Boerrigter, Guido

Background: B-type natriuretic peptide (BNP1–32) is a 32- amino acid cardiac peptide secreted in response to cardiac stress and plays an important compensatory role in cardiorenal homeostasis. Studies have recently reported that a mRNA encoding an alternatively spliced variant of BNP (ASBNP) exists in the human heart and like BNP1–32 is increased in the failing heart. While ASBNP and BNP1–32 have identical ring structures and amino termini, ASBNP and BNP1–32 differ in their carboxy terminus (34 vs. 6 amino acids, respectively). Studies have also reported that ASBNP activates cGMP, the second messenger of BNP1–32, in isolated glomeruli but not in endothelial cells. Also, unlike BNP1–32 ASBNP does not vasorelax isolated arteries or veins. The cardiorenal actions in vivo of ASBNP are unknown. We hypothesized that ASBNP in vivo would have selective renal actions yet as suggested by studies in isolated vessels would be devoid of blood pressure effects. Methods: We therefore determined for the first time the cardiorenal actions of three subsequent intravenous doses of ASBNP (10, 50, and 150 pmol/kg/min) in 5 healthy canines in an acute study under anesthesia in 30-minute clearances at baseline, during, and following infusion. Values provided are mean±SEM for baseline vs. 150 pmol/kg/min. *p<0.05. Results: ASBNP dose-dependently increased plasma levels of cGMP (8.6±1.0 vs. 11.5±1.3* pmol/mL), and the same was true for urinary cGMP excretion (878±191 vs. 2028±551* pmol/min). Mean arterial pressure (p=0.83) and heart rate (p=0.43) remained unchanged. Urine flow increased (0.38±0.05 vs. 1.32±0.20* mL/min) as did urinary sodium excretion (38±15 vs. 133±38* μ Eq/min). Renal blood flow (p=0.51) and glomerular filtration rate (p=0.99) remained unchanged. BNP-immunoreactivity as measured by the Biosite BNP assay increased (5±4 vs. 3807±573* pg/mL). Conclusion: ASBNP, an alternatively spliced form of BNP produced in the human heart, activates cGMP and has diuretic and natriuretic actions but is without blood pressure lowering actions. Thus, ASBNP represents a novel renal selective, non-hypotensive, natriuretic, and diuretic peptide.

Background Sustained Acting Atrial Natriuretic Peptide (SA-ANP) is a 40 AA peptide consisting of native ANP (1–28) with a 12 AA C-terminus extension which we synthesized based upon a familial ANP gene frameshift mutation. SA-ANP possesses markedly greater and more sustained mean arterial pressure (MAP) lowering, diuretic/natriuretic, angiotensin-aldosterone suppressing, and cGMP activating properties compared to ANP in normal dogs consistent with a super agonist of the ANP receptor (NPR-A) ( JACC in press ). Here we evaluated SA-ANP in a model of acute hypertension with the hypothesis that SA-ANP would have potent MAP lowering and renal enhancing properties. Methods Integrated physiological studies were performed in a canine model of ANG induced hypertension (20 ng/kg/min continuous infusion) before, during, and up to 3 hours following a 45 minute infusion of SA-ANP (30 pmol/kg/min) (n=6) or vehicle (n=6). Results MAP is shown in Figure 1 . SVR significantly decreased and despite a significant reduction in MAP both renal blood flow (RBF) and GFR were significantly increased following SA-ANP infusion. There were significant reductions in proximal and distal tubule sodium reabsorption resulting in a significant increase (>50 fold) in diuresis and natriuresis with SA-ANP. These renal actions were associated with significant increases in plasma and urine cGMP and a significant inhibition of aldosterone activation. Discussion We report for the first time in an acute model of hypertension that SA-ANP reduced MAP while enhancing RBF, GFR, and sodium/water excretion and thus may serve as a potential therapeutic agent in the treatment of hypertension in the setting of volume overload.

