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Original Studies |
Departments of Medicine (J.G.L., A.M.R., M.G.N.) and Endocrinology (E.A.E., T.G.Y.), Christchurch Hospital, Christchurch, New Zealand
Address all correspondence and requests for reprints to: Professor A. Mark Richards, Department of Medicine, Christchurch Hospital, Riccarton Avenue, P.O. Box 4345, Christchurch, New Zealand.
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Abstract |
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BNP infusion and NEP inhibition both induced significant hemodynamic and renal responses. The augmented hypotensive effect of combined treatments, and consequent fall in renal perfusion pressure, may underly the observed blunting of the natriuretic response that occurred despite greater than additive increments in plasma BNP, ANP, and cGMP.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous reports suggest that BNP has better-preserved natriuretic effects than ANP in heart failure (6, 7). It has a longer half-life than ANP and has more powerful blood pressure-lowering actions than ANP in hypertension (8). Neutral endopeptidase 24.11 (NEP) contributes to the initial degradation of both ANP and BNP (9, 10). Although NEP inhibition has been demonstrated to markedly impair metabolism of exogenous ANP in men, the effects on BNP clearance and its biological effects are less well known (11).
This study expands the limited published data on the effects of pathophysiological increments in plasma BNP in those with left ventricular impairment, allows comparison of these effects with those obtained with a single dose of NEP inhibitor, and provides the first report of the effects of BNP and NEP inhibition in combination.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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35% (range
21–35%) as documented by echocardiography, were in New York Heart
Association classes 2 or 3, and had no other major systemic
disorders (including no renal impairment). All were treated with an
angiotensin-converting enzyme inhibitor and furosemide. Five patients
were receiving aspirin, and two were receiving digoxin. Subjects were studied on four occasions, on the third and fifth days of two 5-day constant sodium (100 mmol/day) and potassium (60 mmol/day) diets. The 5-day diet periods were separated by at least 1 week. Patients were studied in a placebo-controlled single-blind fashion. On day 3 of each diet phase, the patients received in balanced, randomized order either BNP plus placebo or placebo alone, and on the fifth day of each diet phase the patients received at random either NEP inhibitor alone or the combination of NEP inhibitor and BNP.
On the morning of each study day patients ate breakfast, presented to the study room, and completed a 24-h urine collection at 0800 h. All medications were withheld for the previous 24 h. On each study day patients drank 150 ml water every 2 h between 0800 and 1600 h and remained seated throughout the day except for standing to pass urine.
Venous cannulae were placed (one in each forearm) for infusion of BNP/placebo and venous sampling. A standard blood pressure cuff was attached to the arm, and blood pressure and heart rate were monitored at 30-min intervals throughout the study by a fully automated oscillometric sphygmomanometer (PP203 MII, Nippon Colin Company, Tokyo, Japan). The mean of two consecutive recordings was used in analysis. Cardiac output was measured noninvasively using the Minnesota Impedance Cardiograph model 304B (Instrumentation For Medicine Incorporated, Greenwich, CT) with measurements at 30-min intervals (12).
After a baseline observation period (60 min), a 4-h infusion of synthetic human BNP (Bachem, Torrance, CA; 3.0 pmol/kg min) in hemaccel (Behring, Hamburg, Germany; 10 ml/h) or vehicle alone was commenced, and patients also received a single oral dose of NEP inhibitor (SCH 42495 [Schering, Bloomfield, NJ]; 300 mg) or placebo tablets. Monitoring and sampling were continued for 2 h after the completion of the 4-h BNP/vehicle infusions.
Venous blood samples were taken for measurement of plasma BNP, ANP, cGMP, plasma renin activity, aldosterone, cortisol, and catecholamines at 30- to 60-min intervals throughout the study day. Plasma NEP activity was measured before the dose of inhibitor or placebo, and 4 h later at the end of the BNP or vehicle infusion phase. Plasma sodium, potassium, and creatinine were measured on venous blood taken before and at the conclusion of the infusion phase. Packed-cell volume was measured by the micro-hematocrit method at 2-h intervals throughout the study day. Patients passed urine at set times (beginning and end of the infusion phase and at the end of recordings) and at other times as required. The urine collections during and for the 2 h after the infusion phase (i.e. 6-h collections) were pooled for measurement of sodium, potassium, creatinine, and cGMP.
