Introduction

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Human brain natriuretic peptide (hBNP) is a cardiac derived peptide hormone that regulates cardiovascular and renal function (Lewicki, 1995). A synthetic version of endogenous hBNP with an identical amino acid sequence, termed Natrecor, has beneficial effects in patients with congestive heart failure reflected by reduced pulmonary capillary wedge pressure, reduced right atrial pressure and increased cardiac output (Hobbs, 1996; Marcus, 1996; Mills, 1997; Abraham, 1998). The purpose of the studies described here was to assess pathways involved in the elimination of exogenous hBNP from the plasma compartment. As renal blood flow may be impaired in some congestive heart failure patients who may receive Natrecor, the role of the kidney in the elimination of plasma hBNP may be particularly important and was included in this study.

There are at least two biochemical mechanisms that might mediate the elimination of hBNP from the plasma compartment: the natriuretic peptide clearance (NP-C) receptor and peptidase digestion. The NP-C receptor mediates the cellular internalization and subsequent lysosomal degradation of the peptide hormone atrial natriuretic peptide (ANP; Nussenzveig, 1990) and presumably plays a similar role for the structurally related peptide hBNP. It is known that hBNP binds with high affinity to the NP-C receptor (Bennett, 1991). It has been shown that truncated analogs of rat ANP bind with significantly higher affinity to the NP-C receptor than to the guanylyl cyclase-A receptor (Maack, 1987), a known biological receptor for both hBNP and ANP. One NP-C receptor-specific peptide, rat ANP(4-23), has been used to assess the role of the NP-C receptor in the metabolism of ANP in animals (Almeida, 1989). In this report we describe studies that use an NP-C receptor specific analog, [des(18-22)]human ANP-(4-23)-NH₂ (Scarborough, 1986) to assess the role of the NP-C receptor in the metabolism of hBNP in rabbits. It is known that rabbit tissue expresses the NP-C receptor (Murthy, 1998). Previous studies in sheep with this NP-C receptor ligand have shown that it increases plasma levels of endogenous ANP and BNP (Charles, 1996; Rademaker, 1997).

It is known that hBNP is a substrate for neutral endopeptidase (NEP) 24.11 purified from rat (Norman, 1991; Kenny, 1993). In this report we assess the role of peptidases in the elimination of plasma hBNP in rabbits using the peptidase inhibitor phosphoramidon. Phosphoramidon has been shown to be an inhibitor of NEP24.11 (Roques, 1990).

The role of the kidney in the elimination of plasma hBNP was investigated as this organ might clear the peptide by filtration or by metabolism. It is known that the NP-C receptor (Maack, 1987) and NEP24.11 (Sonnenberg, 1988) are abundant in the kidney and might be involved in metabolism of hBNP in this organ. The effect of complete renal blood flow restriction induced by bilateral renal artery suture ligation on the pharmacokinetics of hBNP in rabbits was assessed.

	Experimental Procedures

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Materials. The NP-C receptor-specific peptide used in these studies is an analog of human ANP in which part of the ring structure as well as the carboxy-terminal tail portion is deleted: Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Cys-amide (Scarborough, 1986), termed C-ANP, and was prepared at Scios Inc. using standard peptide synthesis methodologies. Aprotinin and phosphoramidon(N-((-rhamnopyranosyloxyhydroxyphosphinyl)-Leu-Trp, were purchased from Sigma Chemical Co. (St. Louis, MO) and lidocaine HCl, (Xylocaine Jelly) was purchased from Astra (Westborough, MA).

Methods. Male New Zealand White rabbits (R & R Rabbitry, Standwood, WA) weighing between 2.5 and 3.0 kg were used in the study. The animals were allowed to acclimate at the animal facility for at least 1 week before use. On the day of each study, the rabbits were weighed and placed in Plexiglas restrainers, and both ears were shaved and swabbed with 70% isopropyl alcohol and then 2% lidocaine HCl before catheter placement. Venous catheters (Becton Dickinson, Sandy, UT) fitted with a heparin lock were inserted into the intermedial branch of the peripheral ear vein of both ears for drug administration and initiation and maintenance of anesthesia, respectively. A catheter was also placed in the central ear artery for blood sampling.

Pharmacokinetics of hBNP in Rabbits. Conscious rabbits (n = 6) received an i.v. infusion of vehicle for a 60-min control period followed by an escalating dose of hBNP (0.05, 0.1, and 0.2 µg/kg/min) for 60 min at each dose. Blood samples (2 ml) were taken during the control period (time 0) and 50, 55, and 60 min after the initiation of each dose of hBNP. Thus, blood samples were taken at 0 min (vehicle), 50, 55, and 60 min (0.05 µg/kg/min hBNP), 110, 115, 120 min (0.1 µg/kg/min hBNP), and 170, 175, and 180 min (0.2 µg/kg/min hBNP). Blood was collected into EDTA tubes containing 150 kallikrein-inactivating units aprotinin, centrifuged immediately, and plasma samples were stored frozen at 80°C until assayed for hBNP. Plasma hBNP was determined as described below. An estimated plasma steady-state value associated with each dose was the mean of the values derived from blood drawn at the three time points.