Background: Water and sodium (Na) retention are hallmarks of congestive heart failure (CHF). While widely employed, conventional diuretics may adversely affect renal function. Therefore, physiological strategies that enhance water and Na excretion while maintaining renal function are a high priority. Arginine vasopressin (AVP) increases water reabsorption after binding to the V2 receptor in the collecting duct (CD). In contrast, B-type natriuretic peptide (BNP) decreases Na reabsorption via activation of the NPR-A receptor also in the CD and at higher doses also in the proximal tubule. We hypothesized that coadministration of the V2-receptor antagonist tolvaptan (TLV) and BNP would mediate a potent diuresis and natriuresis without adversely affecting renal hemodynamics in experimental CHF. We also assessed plasma and urinary excretion of AVP. Methods: Severe CHF was induced in 2 groups of dogs by tachypacing. On day 11 cardiorenal function was assessed. One group received TLV alone (0.1 mg/kg IV bolus; n=6), whereas the other group received infusion of BNP (50 ng/kg/min) in addition to TLV (n=5). Results: Renal hemodynamics were preserved in both groups. TLV alone increased urine flow from 0.07±0.01 to 0.48±0.09 mL/min, while urinary Na excretion (UNaV) remained unchanged. TLV+BNP-50 increased urine flow from 0.17±0.07 to 2.58±0.47 mL/min and UNaV from 1±1 to 77±23 uEq/min while decreasing proximal fractional Na reabsorption (all p<0.05 vs. TLV alone). TLV alone increased plasma aldosterone and distal fractional Na reabsorption, both of which were prevented with BNP-50. In addition, while plasma AVP did not change with TLV or TLV+BNP, urinary AVP excretion markedly increased with TLV+BNP (from 16±5 to 694±196 pg/min) as compared to TLV alone (from 1±1 to 69±23 pg/min). Conclusions: TLV and BNP coadministration enhances aquaresis and natriuresis through proximal and distal tubular actions without changing renal hemodynamics. Further, TLV with BNP uniquely enhances urinary elimination of AVP warranting further study. We conclude from these findings in experimental CHF that simultaneously antagonizing the V2 receptor by TLV and activating the NPR-A receptor with BNP may be a novel and physiologic diuretic and natriuretic strategy in overt CHF.

Background: C-type natriuretic peptide (CNP), a 22-amino-acid peptide and a ligand of the natriuretic peptide receptor-B (NPR-B), exhibits venodilating, anti-fibrotic, and vascular-regenerating properties but limited renal actions. A Mayo-designed chimeric peptide, CD-NP, which consists of CNP and the C-terminus of Dendroaspis NP, was synthesized to test the hypothesis that CD-NP would possess CNP-like properties with an improved pharmacodynamic profile. Methods: Normal anesthetized dogs were given CD-NP 50 ng/kg/min (n = 10) or an equimolar dose of CNP 29.3 ng/kg/min (n = 9) i.v. for 75 min. Hemodynamic, renal, and hormonal data were obtained pre-infusion (pre-I), at 30 and 60 min I, and post-I, and were compared both within group vs pre-I (mean ± SE, P< 0.05*, < 0.01 † ) and between groups ( P < 0.05 ‡ , < 0.01 § , < 0.001 ¶ ). Plasma BNP was measured by radioimmunoassay. Results: CD-NP resulted in greater increases in plasma cGMP (7±.4 to 25±3 †¶ to 36±3 †¶ to 23±3 †¶ pmol/ml), urinary cGMP excretion (978±145, 3170±205 †¶ , 5919±616 †¶ , 3077±298 †¶ pmol/min) vs CNP (8±.8, 10±.6 † , 11±.7 † , 8±.5 pmol/ml; 976±110, 1104±115, 1317±103, 998±101 pmol/min, respectively). CD-NP also increased urine Na + excretion (19±4, 168±24 †§ , 237±26 †¶ , 96±12 † μEq/min), urine flow (0.2±.1, 1.3±.2 † , 1.8±.3 † , 0.8±.2 † ml/min), and GFR (37±2 ‡ , 48±3 † , 51±3 † , 53±4 † ml/min) vs CNP (36±12, 68±10, 85±26, 81±25 μEq/min; 0.4±.1, 0.9±.2, 1.2±.3*, 0.9±.3 ml/min; 52±5, 53±7, 50±4, 49±6 ml/min, respectively). CD-NP reduced PCWP (5.7±.7, 4.1±1*, 3.2±.7 † , 4.3±.8 mmHg), RAP (1.8±.4, 1.1±.4 † , 0.9±.5 †§ , 1.3±.5 § mmHg), PAP (12±.6, 10±.4*, 10±.6, 11±.7 mmHg) vs CNP (6±.6, 5±.8, 6±.8, 7±.9 † mmHg; 2.6±.3, 2.5±.3 to 2.9±.3 to 3.5±.5 † mmHg; 13±1, 12±1, 12±1, 13±1 mmHg, respectively). Mean blood pressure was unchanged in either group. CD-NP increased circulating BNP during infusion vs CNP (40±4 vs 18±2 pg/ml, P<0.001). Conclusion: This study demonstrates the successful transformation of CNP to a CNP-like peptide with enhanced natriuretic, diuretic, GFR-enhancing, and cardiac-unloading actions. CD-NP may also promote increases in plasma BNP which via the NPR-A receptor may contribute to its biological actions. The therapeutic potential of CD-NP in heart failure warrants further studies.