Hormone, cGMP concentrations, and neutral endopeptidase activity were measured as previously described (5, 13, 14, 15, 16, 17). To eliminate interassay variation, all samples from all study phases for an individual patient were assayed together. The intraassay coefficients of variation fell between 5 and 9%.
The metabolic clearance rate for BNP was calculated for each individual using the formula metabolic clearance rate = measured infusion rate/(steady-state-baseline BNP level). The mean of the 120-, 180-, and 240-min values of plasma BNP was used as the "steady-state" value.
Net renal generation of cGMP was calculated using the formula net renal generation of cGMP = (urinary cGMP x urine flow rate) - (plasma cGMP x creatinine clearance).
Echocardiographic examinations were performed by a single technician, who was blinded to the study phase, at the midpoint of the infusion phase on each study day for measurement of left ventricular dimensions and ejection fraction by M-mode and mitral transvalvar diastolic flow by pulsed-wave Doppler.
The data were analyzed by two-way ANOVA with treatment and time as repeated measures (18). P < 0.05 was considered significant. Values are expressed as mean ± SEM.
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Results |
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Twenty-four-hour urine indices, collected just before each study day, were not significantly different (results before BNP and placebo, inhibitor and placebo, combined BNP and NEP inhibitor, and placebo, respectively; volume: 1791 ± 273, 1463 ± 240, 1892 ± 291, and 1678 ± 266 ml/24-h; sodium: 86.75 ± 9, 99.9 ± 11, 71.6 ± 7, and 101 ± 15 mmol/24 h; potassium: 53 ± 4, 51.6 ± 5, 60.8 ± 5, and 61.7 ± 6 mmol/24 h; creatinine: 12.1 ± 1.1, 13.0 ± 1.2, 11.1 ± 1, and 12.2 ± 1.1 mmol/24 h).
Baseline plasma BNP was similar for each study day and was stable
before the experimental studies (Fig. 1
).
Mean baseline plasma BNP levels (19.5 ± 3.0 pmol/L) were
approximately 4-fold the mean for normals (4.8 pmol/L, n = 168) in
our laboratory. Mean rate of BNP infusion confirmed by RIA of infusates
was 3.0 ± 0.2 pmol/kg min and 3.1 ± 0.3 pmol/kg min for BNP
given alone and in combination with NEP inhibition, respectively (not
significant). Plasma NEP activity was markedly suppressed on both
occasions when inhibitor was given but was unaffected by BNP infusion
alone or by placebo (Table 1). The
greatest increase in plasma BNP was seen with combined BNP and NEP
inhibition (P < 0.0001) (Fig. 1). Plasma BNP was also
significantly increased by BNP administration alone (P
< 0.001), and a smaller increase was induced by inhibitor alone
(P = 0.001). At the completion of recordings, plasma
BNP remained significantly elevated compared with placebo after both
NEP inhibition alone (P = 0.001) and the combined
treatment (P < 0.001). The metabolic clearance rate of
BNP for infusion of the peptide alone and for combined phases was
5.97 ± 0.46 L/min and 4.49 ± 0.38 L/min, respectively
(P = 0.03).
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) but were
significantly elevated by both NEP inhibitor alone (P =
0.006) and combined BNP infusion and NEP inhibition (P
= 0.003). By the end of the recordings, the plasma ANP remained
significantly elevated after combination treatment and inhibitor alone
(P < 0.01 for both). The increase in endogenous plasma
ANP levels induced by NEP inhibition alone (approximately 55 pmol) was
more than 10-fold the concomitant increase in endogenous plasma
BNP.
Plasma cGMP was elevated by both BNP alone (peak = 16.6 ±
2.1 pmol/ml, P = 0.006) and the inhibitor alone
(peak = 18.6 ± 4.9 pmol/ml, P = 0.01)
compared with placebo (peak = 6.9 ± 1.1 pmol/ml) (Fig. 1).
