A NOVEL ATRIAL NATRIURETIC PEPTIDE BASED THERAPEUTIC IN EXPERIMENTAL ANGIOTENSIN II MEDIATED ACUTE HYPERTENSION

Abstract

M-atrial natriuretic peptide (M-ANP) is a novel next generation 40 amino acid peptide based on atrial natriuretic peptide (ANP), which is highly resistant to enzymatic degradation and has greater and more sustained beneficial actions compared to ANP. The current study was designed to advance our understanding of the therapeutic potential of M-ANP in a canine model of acute angiotensin II induced hypertension with elevated cardiac filling pressures and aldosterone activation. We compare M-ANP to vehicle and equimolar human B-type natriuretic peptide (BNP), which possesses the most potent in vivo actions of the native natriuretic peptides. M-ANP significantly lowered mean arterial pressure and systemic vascular resistance. Importantly, despite a reduction in blood pressure, renal function was enhanced with significant increases in renal blood flow, glomerular filtration rate, diuresis, and natriuresis following M-ANP infusion. While angiotensin II induced an acute increase in pulmonary capillary wedge pressure, M-ANP significantly lowered pulmonary capillary wedge pressure, pulmonary artery pressure, and right atrial pressure. Further, M-ANP significantly suppressed angiotensin II induced activation of aldosterone. These cardiovascular and renal enhancing actions of M-ANP were accompanied by significant increases in plasma and urinary cGMP, the second messenger molecule of the natriuretic peptide system. When compared to human BNP, M-ANP had comparable cardiovascular actions but resulted in a greater natriuretic effect. These results suggest that M-ANP, which is more potent than ANP in normal canines, has potent blood pressure lowering and renal enhancing properties and may therefore serve as an ANP based therapeutic for acute hypertension.

INTRODUCTION

Continuing research has established the importance of the cardiac hormone atrial natriuretic peptide (ANP) in the regulation of blood pressure. This role in blood pressure regulation reflects the pleuripotent cardiorenal actions of ANP, which functions via the guanylyl cyclase (GC) A receptor and the second messenger cyclic guanosine monophosphate (cGMP). The actions of ANP include natriuresis, vasodilatation, suppression of aldosterone, inhibition of myocardial hypertrophy, and suppression of cardiac fibrosis.^1,2 Importantly, Newton-Cheh and coworkers have recently reported that genetic variants of the ANP gene, which result in increased circulating ANP, are associated with lower blood pressure and reduced risk of hypertension.^3,4 The clinical phenotype of human genetic variants of the ANP gene as well as mouse models of ANP gene disruption⁵ or overexpression⁶ together with the cardiorenal enhancing actions of ANP lay the foundation for ANP based therapeutics for cardiovascular diseases such as hypertension.

We recently reported the design of a novel ANP based therapeutic synthetic peptide, M-atrial natriuretic peptide (M-ANP), which is a 40 amino acid (AA) peptide consisting of native 28 AA ANP with a 12 AA C-terminus extension.⁷ M-ANP possesses markedly greater and more sustained mean arterial pressure (MAP) lowering, diuretic and natriuretic, glomerular filtration rate (GFR) enhancing, renin-angiotensin-aldosterone system (RAAS) suppressing, and cGMP activating properties compared to native ANP making it highly attractive as a cardiovascular therapeutic agent. Dickey and co-workers⁸ have shown that M-ANP, compared to ANP, is highly resistant to degradation by neprilysin (NEP) while retaining GC-A activating properties.^7,9–11

To date, the actions of intravenous M-ANP in a model of cardiovascular disease such as acute hypertension remain undefined. Importantly, a recent report from Studying the Treatment of Acute Hypertension (STAT)¹² registry reported a high incidence of acute kidney injury associated with severe acute hypertension, which was associated with greater risk for heart failure (HF), cardiac arrest, and death. These and other studies underscore the need for acute hypertension therapeutic agents which possess renal enhancing in addition to blood pressure lowering properties.

The current study was designed to advance our understanding of the therapeutic potential of M-ANP in a model of acute hypertension. We hypothesized that M-ANP would have potent MAP lowering, cardiac unloading, renal enhancing, and aldosterone suppressing properties in acute hypertension. We further hypothesized that M-ANP, with its extended C-terminus, would have equivalent biological actions in vivo compared to human BNP (nesiritide) which is approved for acute HF in the United States and which possesses greater cardiorenal actions than ANP (carpertide)¹³ which is approved for HF treatment in Japan. Thus the current studies were designed to establish the potential therapeutic efficacy of M-ANP for cardiovascular disease to complement BNP.