Background: B-type natriuretic peptide (BNP) signals via particulate guanylyl cyclase (pGC) and cyclic guanosine monophosphate (cGMP). In heart failure (HF), BNP reduces cardiac filling pressures and in selected patients augments sodium excretion and glomerular filtration rate (GFR). Studies have established that pGC and soluble guanylyl cyclase (sGC), the main target of nitric oxide (NO), can be compartmentalized with both enzymes affecting distinct intracellular cGMP pools, resulting in different biological actions. Importantly, sGC may be oxidized in disease states like HF making it unresponsive to NO and nitrovasodilators. BAY 58 –2667 (BAY) is a novel NO and heme independent sGC activator that preferentially stimulates oxidized sGC. We hypothesized that adding direct sGC stimulation with BAY to pGC activation with BNP in HF would enhance vasodilation and cardiac unloading without attenuating the distinct renal actions of BNP. Methods: Severe HF was induced by tachypacing in 13 dogs. On day 11, cardiorenal function was assessed in an acute study under anesthesia at baseline and with intravenous BNP (50 ng/kg/min) alone or in combination with BAY (0.3 ug/kg/min). Results: BNP significantly increased urine flow (UV), urinary sodium excretion (UNaV), GFR, renal blood flow (RBF) and systemic vascular resistance (SVR) and decreased cardiac output (CO), mean arterial pressure (MAP), right atrial pressure (RAP) and pulmonary capillary wedge pressure (PCWP). Despite a greater decrease in MAP compared to BNP, BAY+BNP resulted in similar increases in UNaV, UV and GFR. Dual pGC and sGC stimulation resulted in greater decreases in SVR, RAP and PCWP together with an augmentation of CO compared to BNP alone. Conclusion: Particulate GC activation with BNP in experimental HF enhances renal function and leads to renal but not systemic vasodilation. Addition of the sGC stimulator BAY provides systemic vasodilation, greater cardiac unloading, and augmented CO. Despite a larger decrease in MAP, additional sGC stimulation does not impair the renal actions observed with pGC stimulation alone. These findings support coactivation of sGC with BAY and pGC with BNP as a novel and beneficial therapeutic strategy in HF to optimize activation of distinct GC enzymes and cGMP.