By far the greatest increase in plasma cGMP was seen with combined BNP
and NEP inhibition (peak = 44.8 ± 8.9 pmol/ml,
P = 0.002, 0.005, 0.002 compared with placebo, BNP
alone, and inhibitor alone, respectively). At the end of recordings
plasma cGMP remained elevated compared with placebo after both
inhibitor alone and combined treatment (P = 0.02 and
0.009, respectively), but had returned to placebo levels within 2
h after cessation of infusions of BNP alone.
Plasma aldosterone levels tended to fall but were not significantly
different from placebo during the infusion phase on all four study days
but rose markedly above time-matched placebo values after both combined
treatment and infusions of BNP alone (P = 0.02 and
0.05, respectively) (Fig. 2). Plasma
renin activity (Fig. 2) and norepinephrine did not differ significantly
between study phases (Table 1). Epinephrine was slightly raised by
inhibitor alone compared with placebo (P = 0.05) (Table 1). Plasma levels of cortisol were elevated by all three active
treatments compared with placebo (P = 0.04, 0.01, and
0.02 for BNP, NEP inhibition, and combined agents, respectively) (Table 1). There were no significant effects on plasma sodium, potassium, and
creatinine (data not shown).
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). Urine volume was significantly
increased by the NEP inhibitor (164.7 ± 20.6 vs.
112.3 ± 13.2 ml/h, P = 0.01), but did not differ
significantly from placebo on the other study days (Fig. 3).
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) was significantly increased by each
active treatment (P = 0.01, 0.001, and 0.01 for
combined agents, BNP, and inhibitor, respectively), with greater
augmentation by inhibitor alone and the combined treatment than by BNP
alone (P < 0.01 for both comparisons). Calculated net
renal generation of cGMP was significantly higher than placebo with
inhibitor alone (P = 0.02) but was not significantly
increased by the combined agents or by BNP alone (Fig. 3).
Systolic and diastolic arterial pressures were significantly reduced by
all active treatments (P < 0.01 for all comparisons)
(Fig. 4). The greatest reductions in both
systolic and diastolic pressures were seen with the combined treatment,
the fall in both being significantly greater than that induced by BNP
alone or inhibitor alone (P < 0.05 for all
comparisons). At the end of the recordings (6 h after initiation of BNP
infusion and administration of inhibitor dose), systolic and diastolic
blood pressures remained significantly below time-matched placebo
levels after combined treatments and after BNP alone (P
< 0.05 for all comparisons).
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). Cardiac output was
unaffected by any treatment (Fig. 4).
Hematocrit was elevated, compared with placebo, by BNP alone
(P = 0.04) (Table 1).
M-mode left ventricular systolic dimensions were reduced significantly compared with placebo by both combined agents (56.6 ± 3.1 vs. 52.7 ± 2.9 mm, P = 0.006) and inhibitor alone (56.6 ± 3.1 vs. 48.7 ± 2.9 mm, P = 0.009) and tended to be reduced by BNP alone (56.6 ± 3.1 vs. 50.6 ± 3 mm, P = 0.06). Diastolic dimension was significantly reduced compared with placebo by both BNP alone (66.7 ± 3.8 vs. 61.6 ± 3.2 mm, P = 0.04) and inhibitor alone (66.7 ± 3.8 vs. 63.6 ± 3 mm, P = 0.04) and tended to be reduced by the combined agents (66.7 ± 3.8 vs. 63 ± 3 mm, P = 0.09). Calculated ejection fraction and mitral valve diastolic flow values were not altered on any study day. (M-mode ejection fraction for placebo, BNP, inhibitor, and combined agents, respectively, = 38 ± 0.04%, 36 ± 0.04%, 35 ± 0.03%, and 32 ± 0.03%, not significant; E:A ratios for placebo, BNP, inhibitor, and combined agents respectively = 0.89 ± 0.12, 0.80 ± 0.05, 0.84 ± 0.04, and 0.92 ± 0.10, not significant).