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European Journal of Heart Failure 2006 8(7):681-686; doi:10.1016/j.ejheart.2005.12.005
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© 2006 European Society of Cardiology

Intact acute cardiorenal and humoral responsiveness following chronic subcutaneous administration of the cardiac peptide BNP in experimental heart failure

Horng H. Chen*, John A. Schirger, Alessandro Cataliotti and John C. Burnett, Jr.

Cardiorenal Research Laboratory, Division of Cardiovascular Diseases, Department of Internal Medicine and Department of Physiology, Mayo Clinic College of Medicine, MN, United States

ast; Corresponding author. Cardiorenal Research Laboratory, Guggenheim 915, Mayo Clinic and Foundation, 200 First St. SW, Rochester, MN 55905, United States. Tel.: +1 507 284 4343; fax: +1 507 266 4710. E-mail address: Chen.homg{at}mayo.edu (H.H. Chen).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
Background: BNP is a cardiac peptide with vasodilating, lusitropic and natriuretic properties mediated by the second messenger cGMP. We have previously shown that chronic subcutaneous (SQ) administration of BNP in experimental CHF resulted in improved haemodynamics and unloading of the heart. However, it is unknown if this will lead to the development of tolerance to exogenous BNP.

Methods: The current study extends our previous study and compares the cardiorenal effects of acute administration of SQ BNP (5 µg/kg) in a group of dogs (n = 5) with rapid ventricular pacing induced CHF (180 bpm for 10 days) to a separate group of CHF dogs (n = 6), who received chronic SQ BNP (5 µg/kg) three times a day for 10 days.

Results: Acute administration of SQ BNP resulted in similar increases in both plasma cGMP (35 ± 5 vs. 29 + 2 pmol/ml) and urinary cGMP excretion (UcGMPV) (6000 ±1000 vs. 4000 ± 600 pmol/min) in both the Chronic SQ BNP treated and the Untreated CHF groups (P>0.05). These were associated with decreased cardiac filling pressures and increased urine flow, which were also similar in both groups.

Conclusion: In experimental CHF, chronic SQ BNP administration did not result in the development of tolerance as demonstrated by increases in both plasma cGMP and UcGMPV following acute administration of SQ BNP. This may have important clinical implications, suggesting that chronic BNP administration does not lead to the development of tolerance to acute BNP administration.

Key Words: Heart failure • Natriuretic peptides • Kidney

Received February 24, 2005; Revised December 2, 2005; Accepted December 15, 2005


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 
Brain natriuretic peptide (BNP) is a cardiac peptide with natriuretic, renin-inhibiting, vasodilating, anti-fibrotic and lusitropic properties [1-3]. Studies have established that BNP binds to the natriuretic peptide A receptor (NPR-A) which, via 3',5'-cyclic guanosine monophosphate (cGMP), mediates its biological actions in maintaining cardiorenal homeostasis [4,5]. In the United States, the Food and Drug Administration has recently approved intravenous therapy with a recombinant form of human BNP (nesiritide, Scios Inc. Fremont, CA), as a therapeutic approach for acutely decompensated human chronic heart failure (CHF). These investigations have demonstrated that acute intravenous BNP administration decreases pulmonary artery pressure (PAP), pulmonary capillary wedge pressure (PCWP), right atrial pressure (RAP), mean arterial pressure (MAP) and increases cardiac index without associated neurohumoral activation [6,7]. However, the need for intravenous administration has limited the clinical use of BNP as a long-term therapeutic strategy for the management of CHF.