METHODS

M-Atrial and B-type Natriuretic Peptides

M-ANP and human BNP were synthesized by Phoenix Laboratories (Mountain View, CA). Structure was confirmed by mass spectrometry and high performance liquid chromatography analysis confirmed purity to be >95%.

Study Protocol

We investigated the effects of M-ANP, vehicle (0.9% normal saline), and human BNP (n=6 for each group) in a canine model of ANG II induced acute hypertension. Studies were performed in accordance with the Animal Welfare Act and with approval of the Mayo Clinic Institutional Animal Care and Use Committee.

Experimental procedures have been extensively described previously^7,13 and are available in the online supplement (please see http://submit-hyper.ahajournals.org). After completion of the procedural set up a baseline (BL) clearance was performed. All clearances lasted 30 minutes and consisted of arterial blood sampling, hemodynamic measurements, and urine collection over 30 minutes. Immediately following the BL clearance, the saline infusion was replaced by continuous ANG II infusion (20 pmol·kg⁻¹ ·min⁻¹, 1 mL/min) (Phoenix Pharmaceuticals, Mountain View, CA), which was continued throughout the experimental protocol. After a 15 minute lead in of the ANG II infusion, a 30 minute ANG II clearance (ANG II CL) was performed. Following the ANG II infusion lead in period (15 minutes) and ANG II CL (30 minutes), either M-ANP (30 pmol/kg/min), vehicle (0.9% normal saline), or BNP (30 pmol/kg/min) was infused at rate of 1 ml/min. M-ANP, vehicle, or BNP was infused for a total of 45 minutes which included a 15 minute lead-in period followed by a 30 minute clearance. M-ANP, vehicle, or BNP infusion was then discontinued and four 30 minute clearances were performed which were 0–30, 31–60, 61–90, and 91–120 minutes post M-ANP, vehicle, or BNP infusion.

Neurohormonal and Electrolyte Analysis

Plasma and urine ANP were measured by radioimmunoassay (RIA) (Phoenix Pharmaceuticals, Mountain View, CA) as previously described.¹⁴ The cross reactivity for M-ANP with the above assay is 100%. Plasma/urine cGMP¹⁵ (PerkinElmer, Shelton, CA), renin¹⁶ (Diasorin, Stillwater, MN), angiotensin II¹⁷ (Phoenix Pharmaceuticals, Mountain View, CA), and aldosterone¹⁸ (Siemens Healthcare Diagnostics, Deerfield, IL) were determined by RIA as described previously. Inulin concentrations were measured using the anthrone method as previously described.¹⁹ Electrolytes including lithium were measured by flame photometry (IL943, Instrumentation Laboratory, Bedford, MA). GFR was measured by inulin clearance. Proximal fractional reabsorption of sodium (PFR_Na) and distal fractional reabsorption of sodium (DFR_Na) were calculated using the lithium clearance technique as previously described.^7,20,21 Net renal generation of cGMP and plasma filtered cGMP were calculated as previously described.²²

Statistical Analysis

Descriptive statistics are reported as mean ± standard error. Comparisons within a group were made by one-way analysis of variance (ANOVA) for repeated measures followed by Bonferroni’s multiple comparison posttest analysis when the global test was significant. Two-way ANOVA was used to compare the main group effects of M-ANP, vehicle, and BNP followed by Bonferroni posttests. GraphPad Prism 5 (GraphPad Software, La Jolla, Ca) was used for the above calculations. Statistical significance was accepted as P < 0.05.

RESULTS

Systemic Hemodynamics

All animals received a continuous infusion of ANG II resulting in a significant increase in systemic vascular resistance (SVR) (Table 1) and MAP (Figure 1A) compared to BL measurements. There was a significant increase in pulmonary capillary wedge pressure (PCWP) (Figure 1B) following ANG II infusion.

Table 1.