Background: Renal dysfunction has emerged as an important independent prognostic factor in patients with heart failure (HF). Cardiac resynchronization therapy (CRT) improves functional status and left ventricular (LV) function in HF patients with ventricular dyssynchrony. We hypothesized that CRT would also improve glomerular filtration rate as estimated by the abbreviated MDRD equation (eGFR) in patients with reduced eGFR. Methods: This is a retrospective analysis of the Multicenter InSync Randomized Clinical Evaluation (MIRACLE) study, which evaluated CRT in HF patients with NYHA class III–IV, ejection fraction ≤35%, QRS ≥130 ms, and stable medical regimen. Patients were evaluated before implantation and 6 months after randomization to control (CRT device OFF, n=225) or CRT (n=228). Patients were categorized according to their baseline eGFR: ≥90 (category A), 60≤eGFR<90 (category B), and 30≤eGFR<60 (category C) mL/min per 1.73m 2 . Changes in eGFR were also correlated with changes in other parameters in univariate analysis. Results: At baseline, reduced eGFR was associated with ischemic etiology, increased age, mitral regurgitation, and neurohumoral activation. CRT increased LV ejection fraction and decreased LV volumes and mitral regurgitation in all categories. Importantly, CRT improved eGFR (p=0.003) and reduced blood urea nitrogen (p=0.008) compared to control in category C, but not A (p=0.75/p=0.41) and B (p=0.43/p=0.89). In addition, only in category C did atrial natriuretic peptide decrease, suggesting reduced atrial distention and filling pressures with CRT in this group with impaired eGFR. In univariate analysis, changes in eGFR correlated weakly but significantly with changes in aldosterone (r=−0.22), plasma renin activity (r=−0.19, norepinephrine (r=−0.16), and mean blood pressure (r=+0.14), a pattern consistent with reduced neurohumoral activation due to increased systemic and renal perfusion pressure. Conclusions: CRT increased eGFR and reduced blood urea nitrogen in HF patients with reduced baseline eGFR. By improving cardiac function, CRT can indirectly improve renal function, underscoring the importance of cardiorenal interaction and providing another mechanism for the beneficial effects of CRT.

C-type natriuretic peptide (CNP) possesses well-established cardiovascular properties. Although present in the mammalian kidney, CNP production in human kidney and its modulation in human renal disease remain less defined. We investigated the presence of CNP in normal human kidney and in patients with nephrotic syndrome (NS). We also addressed whether or not a low-protein diet (LPD) alters plasma CNP and urinary CNP excretion in NS. In situ hybridization studies demonstrated CNP mRNA expression in tubular cells and glomeruli of normal human kidneys. CNP immunoreactivity was positive in proximal, distal, and medullary collecting duct tubular cells in both controls and patients with NS. The ratios of plasma CNP and urinary CNP to creatinine were significantly higher in patients with NS compared with controls. Urinary CNP, but not plasma CNP, was significantly lowered in patients with NS after an LPD. Similarly, the ratios of urinary protein to creatinine and urinary albumin to creatinine, but not urinary guanosine 3′,5′-cyclic monophosphate to creatinine, decreased significantly with an LPD. These data confirm and extend previous reports and demonstrate for the first time the presence of CNP in human kidney with NS. We also report increased plasma CNP concentration and urinary CNP excretion in NS patients and a significant reduction of CNP excretion with an LPD. Our findings demonstrate that CNP metabolism is altered in patients with NS and support the hypothesis that activation of renal CNP can be partially offset by an LPD. These results underscore that the beneficial effect of an LPD on protein excretion is paralleled by a substantial reduction in intrarenal CNP release.

In heart failure (HF), the cardiac hormone natriuretic peptides (NPs) atrial (ANP), B-type (BNP), and C-type (CNP) play a key role to protect cardiac remodeling. The proprotein convertases corin and furin process their respective pro-NPs into active NPs. Here we define in a canine model of HF furin and corin gene and protein expression in normal and failing left atrium (LA) or ventricle (LV) testing the hypothesis that the NP proproteins convertases production is altered in experimental HF. Experimental canine HF was produced by rapid right ventricular pacing for 10 days. NPs, furin, and corin mRNA expression were determined by quantitative RT-PCR. Protein concentration or expression was determined by immunostaining, radioimmunoassay, or Western blot. Furin and corin proteins were present in normal canine LA and LV myocardium and vasculature and in smooth muscle cells. In normal canines, expression of NPs was dominant in the atrium compared with the ventricle. In experimental early stage HF characterized with marked atrial fibrosis, ANP, BNP, and CNP mRNA, and protein concentrations were higher in HF LA but not HF LV compared with normals. In LA, corin mRNA and protein expressions in HF were lower, whereas furin mRNA and protein expressions were higher than normals. NPs and furin expressions were augmented in the atrium in experimental early stage HF and, conversely, corin mRNA and protein expressions were decreased with atrial remodeling. Selective changes of these NP convertases may have significance in the regulation of pro-NP processing and atrial remodeling in early stage HF.