We have previously demonstrated that acute subcutaneous (SQ) administration of BNP in experimental heart failure (HF) results in elevation of plasma BNP and its second messenger cGMP with natriuresis and a reduction in cardiac filling pressures in the absence of activation of renin angiotensin aldosterone system (RAAS) [8]. Furthermore, chronic 10-day SQ BNP administration in experimental CHF resulted in increases in cardiac output (CO) and decreases in pulmonary capillary wedge pressure and systemic vascular resistance [8]. To date, it is unclear if chronic SQ BNP administration would lead to the development of tolerance to exogenous BNP, particularly with a reduced ability to generate the second messenger cGMP and associated biological responses. This question is clinically relevant as there are ongoing clinical studies to determine if strategies to administer BNP on a more long term basis such as chronic SQ administration of BNP [9] or repeated out-patient infusion (FUSION II Study) [10] may have clinical benefit. Therefore the issue of whether it would lead to the development of tolerance and the lack of therapeutic efficacy needs to be clarified.

The objective of the current study was to extend out previous study and define the cardiorenal effects of acute administration of SQ BNP (5 µg/kg) in a group of Untreated CHF dogs (n=5) with rapid ventricular pacing induced CHF (180 bpm for 10 days) to a separate group of CHF dogs (n=6), who received Chronic SQ BNP (5 µg/kg) three times a day during the 10 days of pacing.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgment
 References
 
Studies were conducted in two groups of anesthetized male mongrel dogs (18-23 kg) with chronic heart failure (CHF) produced by rapid ventricular pacing at 180 beats per minute (bpm) for 10 days on a fixed sodium diet [11]. The two groups consisted of an Untreated CHF Group (n=5), and Chronic SQ BNP Group (n=6). We have previously reported the cardiorenal and humoral function of the Untreated CHF Group in response to acute SQ BNP and the cardiorenal function of Chronic BNP Group after 10 days of chronic SQ BNP administration [8]. In the current paper, we compare the cardiorenal and humoral response to acute SQ BNP administered on day 11 in both the Untreated CHF group and the Chronic BNP Group, which has not been published previously. Studies were performed in accordance with the Animal Welfare Act and with approval of the Mayo Clinic Institutional Animal Care and Use Committee.

2.1. Model of pacing-induced chronic heart failure
All dogs underwent implantation of a programmable cardiac pacemaker (Medtronic, Minneapolis, MN). Under pentobarbital sodium anesthesia (30 mg/kg, intravenous) and artificial ventilation (Harvard respirator, Harvard Apparatus, Millis, MA) with 5 l/min supplemental oxygen, a left lateral thoracotomy and pericardiotomy were performed. With the heart exposed, a screw-in epicardial pacemaker lead was implanted into the right ventricle. The pacemaker generator was implanted subcutaneously into the left chest wall and connected to the pacemaker lead. Dogs received pre- and post-operative prophylactic antibiotic treatment with 225 mg clindamycin subcutaneously and 400,000 U procaine penicillin G plus 500 mg dihydrostreptomycin intramuscularly (Combiotic, Pfizer, Inc., New York, NY). Post-operative prophylactic antibiotic was continued through the first two post-operative days. Dogs were fed a fixed sodium diet (58 mEq/day, Hill's ID) and allowed water ad lib. All dogs were walked daily. Appetite, activity, body temperature and condition of surgical skin sites were documented.

2.2. Experimental protocol
Following a 14-day post-operative recovery period, the pacemaker was turned on at 180 bpm. Group 1 (Untreated CHF Group, n=5) received no therapy during the 10 days of pacing. Group 2 (Chronic SQ BNP Group, n=6) received SQ administration of canine BNP at a dose of 5 µg/kg every 8 h throughout the 10 days of pacing.

On day 11 of rapid ventricular pacing at 180 bpm acute SQ BNP (5 µg/kg) was administered to both groups of dogs. On the night before the acute experiment, animals were fasted. For the chronic SQ BNP group, the acute experiment was carried out 8 h after the last dose of SQ BNP. On the day of the acute experiment, dogs were anesthetized with sodium pentobarbital (15 mg/kg, i.v.), intubated and mechanically ventilated with supplemental oxygen (Harvard respirator, Amersham, MA) at 20 cycles per minute. A flow-directed balloon-tipped thermodilution catheter (Ohmeda, Criticath, Madison, WI) was advanced into the pulmonary artery via the external jugular vein for cardiac haemodynamic measurement. The femoral artery was cannulated for blood pressure monitoring and blood sampling. The femoral vein was also cannulated for inulin and normal saline infusion. The left kidney was exposed via a flank incision and the ureter was cannulated for urine collection.