Cardiovascular and Renal Hemodynamics: M-Atrial Natriuretic Peptide Compared to Vehicle

Variable	Peptide	Baseline	ANG II	M-ANP or Vehicle Infusion	Post M-ANP or Vehicle Infusion
					30 min	60 min	120 min
CO, L/min	Vehicle	3.5±0.2	2.7±0.2*	2.8±0.3*	2.8±0.2*	2.8±0.2*	2.9±0.3*
	M-ANP	3.1±0.2	2.6±0.2	2.9±0.1	2.5±0.1*	2.5±0.1*	2.6±0.1
SVR, mmHg• L−1•min−1	Vehicle	38.1±2.5	57.6±11.7*	56.0±3.7*	56.1±4.0*	54.6±4.0*	51.9±3.9*
	M-ANP	43.3±2.6	57.4±2.8*	46.2±2.0†	51.6±1.9*	54.9±2.7*	49.0±2.7*
PAP‡, mmHG	Vehicle	13.6±0.5	14.7±0.7	15.2±0.9	15.5±1.0	15.5±1.0	15.8±1.1*
	M-ANP	14.2±1.0	14.8±1.0	12.0±0.8*†	11.4±0.8*†	11.7±0.9*†	12.6±1.3†
RAP‡, mmHg	Vehicle	1.3±0.5	1.9±0.7	2.0±0.8	2.1±0.9	2.4±1.2	2.5±1.4
	M-ANP	1.5±0.3	2.0±0.3	−0.4±0.3*†	−0.8±0.4*†	−0.3±0.3*†	0.3±0.3*†
PVR, mmHg•L−1•min−1	Vehicle	2.4±0.2	2.8±0.3	2.8±0.2	2.9±0.2*	3.0±0.2*	2.9±0.1
	M-ANP	2.9±0.2	2.9±0.3	3.3±0.2	3.7±0.2	3.4±0.3	3.7±0.6
RVR‡ mmHg•L−1•min−1	Vehicle	729±51	995±118*	936±117*	880±95*	851±99	863±88
	M-ANP	716±61	955±69*	642±147†	729±154†	705±21†	760±31
Heart Rate, beats/min	Vehicle	100±5	115±4*	124±4*	129±5*	129±6*	137±7*†
	M-ANP	93±4	112±8*	130±10*†	132±7*†	129±7*†	134±6*†

^*P<0.05 vs BL and

^†P<0.05 vs ANG II, 1-way ANOVA with Bonferroni’s multiple-comparison test.

^‡P < 0.05 for main group effect of M-ANP vs. vehicle, 2-way ANOVA.

ANG II CL, angiotensin II clearance; M-ANP, M-atrial natriuretic peptide; CO, cardiac output; SVR, systemic vascular resistance; PAP, pulmonary artery pressure; RAP, right atrial pressure; RVR renal vascular resistance; PVR, pulmonary vascular resistance.

Figure 1.

Mean arterial pressure (MAP) (A) and pulmonary capillary wedge pressure (PCWP) (B) at baseline (BL), during ANG II clearance (ANG II CL), during M-ANP or vehicle infusion, and 30, 60, 90 and 120 minutes post M-ANP or vehicle infusion. *P<0.05 vs BL, †P<0.05 vs ANG II, 1-way ANOVA with Bonferroni’s multiple-comparison test. P<0.001 between M-ANP and vehicle groups for both MAP and PCWP (2-way ANOVA). ^‡P<0.05 for M-ANP vs vehicle at a specific time point, 2-way ANOVA with Bonferroni posttests.

Following M-ANP infusion, and while receiving concomitant continuous ANG II infusion, MAP significantly decreased compared to ANG II infusion measurements and remained significantly decreased up to 120 minutes following M-ANP infusion (Figure 1A). In contrast there was no change in MAP following vehicle infusion. The significant reduction in MAP was associated with a significant decrease in SVR following M-ANP infusion compared to no change in SVR with vehicle infusion (see Table 1 for SVR and other hemodynamic parameters). There was a significant and sustained decrease in PCWP following M-ANP infusion (Figure 1B) consistent with reduction in cardiac filling pressures as there was no significant change in pulmonary vascular resistance (PVR). In addition, both pulmonary artery pressure (PAP) and right atrial pressure (RAP) significantly decreased following M-ANP versus no change with vehicle infusion. The actions of M-ANP on PAP and RAP were independent of ANG II pretreatment as ANG II did not affect these parameters. Heart rate significantly increased in response to ANG II in both groups and there was no difference in heart rate response between M-ANP and vehicle groups (Table 1).