Complete Restriction of Renal Blood Flow. For studies involving bilateral renal artery ligation, the animals were anesthetized with 40 to 60 mg/kg pentobarbital. Once under anesthesia, the animals were removed from the restrainers, the abdominal area was shaved and the animal was positioned on its side on a table lined with a heating pad. The temperature was maintained at 37°C for the duration of the experiment. A surgical plane of anesthesia was attained by a constant infusion of 10 mg/kg/h of pentobarbital. Access to the kidneys was through an incision made on the dorsal lateral side of the abdomen. The abdominal muscle was bluntly dissected to gain entry into the peritoneal cavity and expose the kidney. The renal artery was dissected free of fatty tissue and ligated proximally and distally with 3 to 0 suture. The kidney was positioned back into the peritoneal cavity and the wound closed with stainless steel staples. The same procedure was performed on the contralateral kidney. Control animals underwent the same surgical manipulations with the exception of actual renal artery ligation (sham procedure). hBNP was administered as a continuous infusion at a dose of 0.1 µg/kg/min, which was initiated 60 min before the renal artery ligation and continued for another 60 min after the surgical procedure was completed. Blood samples were drawn at 20, 0, 50, 55, 60, 110, 115, and 120 min into the infusion to measure steady-state plasma levels of hBNP before and after ligation of the renal arteries. Blood volume was replaced at each time point with an equal volume of 0.9% NaCl. Plasma was collected as described above and immunoreactive-hBNP determined as described below. An estimated plasma hBNP steady-state value for each animal before and after the surgical procedure (ligation or sham) was derived from blood drawn at the three time points. There were six animals per experimental group.

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NP-C Receptor and Peptidase Inhibition. The roles of NP-C receptor and peptidases in the elimination of plasma hBNP were evaluated in conscious rabbits. hBNP was administered to rabbits as a continuous infusion at a dose of 0.02 µg/kg/min for a total of 4 h. One hour after the initiation of the hBNP infusion, C-ANP was infused at escalating doses of 0.1, 1.0, and 10.0 µg/kg/min for 1 h at each dose. Blood samples were drawn at 20, 0, 80, 85, 90, 140, 145, 150, 200, 205, 210, 260, 265, and 270 min into the hBNP plus C-ANP infusion.

Plasma hBNP Assay. Plasma immunoreactive hBNP was determined by immunoassay as described previously (Clemens, 1998). Briefly, plasma samples were diluted appropriately with pooled normal rabbit plasma. hBNP-specific monoclonal antibodies were added to the samples and incubated overnight in microtiter wells precoated with Fc-specific antimurine monoclonal antibodies. The amount of hBNP bound relative to the total binding capacity of the monoclonal antibody was determined by measuring the binding of biotinylated hBNP. Biotinylated hBNP was quantitated using avidin-horseradish peroxidase with a tetramethyl benzidine substrate, which allows for a colorimetric endpoint.

Statistical Analysis. Plasma hBNP concentrations and steady-state values are expressed as the mean ± S.D. The significance of complete renal blood flow restriction or the sham procedure on steady-state plasma hBNP was assessed by comparing steady-state levels achieved by continuous hBNP infusion before and after these procedures using a paired Student's t test with P < .05 considered statistically significant. The effect of complete renal blood flow restriction on the fold increase in steady-state hBNP values relative to sham was assessed using an unpaired Student's t test with P < .05 considered statistically significant.

	Results

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Pharmacokinetics of hBNP in Rabbits. Plasma levels of hBNP resulting from a dose-escalating protocol involving continuous i.v. infusion of 0.05, 0.1, and 0.2 µg/kg/min were determined at the times indicated (see Table 1). There was no significant difference between the plasma hBNP levels taken 50, 55, or 60 min after initiation of each infusion dose, suggesting a steady-state level was achieved within 50 min. There was a linear relationship (R² = 0.9999) between the estimated steady-state values of hBNP and the infusion dose of hBNP (see Fig. 1), with steady-state values of 2.5 ± 0.2, 5.7 ± 0.8, and 11.9 ± 1.7 ng/ml for hBNP continuous infusion doses of 0.05, 0.1, and 0.2 µg/kg/min, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Effect of hBNP infusion rate on the steady-state plasma level of hBNP. Conscious rabbits (n = 6) received i.v. hBNP by a continuous dose escalating protocol for 60 min per dose (0.05, 0.1, and 0.2 µg/kg/min). Steady-state plasma hBNP values ± S.D. are indicated.