Background— TRV120027 is a novel β-arrestin biased ligand of the angiotensin II type 1 receptor; it antagonizes canonical G-protein–mediated coupling while, in contrast to classical angiotensin II type 1 receptor antagonists, it engages β-arrestin–mediated signaling. Consequently, TRV120027 inhibits angiotensin II–mediated vasoconstriction while, via β-arrestin coupling, it increases cardiomyocyte contractility. We hypothesized that TRV120027 would elicit beneficial cardiorenal actions when added to furosemide in experimental heart failure. Methods and Results— Two groups of anesthetized dogs (n=6 each) with tachypacing-induced heart failure were studied. After a baseline clearance, 1 group (F+V) received furosemide (1 mg/kg per hour) plus saline for 90 minutes, whereas the other (F+T) received the same dose of furosemide plus TRV120027 (0.3 and 1.5 µg/kg per minute for 45 minutes each); 2 clearances were done during drug infusion. After a washout, a postinfusion clearance was done; * P <0.05 between groups. F+V and F+T increased diuresis and natriuresis to a similar extent during drug administration, but urine flow* and urinary sodium excretion* were higher in the postinfusion clearance with F+T. Glomerular filtration rate was preserved in both groups. Renal blood flow increased with F+T but this was not significant versus F+V. Compared with F+V, F+T decreased mean arterial pressure*, systemic* and pulmonary* vascular resistances, and atrial natriuretic peptide*. Pulmonary capillary wedge pressure* decreased to a larger extent with F+T than with F+V. Conclusions— When added to furosemide, TRV120027, a novel β-arrestin biased angiotensin II type 1 receptor ligand, preserved furosemide-mediated natriuresis and diuresis, while reducing cardiac preload and afterload. These results provide support for TRV120027 as a promising novel therapeutic for the treatment of heart failure.

Background— The angiotensin II type 1 receptor (AT1R) plays a key role in regulating cardiorenal function. Classic “unbiased” AT1R antagonists block receptor coupling to both G αq and ß-arrestin–mediated signals, which desensitize G-protein signaling as well as transduce G-protein–independent signals. TRV120027 is a novel ß-arrestin–biased AT1R ligand, which engages ß-arrestins while blocking G-protein signaling. At the AT1R, TRV120027 can inhibit angiotensin II–mediated vasoconstriction, whereas, through ß-arrestin coupling, increase cardiomyocyte contractility. We defined for the first time the acute cardiorenal actions of TRV120027 in healthy and heart failure (HF) canines. Methods and Results— Healthy and HF canines (induced by tachypacing) were anesthetized. After instrumentation and equilibration, a 30-minute baseline clearance was performed, followed by further clearance with escalating doses of intravenous TRV120027 (0.01, 0.1, 1, 10, and 100 μg/kg per minute) and a 30-minute washout. In healthy canines, TRV120027 decreased pulmonary capillary wedge pressure and systemic and renal vascular resistances, while increasing cardiac output, renal blood flow, glomerular filtration rate, and urinary sodium excretion. In HF canines, TRV120027 decreased mean arterial pressure, right atrial pressure, and pulmonary capillary wedge pressure, systemic and renal vascular resistances and increased cardiac output and renal blood flow. Glomerular filtration rate and urinary sodium excretion were maintained. Conclusions— We report for the first time the cardiorenal actions of the novel ß-arrestin–biased AT1R ligand TRV120027. In both normal and HF canines, TRV120027 demonstrated cardiac unloading actions while preserving renal function. With this beneficial pharmacological profile, TRV120027 represents a novel strategy for the treatment of HF.