The experiment began after a 60-min equilibration period, with a 30-min baseline urinary clearance. After the 30-min baseline urinary clearance, canine BNP 5 µg/kg dissolved in 1 ml of normal saline was given subcutaneously in the left hind limb. Following a 15-min lead in period, a 30-min urinary clearance period was performed. Cardiovascular parameters measured during the acute experiment included MAP, RAP, PAP, cardiac output (CO) and PCWP. CO was determined by thermodilution in triplicate and averaged (Cardiac Output model 9510-A computer, American Edwards Laboratories, Irvine, CA). MAP was assessed via direct measurement from the femoral arterial catheter. Systemic vascular resistance (SVR) was calculated as [SVR=(MAP–RAP)/CO]. Inulin was administered intravenously at the start of the equilibration period as a calculated bolus, followed by a 1 ml/min continuous infusion to achieve plasma levels of 40-60 mg/dl. Glomerular filtration rate (GFR) was measured by inulin clearance.

Cardiovascular haemodynamics were measured at the start of each urinary clearance. Arterial blood was collected in heparin and EDTA tubes and immediately placed on ice midway through each clearance. After centrifugation at 2500 rpm at 4 °C, plasma was decanted and stored at –20 °C until analysis. Urine was collected on ice during the entire period of each clearance for assessment of urine volume, electrolytes and inulin. Urine collected for cGMP analysis was heated to more than 90 °C before storage.

2.3. Hormonal and electrolyte analysis
After extraction, plasma BNP was measured by radioimmunoassay (RIA) as previously described [8,12]. Plasma and urinary samples for cGMP were measured by RIA using the method of Steiner et al. [13]. Urinary and plasma inulin concentrations were measured by the anthrone method.

2.4. Statistical analysis
Results are expressed as mean±SEM. Student's unpaired t-test for single comparison between any two groups and Student's paired t-tests were performed for single comparison within each group using GraphPad Prism software. Statistical significance was accepted as P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgment
 References
 
3.1. Baseline cardiorenal characteristics on day 11 of rapid ventricular pacing at 180 bpm
Table 1 reports the baseline cardiorenal characteristics on day 11 of rapid ventricular pacing at 180 bpm prior to the administration of acute SQ BNP in the Untreated CHF Group and the Chronic SQ BNP Group. As reported previously, 10 days of chronic SQ BNP administration increased CO and decreased PCWP and SVR, compared to Untreated CHF Group [8].


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  		Table 1 Baseline characteristics on day 11 of rapid ventricular pacing at 180 bpm prior to the administration of acute SQ BNP in the untreated CHF group and the chronic SQ BNP group

 
3.2. Acute SQ BNP administration on day 11of rapid ventricular pacing at 180 bpm
Fig. 1 illustrates the responses in plasma BNP, cGMP and urinary cGMP excretion (UcGMPV) to acute SQ BNP administration in the Untreated CHF and the Chronic SQ BNP Groups on day 11 of rapid ventricular pacing at 180 bpm. Plasma BNP, cGMP and UcGMPV increased significantly and similarly (P>0.05) 30 min after administration of SQ BNP in both the Untreated CHF Group and the Chronic SQ BNP Group. In the chronic SQ BNP group, plasma BNP level 7 h after the last dose (pre-dose) was 50±24 pg/ml and plasma BNP level after 1 h of the acute dose was 222±119 pg/ml. Furthermore, in the chronic SQ BNP group there was a non-significant decrease in both renin (3.2±2.6 to 0.6±0.4 ng/ml/h) and aldosterone (7.3±1.4 to 5.7±0.6 ng/dl) 30 min after the acute SQ BNP administration.