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of BNP infusion alone are reminiscent of those seen with ANP infusions in patients with heart failure (20) and are similar to our previous results with BNP (21). They contrast, however, with data from Yoshimura et al. (6) who reported a substantial natriuretic and diuretic response to BNP without alteration in systemic pressures but with a fall in cardiac filling pressures. A number of differences between studies may reconcile the discrepant findings. Yoshimura et al. (6) provide no time-matched control data, and the natriuresis and diuresis reported were confined to a total duration of 1 h. In addition, mean arterial pressure was relatively high in their heart-failed patients. In comparison with the current experiment, a much higher dose of BNP causing a 20-fold rise in plasma BNP concentration was used. In our own study, falls in blood pressure did not commence for the first hour of BNP infusions, and it is therefore not surprising that Yoshimura et al. (6) recorded no effect on blood pressure during a brief 30-min infusion. The more recent work by Marcus et al. (7) confirms the significant hemodynamic effects of BNP infusion in patients with impaired left ventricular function. They observed significant falls in systemic arterial pressure and cardiac filling pressures with BNP infusion over a range of doses, and although they recorded an increase in cardiac index, this was only seen at infusion rates approximately 3–10 times those used in the present study. Marcus et al. (7) also observed a significant natriuresis and diuresis with BNP infusion, but these effects were much less than those seen in the study by Yoshimura et al. (6). Peak plasma BNP levels were approximately 20-fold greater than placebo in the study by Marcus et al. (7), yet the increase in sodium excretion with BNP infusion was small (urine sodium excretion 2.6 ± 2.4 meq/h and 1.4 ± 1.2 meq/h for BNP and placebo, respectively). These findings plus those in the present study and our previous results (21) suggest that the natriuretic effects of BNP are attenuated in heart failure, particularly when compared with the effects of infused BNP in healthy volunteers (4) and hypertensive patients (22).
The renal hyporesponsiveness to natriuretic peptides in heart failure is not fully understood. It may be related to reduced renal perfusion pressure (23, 24). This mechanism seems likely to have contributed to our finding that combined BNP infusion and NEP inhibition, which clearly produced the greatest falls in arterial pressure, resulted in less natriuresis compared with NEP inhibition alone. Additional factors that may reduce natriuretic responses to BNP in heart failure, but cannot be evaluated in the current study, include down-regulation of guanylate cyclase-linked natriuretic peptide receptors (25), attenuated renal cGMP production (26), an intracellular defect distal to cGMP production (27), reduced delivery of natriuretic peptide to the distal nephron (28), deficiency of intrarenal kinins (29), and the antinatriuretic, antidiuretic actions of angiotensin II, the renal sympathetic system, and vasopressin (30, 31, 32, 33).
The more marked natriuretic and diuretic effects seen with NEP inhibition compared with BNP infusions (in the face of similar falls in blood pressure) suggest a possible action distal to the renal glomerulus. The NEP inhibitor alone caused a marked increase in circulating ANP with a smaller increment in BNP, both of which would be filtered by renal glomeruli and then possibly protected from breakdown by the normally rich NEP activity in renal brush border membranes by the presence of the NEP inhibitor, thereby facilitating vigorous stimulation of second messenger levels and altered tubular sodium handling, producing a greater natriuretic and diuretic effect. NEP inhibition may similarly protect the natriuretic peptide urodilatin (produced solely within the kidney), which may contribute to the natriuretic response (34). In concert with greater natriuretic bioactivity in the kidney, there was a marked increase in renal generation of cGMP.
As with other effects of endopeptidase inhibition, it cannot be assumed that the renal response necessarily reflects increased tissue and plasma cardiac peptides alone. Other vasoactive substrates to EC 24.11 (e.g. bradykinin) may alter blood pressure and natriuresis (35). However, animal studies indicate that pretreatment with polyclonal anti-ANP antisera attenuates or abolishes the cGMP, natriuretic, and hypotensive effects of NEP inhibitors, whereas antagonists to other substrates (including bradykinin), at least in some models, do not (36, 37, 38). These findings suggest that ANP is a major mediator of the end-organ effects of NEP inhibition.
