be that NEP inhibition increases the intratubular concentration of ANP, allowing it to stimulate ANP receptors in the distal nephron,21 whereas increasing circulatory levels of ANP may not have such an effect because any filtered ANP into the renal tubules is subject to inactivation by the high NEP levels in the brush border. Moreover, Cavero et al21 demonstrated that infusion of SQ-28,603 into dogs with CHF resulted in diuresis and natriuresis, with an increase in the fractional excretion of lithium, a marker for sodium reabsorption in the proximal tubule. These effects were independent of GFR or renal blood flow, suggesting that NEP inhibition exerts an increase in sodium excretion by a tubular mechanism. Potentiation of ANP could be implicated in such a mechanism because its natriuretic action is also due to direct tubular effects.40 41
The reasons for the different natriuretic responses to NEP inhibition in decompensated and compensated rats remain unclear. However, like ANP, activation of the RAAS in the former may contribute to the blunted response to NEP inhibition. Long-term inhibition of the RAAS potentiated the renal effects of NEP inhibition in dogs with severe CHF.38 Furthermore, Bralet et al42 reported that Alatriopril, a mixed inhibitor of angiotensin-converting enzyme and NEP, had a more potent antihypertrophic effect than captopril in rats with myocardial infarction. Taken together, these findings suggest that dual inhibition of the RAAS and NEP is a better therapeutic approach to severe CHF than either one alone.
Although the hemodynamic and renal effects of NEP inhibition have been studied extensively, little is known about the modulation of the expression and activity of this enzyme. Based on the results obtained from experiments with NEP inhibitors in CHF, it was tempting to speculate that either the expression or activity of NEP is elevated in this condition. However, the results of the present study show unequivocally that this is not the case. NEP expression in decompensated CHF was unaltered in the kidney and even decreased in the lungs compared with that in either controls or rats with compensated CHF. This finding is in agreement with a preliminary communication by Trippodo et al,43 who reported that neither NEP gene expression nor NEP activity was upregulated in rats with heart failure induced by ligation of the left main coronary artery. Most recently, Huang et al44 reported that pulmonary NEP activity was not different between normal rats and rats with myocardial infarctions of different severities. The differences between the results of Huang et al and the present study may be due to differences in the severity of the heart failure. The rats of Huang et al with severe CHF lived 3 months, whereas the rats with severe CHF in the present study survived for only 7 to 10 days.
Our finding of reduced NEP levels in the lungs of rats with CHF provides an additional explanation for the high levels of circulating ANP in this disorder, in which atrial production of the peptide is increased. Pulmonary NEP probably modulates circulatory levels of ANP and other peptides. Renal NEP, because of its extracirculatory location on the brush border of the proximal tubule probably acts to prevent these peptides from reaching the distal nephron, as well as to prevent their urinary excretion. The decrease in the levels of pulmonary NEP found in the present study paralleled the severity of the CHF, consistent with the increased levels of circulatory ANP found in severe CHF. In contrast, renal NEP was not altered in CHF, suggesting that renal NEP does not play a key role in the modulation of circulatory ANP in CHF.
In summary, we found that neither NEP expression nor activity was increased in CHF. This rules out the possibility that the sodium retention and blunted response to ANP in CHF are due to elevated levels of the enzyme. The mechanism for the beneficial renal effects of NEP inhibition in this disorder do not depend on an upregulated renal NEP.
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Acknowledgments |
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The authors wish to thank Dr Louis Hersh (University of Kentucky) for the gift of the antibody to NEP, Dr Robert L. Bridenbaugh (Genentech) for the gift of recombinant NEP, and Bristol-Myers Squibb Co for the gift of SQ-28,603.
Received November 9, 1994; first decision December 9, 1994; accepted January 25, 1995.
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