Renal Function and Hemodynamics

GFR, RBF, urinary sodium excretion, and water excretion are shown in Figure 2. ANG II infusion significantly increased renal vascular resistance (RVR) (Table 1) and reduced RBF. ANG II infusion did not significantly alter diuresis, natriuresis, or GFR. Despite a significant reduction in MAP, both RBF (Figure 2A) and GFR (Figure 2B) significantly increased following M-ANP in contrast to no change with vehicle. RVR significantly decreased following M-ANP infusion. In addition, there was a marked increase in both urinary water excretion (Figure 2C) and urinary sodium excretion (Figure 2D) following M-ANP infusion. Consistent with the increase in urinary sodium and water excretion both PFR_Na and DFR_Na significantly decreased from 88.9±1.1 and 99.5±0.1 percent respectively at BL to 58.9±4.5 and 72.1±2.3 percent respectively with M-ANP infusion. Both PFR_Na and DFR_Na returned to baseline 60 minutes post M-ANP infusion. There was no significant change in either PFR_Na or DFR_Na during or following vehicle infusion (data not shown).

Figure 2.

Renal blood flow (RBF) (A), glomerular filtration rate (GFR) (B), urinary water excretion (UV) (C), and urinary sodium excretion (UNaV) (D) at baseline (BL), during M-ANP or vehicle infusion, and 30, 60, 90 and 120 minutes post M-ANP or vehicle infusion. *P<0.05 vs BL, †P<0.05 vs ANG II, 1-way ANOVA with Bonferroni’s multiple-comparison test. P<0.001 between M-ANP and vehicle groups for RBF, GFR, UV, and UNaV (2-way ANOVA). ^‡P<0.05 for M-ANP vs vehicle at a specific time point, 2-way ANOVA with Bonferroni posttests.

Neurohumoral Analysis

Neurohumoral parameters are shown in Table 2 and Figure 3. Plasma ANG II and aldosterone levels were similarly increased and renin levels similarly suppressed in the M-ANP and vehicle groups in response to ANG II infusion. Plasma and urinary ANP and cGMP were not significantly changed by ANG II infusion. M-ANP infusion resulted in significant increases in plasma and urinary ANP immunoreactivity. Consistent with increased plasma and urinary ANP immunoreactivity levels, both plasma cGMP (Figure 3A) and urinary cGMP excretion (Figure 3B) were markedly increased following M-ANP infusion. Both renal generation and plasma filtration of cGMP were significantly increased (Table 2). The significant increase in plasma and urinary cGMP following M-ANP infusion persisted for 60 minutes post infusion. There was no increase in plasma or urinary cGMP with vehicle infusion. Despite the continuous infusion of ANG II and significant increase in plasma ANG II levels, aldosterone levels were significantly suppressed following M-ANP infusion (Figure 3C).

Table 2.

Neurohumoral Data: M-Atrial Natriuretic Peptide Compared to Vehicle

Variable	Peptide	Baseline	ANG II CL	M-ANP or Vehicle Infusion	Post M-ANP or Vehicle Infusion
					30 min	60 min	120 min
ANG II, pg/ml	Vehicle	4.0±0.8	70.6±8.2*	66.7±8.1*	80.5±13.3*	75.7±10.4*	66.9±10.3*
	M-ANP	7.1±2.0	65.0±12.5*	82.2±15.8*	88.6±13.5*	79.0±15.8*	72.6±15.9*
Plasma Renin, ng/ml/hr	Vehicle	1.8±0.5	0.7±0.2	0.6±0.1	0.3±0.1*	0.3±0.04*	0.3±0.1*
	M-ANP	2.2±0.3	1.1±0.1	0.8±0.1	0.7±0.1	0.5±0.2	2.0±0.8
Plasma ANP§‡, pg/ml	Vehicle	53±7	66±5	69±10	83±10	81±9	78±8
	M-ANP	30±2	48±1	1735±185*†	426±66*†	82±9	51±4
Urine ANP§‡, pg/min	Vehicle	19±2	24±4	26±4	31±6*	35±3	22±2.8
	M-ANP	14±2	17±2	134±28*†	122±12*†	35±3	22±3
Renal cGMP Generation‡, pmol/min	Vehicle	661±120	805±215	781±132	779±78	711±110	571±96
	M-ANP	486±62	550±114	5827±1246*†	8303±1769*†	2203±347*†	847±176
Filtered cGMP‡, pmol/min	Vehicle	303±77	322±58	314±65	393±81	417±68	509±115
	M-ANP	365±53	461±86	3304±187*†	1995±330*†	1034±116*†	489±58

^*P<0.05 vs BL and

^†P<0.05 vs ANG II, 1-way ANOVA with Bonferroni’s multiple-comparison test.