Endogenous plasma ANP was unaffected by BNP infusions but markedly increased (far more than endogenous BNP) by the endopeptidase inhibitor. NEP has a greater affinity for ANP than BNP (39). It seems likely that the natriuretic effects of NEP inhibition given alone are mediated largely through enhancement of plasma and tissue levels of ANP rather than BNP. The effects of combined BNP infusion and NEP inhibition on plasma ANP clearly exceeded the sum of either alone. This synergism suggests that when peptide levels are elevated, competition for clearance (i.e. for clearance receptors when endopeptidase is inhibited) occurs. This is also in accord with our previous observations in healthy volunteers and patients with hypertension receiving coinfusions of ANP and BNP (40, 41, 42).
Blood pressure fell with infusion of BNP alone and NEP inhibitor alone with an additive effect from combined treatment. The drop in pressure tended to be slightly greater and was more sustained with BNP alone, with a significant reduction in blood pressure persisting at the end of recordings, in contrast to a briefer effect with NEP inhibitor alone. These temporal differences in the blood pressure response to BNP and NEP inhibitor contrast with their effects on plasma cGMP. It is not easy to explain this discrepancy; however, the prolonged effect of natriuretic peptides on hemodynamics has been seen previously in studies of ANP and BNP infusion in heart failure (20, 21). The plasma levels of cGMP may not accurately reflect bioactivity at tissue level.
The hemodynamic effects of endopeptidase inhibition and BNP seen in this study may result from several factors. A direct vasodilator effect of the natriuretic peptides is likely, and in previous studies with administration of ANP and BNP to patients with heart failure, falls in peripheral vascular resistance have been recorded (6, 7, 21, 43). The net loss of fluid volume via the kidneys was trivial. However, fluid shift from the intravascular to the extravascular compartment may have contributed to the fall in blood pressure and the observed reductions in left ventricular dimensions.
The substantial fall in blood pressure (20% fall in systolic pressure) was not accompanied by evidence of increased activity of the renin-angiotensin-aldosterone or sympathetic nervous systems apart from a marginal increase in epinephrine with NEP inhibitor alone. These results suggest relative suppression of these counter-regulatory mechanisms by the active treatments. The postinfusion increase in plasma aldosterone, which rebounds above control levels after release from BNP-containing treatments, would tend to support this concept. There is clearly renin-aldosterone dissociation during this rebound phase at a time when cortisol is increased above placebo levels (by all three active infusions), suggesting that ACTH may be driving the late aldosterone response, which at this time is no longer blunted by high ANP and BNP levels.
An increase in cortisol was seen in this study on each active phase. This has not generally been seen in studies of infused natriuretic peptides or endopeptidase inhibition in heart failure, despite consistent falls in arterial pressure. We cannot rule out hypotension-induced ACTH secretion as the stimulus behind raised plasma cortisol in the current study (indeed, the most hypotensive regime did produce the greatest increment in cortisol), but a fall in cortisol clearance caused by reduced hepatic blood flow is also possible (44).
In summary, low-dose BNP and NEP inhibition both caused clear and additive falls in systemic arterial pressure. NEP inhibition had more effect in elevating ANP than BNP and induced a significant natriuretic and diuretic effect in excess of that observed with BNP infusion. Marked falls in blood pressure with combined treatments probably underlie the concomitant blunted renal response that was observed despite more than additive increments in ANP, BNP, and cGMP. The increased generation of renal cGMP induced by NEP inhibition suggested that protection of natriuretic peptides from degradation in the kidneys may underlie the renal response to such agents. Our findings document the renal effects of BNP and NEP inhibition in left ventricular impairment and suggest that the increases in plasma BNP seen in heart failure have important compensatory effects on hemodynamics, neurohormonal status, and sodium homeostasis. Manipulation of the natriuretic peptide system in heart failure has therapeutic potential and warrants further study.
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Acknowledgments |
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Footnotes |
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Received April 28, 1998.
Revised October 28, 1998.
Accepted November 9, 1998.
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References |
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),
NEP inhibition alone (
), and placebo (
). Plasma BNP was increased
by all active treatments (combined, P < 0.0001; BNP
infusion, P < 0.001; NEP inhibitor, P
= 0.001). Plasma ANP was increased by combined treatment
(P = 0.003) and NEP inhibitor (P =
0.006) but unchanged by BNP infusion. Plasma cGMP was increased by all
active treatments (combined, P < 0.001; BNP,
P < 0.006; NEP inhibitor, P = 0.01).
P