Figure 1
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  		Fig. 1 Plasma BNP and plasma cGMP and urinary cGMP excretion (UcGMPV) at baseline {rectangle} and 30 min {image} after subcutaneous BNP (SQ BNP) administration on day 11 in the Untreated CHF group and the chronic SQ BNP Group. *P<0.05 at 30 min after SQ BNP {image} vs. baseline {rectangle}.

 
Fig. 2 illustrates the responses in PCWP, RAP and PAP to acute SQ BNP administration in the Untreated CHF and the Chronic SQ BNP groups on day 11 of rapid ventricular pacing at 180 bpm. Despite the fact that baseline PCWP was significantly lower in the Chronic SQ BNP Group as compared to the Untreated CHF Group, there was a significant decrease in PCWP (delta: –4.1 mm Hg [SQ BNP group] vs. –2.5 mm Hg [Untreated CHF group]) with the acute administration of SQ BNP. Furthermore, RAP (delta: –1.4 mm Hg [SQ BNP group] vs. –1.0 mm Hg [Untreated CHF group]) and PAP (delta: –4.5 mm Hg [SQ BNP group] vs. –5.5 mm Hg [Untreated CHF group]) deceased similarly in both groups. With administration of acute SQ BNP, there was a non-significant trend for the CO to decrease in the chronic SQ BNP group (3.6±0.4 to 2.8±0.3 l/min) but CO was unchanged in the untreated CHF group (2.5±0.1 to 2.4±0.1 l/min). MAP and SVR did not change significantly with the acute SQ BNP in both groups.


Figure 2
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  		Fig. 2 Pulmonary capillary wedge pressure (PCWP), right atrial pressure (RAP) and mean pulmonary artery pressure (PAP) at baseline {rectangle} and 30 min {image} after subcutaneous BNP (SQ BNP) administration on day 11 in the Untreated CHF group and the chronic SQ BNP Group. *P<0.05 at 30 min after SQ BNP {image} vs. baseline {rectangle}. {dagger}P<0.05 Untreated CHF Group vs. Chronic SQ BNP Group.

 
Fig. 3 illustrates the responses in Urine flow and UNaV to acute SQ BNP administration in the Untreated CHF and the Chronic SQ BNP groups on day 11 of rapid ventricular pacing at 180 bpm. Both urine flow and UNaV significantly increased after SQ BNP in both groups. Baseline UNaV trended to be lower in the Chronic SQ BNP Group and the increase in UNaV was significantly lower in response to acute SQ BNP as compared to the Untreated CHF Group. Both proximal (75±6 to 63±7% SQ BNP group; 75±7 to 64±7% Untreated CHF group) and distal (96±1 to 87±1% SQ BNP group; 97±1 to 87±1% Untreated CHF group) tubular sodium reabsorption decreased similarly in both groups.


Figure 3
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  		Fig. 3 Urine flow and Urinary sodium excretion at baseline {rectangle} and 30 min {image} after subcutaneous BNP (SQ BNP) administration on day 11 in the Untreated CHF group and the chronic SQ BNP Group. *P<0.05 at 30 min after SQ BNP {image} vs. baseline {rectangle}. {dagger}P<0.05 Untreated CHF Group vs. Chronic SQ BNP Group.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgment
 References
 
The objective of the current investigation was to define if chronic SQ BNP administration three times a day over a period of 10 days in experimental CHF would result in the development of tolerance to exogenous BNP administration. We demonstrated that in this model of experimental CHF, chronic SQ BNP administration did not result in the development of tolerance as demonstrated by similar increases in both plasma cGMP and UcGMPV in the chronic BNP treated and the untreated groups with acute administration of SQ BNP. Furthermore, acute administration of SQ BNP resulted in a similar decrease in cardiac filling pressures and increase in urine flow. This may have important clinical implications, suggesting that chronic SQ BNP administration does not result in diminished therapeutic efficacy of acute BNP administration.