The Effect of Kidney on the Elimination of Plasma hBNP. The pharmacokinetics of hBNP derived from continuous i.v. infusion (0.1 ug/kg/min) was evaluated before and after complete restriction of kidney blood flow by bilateral renal artery suture ligation. The plasma hBNP levels are shown in Table 2. The steady-state plasma hBNP values increased in a statistically significant manner (p < .01) from 5.2 ± 2.0 ng/ml before the surgical procedure to 9.7 ± 3.9 ng/ml after suture ligation (1.9 ± 0.4-fold). In contrast, there was no significant change in the steady-state plasma hBNP values before and after the sham procedure, 6.0 ± 1.8 ng/ml and 6.2 ± 2.5 ng/ml, respectively (1.1 ± 0.4-fold).

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View larger version (17K): [in this window] [in a new window]   Fig. 2.   Pharmacokinetics of hBNP after a 10 µg/kg i.v. bolus in rabbits immediately after bilateral renal artery ligation or a sham surgical procedure (n = 4 per group). Plasma hBNP concentrations ± S.D. are indicated. *p < .05 comparing data obtained from the two groups at each time point.

The Effect of the ANP Clearance Receptor on the Elimination of Plasma hBNP. The effect of the NP-C receptor on the elimination of plasma hBNP was evaluated with the use of the NP-C receptor-specific peptide agonist C-ANP. In animals administered i.v. hBNP by continuous infusion (0.02 µg/kg/min), plasma hBNP levels were determined before and after coadministration of C-ANP at doses of 0.1, 1.0, and 10 µg/kg/min. As shown in Fig. 3, when compared with values obtained before C-ANP infusion, the steady-state plasma hBNP values increased in a statistically significant manner after infusion of C-ANP at doses of 1.0 µg/kg/min (1.4 ± 0.2-fold increase) and 10 µg/kg/min (1.9 ± 0.3-fold) but not at a dose of 0.1 µg/kg/min (1.0 ± 0.2-fold).

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Effect of pharmacological blockade of the NP-C receptor on steady-state plasma hBNP. Rabbits (n = 6) infused i.v. with saline followed by hBNP (0.02 µg/kg/min) were then treated with C-ANP using a dose-escalating protocol of 0.1, 1, and 10 µg/kg/min for 60 min/dose. Steady-state plasma hBNP values were derived and are indicated ±S.D. #p < .01 versus no C-ANP treatment, +p < .05 versus 0.1 µg/kg/min C-ANP treatment, and @p < .01 versus 1 µg/kg/min C-ANP treatment.

The Effect of Peptidases on the Elimination of Plasma hBNP. The effect of peptidases on the elimination of plasma hBNP was evaluated with the use of the peptidase inhibitor phosphoramidon. In animals administered i.v. hBNP by continuous infusion (0.02 µg/kg/min), plasma hBNP levels were determined before and after i.v. phosphoramidon treatment and again after combined treatment with phosphoramidon plus C-ANP. The mean steady-state plasma hBNP values increased in a statistically significant manner after 50 µg/kg/min phosphoramidon (p < .01, 1.7 ± 0.4-fold increase), but not 25 µg/kg/min phosphoramidon (see Fig. 4). The mean steady-state plasma hBNP values increased further when animals were treated with both phosphoramidon and C-ANP (p < .01, 2.4 ± 0.3-fold increase over the value derived from hBNP treatment alone; see Fig. 4). Treatment with hBNP plus C-ANP in the absence of phosphoramidon increased the mean steady-state plasma hBNP values in a statistically significant manner, 1.7 ± 0.6-fold over the values derived from hBNP treatment alone (see Fig. 4).

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Effect of peptidase inhibition with and without pharmacological blockade of the NP-C receptor on steady-state plasma hBNP. Group 1 and group 2 rabbits were treated with hBNP (0.02 µg/kg/min) throughout the study. Group 1 animals were treated with either 25 or 50 µg/kg/min of phosphoramidon alone or with the C-ANP peptide (10 µg/kg/min) as indicated. Group 2 animals were treated with the C-ANP peptide (10 µg/kg/min) as indicated. Steady-state plasma hBNP values were derived and are indicated ±S.D. #p < .01 and ##p < .001 versus hBNP only treatment in group 1 animals; *p < .01 versus hBNP plus 50 µg/kg/min of phosphoramidon treatment; @p < .05 versus hBNP only treatment in group 2 animals.