^‡P < 0.05 for main group effect of M-ANP vs. vehicle, 2-way ANOVA.

ANG II CL, angiotensin II clearance; ANP, atrial natriuretic peptide; M-ANP, M-atrial natriuretic peptide.

^§ANP immunoreactivity.

Figure 3.

Plasma 3′,5′-cyclic guanosine monophosphate (cGMP) (A), urinary cGMP excretion (B), and plasma aldosterone (C) at baseline (BL), during M-ANP or vehicle infusion, and 30, 60, 90 and 120 minutes post M-ANP or vehicle infusion. *P<0.05 vs BL, †P<0.05 vs ANG II, 1-way ANOVA with Bonferroni’s multiple-comparison test. P<0.001 between M-ANP and vehicle groups for plasma cGMP, urinary cGMP excretion, and plasma aldosterone (2-way ANOVA). ^‡P<0.05 for M-ANP vs vehicle at a specific time point, 2-way ANOVA with Bonferroni posttests.

M-ANP and BNP Comparison

Equimolar (30 pmol/kg/min) human recombinant BNP (nesiritide) infusion was compared to M-ANP. Important hemodynamic, renal, and neurohumoral data are shown in Table 3. M-ANP infusion resulted in a significantly greater natriuretic effect when compared to human BNP. Other cardiorenal parameters including MAP, PCWP, RBF, GFR, SVR, and aldosterone were not significantly different following M-ANP and BNP infusion.

Table 3.

M-Atrial Natriuretic Peptide and B-type Natriuretic Peptide

Variable	Peptide	Baseline	ANG II CL	M-ANP or BNP Infusion	Post M-ANP or BNP Infusion
					30 min	60 min	120 min
MAP, mmHg	M-ANP	131±6	148±6*	133±6†	129±6†	136±5†	139±6*†
	BNP	125±4	150±4*	130±3†	136±4*†	140±4*†	144±3*
PCWP, mmHg	M-ANP	5.4±0.8	7.4±0.8*†	2.6±0.5*†	2.1±0.5*†	3.1±0.5*†	3.4±0.4*†
	BNP	5.2±0.6	7.1±0.8*	2.4±0.6*†	2.1±0.5*†	2.9±0.7*†	3±0.7*†
SVR, mmHg•L−1•min−1	M-ANP	43.3±2.6	57.4±2.8*	46.2±2.0†	51.6±1.9*	54.9±2.7*	49.0±2.7*
	BNP	38.9±2.5	57.1±4.6*	45.2±3.7†	53.6±4.3*	58.9±5.0*	57.1±4.7*
RBF, ml/min	M-ANP	194±9	155±6*	267±14*†	226±12*†	195±9†	196±7
	BNP	205±16	163±14*	250±10†	219±12†	200±14	198±19
GFR, ml/min	M-ANP	30±4	32±7	57±4*†	41±6	36±4	31±2
	BNP	32±2	40±3	47±2*	41±5	39±2	41±3
UNaV, μEq/min‡	M-ANP	3±1	34±20	891±62*§†	395±106*†	40±12	9±3
	BNP	6±2	16±6	679±33*†	288±64*†	74±25	20±7
Aldosterone, ng/dl	M-ANP	4.8±1.7	21.0±1.8*	9.9±1.5†	10.1±2.0†	22.0±3.8*	33.2±4.3*†
	BNP	3.2±0.4	13.0±2.7*	8.7±2.0	12.7±2.3*	18.9±3.0*	30.1±3.1*†
Plasma cGMP, pmol/ml	M-ANP	12.1±0.7	14.6±0.5	58.6±3.1*†	48.0±3.5*†	29.7±1.3*†	17.0±0.5
	BNP	10.0±2.9	11.7±9.2	56.7±8.2*†	43.5±5.1*†	31.0±3.7*†	16.2±2.2

^*P<0.05 vs BL and

^†P<0.05 vs ANG II, 1-way ANOVA with Bonferroni’s multiple-comparison test.