Within 30 min of acute SQ administration of BNP, plasma BNP significantly increased in both the Untreated CHF and the Chronic SQ BNP Groups. Thus demonstrating that chronic SQ BNP administration did not affect the absorption of subcutaneously administered BNP. More importantly both plasma cGMP and urinary cGMP excretion increased to a similar extent in both groups suggesting that there was no development of tolerance to exogenous BNP in the Chronic SQ BNP Group. Thus, BNP was rapidly absorbed and resulted in activation of NPR-A receptors, as demonstrated by parallel increases in both plasma and urinary cGMP, markers of tissue and renal biological activity of the natriuretic peptides [14]. The exact mechanism for the lack of development of tolerance with chronic SQ BNP is unknown and is most likely multifactorial. We have previously reported that plasma BNP returns to baseline approximately 120 min after SQ BNP administration, therefore one potential mechanism for the lack of development of tolerance could be the fact the NPR-A receptors are stimulated intermittently [8]. Further studies are required to define the expression of the NPR-A receptors with chronic SQ BNP.

Despite the fact that the baseline PCWP was significantly lower in the chronic SQ BNP group, there was a further reduction of PCWP with acute administration of acute SQ BNP. RAP and PAP were also significantly decreased therefore suggesting a preservation of the vascular response to exogenous BNP despite 10 days of chronic SQ BNP.

Baseline UNaV trended to be lower in the Chronic SQ BNP Group as compared to the Untreated CHF Group, however, glomerular filtration rate (GFR) (Table 1) was similar between both groups. The dogs were kept on a fixed sodium diet, we speculate that lower baseline UNaV is most likely due to decreased total body sodium as a result of chronic SQ BNP administration rather then a deterioration in renal function, as GFR was similar. As a result of this decreased total body sodium, the increase in UNaV in response to acute BNP was significantly less in the Chronic SQ BNP Group as compared to the Untreated CHF Group. Furthermore, the diuretic action of exogenous BNP was similar between the two groups.

The findings of the current study may be of clinical relevance. Human recombinant BNP (nesiritide) is used for the management of acute decompensated CHF. Currently, there are ongoing clinical studies to determine if strategies to administer BNP on a more long term basis such as chronic SQ administration of BNP [9] or repeated out-patient infusion (FUSION II Study) [10] may have clinical benefits. Therefore the issue of whether long term administration of BNP would lead to the development of tolerance and the lack of therapeutic efficacy needs to be clarified. Our findings would suggest that with 10 days of SQ BNP administration, there was improvement of baseline haemodynamics and no development of tolerance to exogenous BNP administration. Further studies are needed to reproduce these findings in human CHF.


   Acknowledgment
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgment
References
 
The authors gratefully acknowledge the assistance of Denise M. Heublein, Sharon S. Sandberg and Gail Harty.

This research was supported by grants HL 36634 and HL 07111 from the National Institutes of Health, Miami Heart Research Institute, Mayo Foundation, Joseph P and Jeanne Sullivan Foundation, American Heart Association National Scientist Development Grant and American College of Cardiology Career Development Grant awarded to Dr. Horng Chen.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgment
 References
 

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  8. Chen H.H., Grantham J.A., Schirger J.A., et al. Subcutaneous administration of brain natriuretic peptide in experimental heart failure. J Am Coll Cardiol (2000) 36:1706–1712.[Abstract/Free Full Text]
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  14. Wada A., Tsutamoto T., Matsuda Y., et al. Cardiorenal and neurohumoral effects of endogenous atrial natriuretic peptide in dogs with severe congestive heart failure using a specific antagonist for guanylate cyclase-coupled receptors. Circulation (1994) 89:2232–2240.[Abstract/Free Full Text]

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