	Discussion

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This study assessed factors that influence the elimination of hBNP from the plasma compartment of rabbits including the kidney, the NP-C receptor, and peptidases. Previous studies have demonstrated that the rabbit is a useful species to study hBNP, as doses of hBNP effective in human studies yield similar hemodynamic and renal effects in rabbits (Hobbs, 1996; Clemens, 1997). The plasma concentrations of hBNP in rabbits are linearly related to the pharmacological doses used in this report. In addition, an estimated steady state was achieved within 50 min as there was consistently no significant difference in the plasma concentration of hBNP achieved 50, 55, or 60 min after initiation of infusion at all of the doses tested. The pharmacokinetics of plasma hBNP in rabbits after bolus administration is best fit by a two-compartment model with T_1/2 and T_1/2 values of 5.4 ± 1.3 and 33 ± 5 min, respectively. Similar pharmacokinetic values have been previously published in a different study in rabbits (T_1/2 and T_1/2 values of 5.5 ± 0.9 and 27 ± 10 min; Clemens, 1998). Beneficial effects of hBNP in human studies has been noted with doses ranging from 10 to 100 µg/kg/min (Yoshimura, 1991; Marcus, 1996; Mills, 1997; Abraham, 1998). Thus, the doses used in this animal study can be directly related to therapeutic doses.

Complete restriction of kidney blood flow reduced the clearance of hBNP by one-half, indicating that the kidney is involved in the elimination of hBNP from the blood. If this can be extrapolated to congestive heart failure patients with reduced renal blood flow, these results suggest that at worst, hBNP clearance will be reduced by half in these individuals. There is data that the kidney plays a role in the removal of BNP in humans. In a study that monitored regional plasma levels of endogenous BNP in patients with cardiac disease, a significant arteriovenous concentration gradient was found between the femoral artery and renal vein suggesting the kidney is extracting BNP in these individuals (Richards, 1993). Whether the kidney clears hBNP by filtration, proteolysis, or both, mechanisms cannot be understood from these studies. It is known that the kidney is a rich source of the NP-C receptor (Maack, 1987) and NEP24.11. The role of each of these biochemical pathways in the process of renal clearance of hBNP cannot be understood from the studies presented here.

Treatment of rabbits with the NP-C receptor-specific ligand, C-ANP, significantly increased in a dose-related manner the steady-state plasma levels of hBNP resulting from a continuous i.v. infusion of hBNP. This suggests that the NP-C receptor is involved in the elimination of plasma hBNP. Previous studies utilizing the identical NP-C receptor-specific peptide used in this current study demonstrate that the NP-C receptor is involved in the clearance of endogenous ANP and BNP (Charles, 1996). However, this is the first demonstration showing that the NP-C receptor mediates the elimination of exogenous hBNP when administered at pharmacological doses. Treatment of rabbits with the peptidase inhibitor phosphoramidon significantly increased the steady-state level of hBNP. This suggests that peptidases are involved in the elimination of plasma hBNP. This hypothesis is consistent with the data demonstrating that hBNP is a substrate for NEP24.11 (Norman, 1991; Rademaker, 1997), however, whether NEP24.11 is the primary peptidase involved in this process cannot be determined from this study. The neutral endopeptidase inhibitors SCH-32615 has been shown to increase endogenous hBNP in normal sheep and sheep with experimental heart failure (Charles, 1996; Rademaker, 1997). Furthermore, in cardiac disease patients, administration of SCH-32615 resulted in the elevation of endogenous BNP levels, suggesting that peptidases are involved in the removal of BNP in humans (Lainchbury, 1998). This report demonstrates that peptidase inhibition alters the elimination rate of exogenous hBNP administered at pharmacological doses.

Treatment of rabbits with both the NP-C receptor blocker and peptidase inhibitor increased the steady-state levels of hBNP above what was achieved with phosphoramidon alone, suggesting that both of these pathways are involved in the process of hBNP elimination. An alternative explanation that cannot be excluded is that phosphoramidon reduces the elimination of the NP-C receptor blocker, thereby enhancing its effect. It has been demonstrated that the peptidase inhibitor SCH-32615 increased the steady-state plasma levels of C-ANP when administered to sheep(Charles, 1996). However, a similar additive effect of NP-C receptor blockade and peptidase inhibition on the pharmacokinetics of ANP in rats has been demonstrated (Okolicany, 1992). In general, these data support they hypothesis that these two structurally related peptide hormones share some similar metabolic pathways. However, because hBNP has a lower affinity for the NP-C receptor (Mukoyama, 1991) and is a poorer substrate for NEP24.11 when compared with ANP (Kenny, 1993), it is likely that important metabolic differences exist as well.

In summary, these studies suggest that the kidney, the NP-C receptor, and peptidases (possibly NEP24.11) are involved in the metabolism of hBNP. However, it is not clear whether the NP-C receptor and NEP24.11 expressed in the kidney are responsible for the effects of C-ANP and phosphoramidon demonstrated in vivo as both of these proteins are found in other tissues as well as the kidney.