^‡P < 0.05 for main group effect of M-ANP vs BNP and

^§P<0.05 for M-ANP vs BNP at a specific time point, 2-way ANOVA with Bonferroni posttests.

MAP, mean arterial blood pressure; PCWP, pulmonary capillary wedge pressure; SVR, systemic vascular resistance; RBF, renal blood flow; GFR, glomerular filtration rate; UNaV, urinary sodium excretion; cGMP, cyclic guanosine monophosphate; M-ANP, M-atrial natriuretic peptide; BNP, B-type natriuretic peptide; ANG II CL, angiotensin II clearan

DISCUSSION

The current study is the first to define the potential therapeutic actions of M-ANP in a model of acute hypertension. Importantly, M-ANP has more sustained and greater cardiorenal actions⁷ together with greater resistance to enzymatic degradation⁸ compared to native ANP. In the current study of ANG II induced hypertension with elevated cardiac filling pressures M-ANP lowered MAP. Importantly, despite a reduction in MAP, renal function was enhanced with significant increases in RBF, GFR, natriuresis, and diuresis following M-ANP infusion. While ANG II induced an acute increase in PCWP secondary to systemic vasoconstriction together with effects on the pulmonary circulation, M-ANP significantly lowered PCWP, PAP, and RAP, and SVR. Further, M-ANP significantly suppressed ANG II induced activation of aldosterone. When compared to recombinant human BNP, M-ANP had comparable cardiovascular actions to human BNP but resulted in a greater natriuretic effect. These results suggest that M-ANP, which is more potent than ANP in normal canines⁷, has blood pressure lowering and renal enhancing properties and may therefore serve as an ANP based therapeutic for acute hypertension.

The natriuretic peptides, specifically ANP, are increasingly recognized to play a fundamental role in blood pressure regulation and cardiorenal homeostasis.^3–6,23 These studies support the development of an ANP based therapeutic for hypertension. Indeed, ANP (carperitide) is already approved as an intravenous agent for HF treatment in Japan and was shown to have beneficial renoprotective actions following bypass surgery, reduced infarct size and HF following myocardial infarction, and prevented contrast induced nephropathy.^24–26

M-ANP is novel ANP based peptide with greater diuretic, natriuretic, GFR enhancing, aldosterone suppressing, and MAP lowering properties when compared to native ANP in normal canines.⁷ When we compare the actions of M-ANP in the current study to the actions of ANP in historical studies of ANG II induced acute hypertension there are important differences.²⁷ Specifically, GFR was not enhanced by ANP whereas there was significant increase in GFR by M-ANP in the current study. ANP did not suppress aldosterone activation whereas M-ANP significantly suppresses aldosterone in the current study. Further, the natriuretic effect of M-ANP was approximately 100 percent greater in the current study than with native ANP in the previous study. Recent studies⁸ have shown that M-ANP, compared to ANP, is highly more resistant to degradation by NEP and this may serve as the principal mechanism for its enhanced in vivo cardiorenal actions compared to ANP as there was no change in affinity for its GC-A receptor.^7,9–11

The mechanism of the blood pressure lowering properties observed in the current of M-ANP was multifactorial. First, there was a significant reduction in SVR and it is known that M-ANP activates GC-A⁸ which mediates cGMP related vasodilatation.¹ A second mechanism was the significant natriuretic and diuretic effect of M-ANP. This is an important property of M-ANP, which goes beyond conventional vasodilators which lack diuretic and natriuretic actions. Also, M-ANP suppressed ANG II induced aldosterone production from the zona glomerulosa where there is an abundance of GC-A.^28,29 Finally, it is possible that M-ANP directly inhibits the vasoconstrictive effects of ANG II on the blood vessel wall, however, this is speculative and would require additional studies to confirm.

It is notable that while there was significant reduction in MAP, both RBF and GFR significantly increased after M-ANP. These renal hemodynamic actions most likely were related to significant increases in plasma and urinary cGMP and net renal cGMP generation with subsequent activation of GC-A which is widely distributed in the kidney. The increases in GFR and RBF are also consistent with M-ANP/GC-A mediated cGMP generation⁸ and inhibition of ANG II related renal arterial and afferent arteriolar vasoconstriction.^27,30,31 A therapeutic blood pressure lowering and diuretic/natriuretic agent that concomitantly enhances renal function is a highly attractive characteristic when compared to other conventional diuretic agents and vasodilators which tend to reduce renal perfusion and activate the RAAS.^32–34

In the current study we compared M-ANP to recombinant human BNP testing the hypothesis that M-ANP would have comparable biological actions to BNP. Importantly, in normal human subjects, equimolar BNP is 2–3 times more potent than ANP which may be secondary to the much greater metabolic clearance of ANP compared to BNP.³⁵ Further, in experimental HF, BNP has more potent cardiorenal enhancing actions compared to ANP at equimolar doses.¹³ It should be noted that while human and canine ANP are identical, there is approximately 20 percent amino acid heterogeneity between human and canine BNP.^36,37 Extensive previous studies have demonstrated the ability of human BNP to have potent cardiorenal and humoral actions in the dog.³⁸ Indeed, human BNP activates cGMP generation in isolated canine glomeruli.³⁹ While our results suggest comparable actions between M-ANP and human BNP our conclusions need to be cautious due to differences in human and canine BNP.

We used a model of ANG II induced acute hypertension in the current study to investigate the cardiovascular, neurohumoral, and renal effects of M-ANP and compare to human BNP. This model can be viewed as a limitation, but as it has been widely used in the literature, as it allows for reproducibility and has clinical relevance as the role of the RAAS in hypertension is well established. Indeed, this model of ANG II---mediated acute hypertension has been used extensively in the past and in key studies in the development of antihypertensive agents such as beta blockers, calcium channel blockers, and native natriuretic peptides.^27,40–43

The clinical relevance of the current study is underscored by the recent report from the STAT registry which showed that acute and chronic kidney disease is a common comorbidity in subjects with acute hypertension and is associated with greater risk for HF, cardiac arrest, and death.¹² These findings underscore the need for therapeutics aimed at preventing or mitigating the severity of kidney injury to reduce subsequent increased morbidity and mortality. As recently emphasized by de Bold⁴⁴, the central importance of ANP as a therapeutic agent is its diverse pharmacological properties. Moreover, ANP exerts beneficial cardiorenal effects without activating the RAAS, which often occurs with other agents used in acute hypertension or acute heart failure. It is important to underscore that ANP has been successfully employed in Japan for acute cardiorenal syndromes and has significant renoprotective actions.^24,25 As further stated by de Bold⁴⁴ and as we have undertaken with M-ANP, structural modification of the natriuretic peptides with the purpose of increasing potency represents a new therapeutic direction.

PERSPECTIVES

M-ANP is a novel next generation 40 AA ANP based peptide, which is highly resistant to enzymatic degradation⁸ and has greater and more sustained beneficial actions compared to ANP.⁷ In the current study we report that M-ANP lowers blood pressure in a model of acute ANG II induced hypertension and reduces cardiac filling pressures. Importantly, the cardiovascular properties of this advanced ANP were also associated with an improvement in renal function with significant increases in RBF, GFR, natriuresis, and diuresis together with suppression of aldosterone activation. These characteristics make M-ANP an attractive candidate for the treatment of acute hypertension warranting further studies.

ABBREVIATIONS

AA

amino acid

ANG II

angiotensin II

ANOVA

analysis of varianc

ANP

atrial natriuretic peptide

BL

baseline

BNP

B-type natriuretic peptide

cGMP

cyclic guanosine monophosphate

CL

clearance

CO

cardiac output

DFR_Na

distal fractional reabsorption of sodium

GC

guanylyl cyclase

GFR

glomerular filtration rate

HF

heart failure

M-ANP

M-atrial natriuretic peptide

MAP

mean arterial pressure

NEP

neprilysin

PAP

pulmonary artery pressure

PCWP

pulmonary capillary wedge pressure

PFR_Na

Proximal fractional reabsorption of sodium

PVR

pulmonary vascular resistance

RAAS

renin angiotensin aldosterone system

RAP

right atrial pressure

RBF

renal blood flow

RIA

radioimmunoassay

RVR

renal vascular resistance

STAT

Studying the Treatment of Acute Hypertension

SVR

systemic vascular resistance