Harty, Gail J.

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.

Introduction: Blood pressure (BP) control remains low despite combination therapy in resistant hypertension (RH). Thus, there is an unmet need for novel strategies targeting RH. Recently we have reported that M-ANP, a novel Mayo engineered ANP-based peptide, possesses greater and more sustained biological actions compared to ANP and is more resistant to neprilysin degradation. The current study was designed to investigate for the first time the efficacy of a single daily subcutaneous (SQ) injection of M-ANP using a chronic canine model of hypertension induced by angiotensin II (Ang-II). Hypothesis: We hypothesize that a daily SQ M-ANP injection would have potent BP lowering and renal enhancing properties in our chronic hypertensive canine model. Methods: We induced hypertension in canines (n=5) by continuous Ang II (80 ng/kg/min) osmotic mini-pump administration for 24 days. Continuous 24 hour mean BP (MBP) was measured by telemetry and a 24 hour urine collection was used to assess urinary volume, sodium (Na) and cGMP, the second messenger for the ANP system. M-ANP was administered SQ for 5 days (10μg/kg/day). Data are mean±SE. ΨP<0.05 Ang II Day 16 vs. baseline; *P<0.05 Ang II Day 16 vs. M-ANP (Table 1). Results: Chronic Ang II administration resulted in model of severe hypertension, with an increase in MBP from 112±2 to 157±2 mmHg (day 16). As seen in Table 1, M-ANP significantly reduced MBP, significantly increased plasma cGMP as well as urinary cGMP excretion, urinary volume rate (UV) and urinary Na excretion (UNa), without significant changes in heart rate (HR). Furthermore, there was a trend for plasma ANP to increase and plasma aldosterone to decrease with SQ M-ANP injection. Conclusions: This study demonstrates for the first time that chronic SQ administration of M-ANP reduces BP, activates plasma cGMP, and increases UV and UNa, without significant changes in HR in an experimental model of chronic hypertension.

Introduction: Heart failure (HF) is a major health burden in which renal impairment worsens prognosis. Targeting the vicious cycle between heart and kidney may be a more optimal therapeutic strategy for HF. The cardiac natriuretic peptides have cardiorenal protective effects through their receptor GC-A and GC-B. We sought to determine the actions of chronic administration of the dual GC-A/GC-B agonist cenderitide (CD-NP) upon cardiorenal function in experimental HF. Hypothesis: Chronic cenderitide therapy will improve cardiorenal function in HF. Methods: Experimental canine HF was induced by rapid right ventricular pacing at 240 bpm for 10 days which mimics advanced overt HF in humans. Canines (n=5 of each group) were treated with or without cenderitide (Capricor Therapeutics) subcutaneous injection (SQ) twice per day (SQ-BID, 10 ug/kg/day), cenderitide continuous SQ pump (SQ-pump, 5 ng/kg/min), or oral enalapril (ACEi, 0.5 mg/kg/day) for 10 days from the beginning of pacing. We defined hemodynamics, echo parameters, circulating neurohumoral factors at day 11, and global gene profiles using RT-PCR microarrays related to the NP system, fibrosis, growth factors, and inflammation in left atrium which undergoes early remodeling in this model. Results: The SQ-pump group showed significantly less atrial and renal weights, higher cardiac output, glomerular filtration rate, and % ejection fraction, and lower systemic vascular resistance and plasma renin activity (all p<0.05) without lowering mean arterial pressure (MAP), whereas SQ-BID and ACEi lacked these effects together with lowering MAP compared to untreated canines. Gene profile analyses showed gene related to inflammatory cytokines such as TNF-alpha, IL-1 beta, and ROCK-2, and apoptosis such as caspase 7, Fas ligand, C-Myc and TP53 were suppressed (all p<0.05) in SQ-pump but not SQ-BID or ACEi compare to un-treated group. Conclusions: Continuous SQ administration of cenderitide demonstrated superior beneficial effects on cardiorenal function and remodeling together with inhibition of circulating renin and tissue inflammatory and apoptosis pathways. Our studies suggest that continuous administration of cenderitide may provide cardiorenal protection in the setting of advanced HF.

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.

Introduction: The cardiac atrium is an important endocrine organ which also releases natriuretic peptides (NPs). We reported that dysregulation of the proNP convertases, furin and corin, may disrupt NP progressing, causing atrial fibrosis in experimental heart failure (HF). Atrial fibrosis results in atrial arrhythmias and impairs the prognosis of human HF, therefore understanding mechanisms of atrial fibrosis may help in designing treatment strategies for HF. We sought to extend our studies into the entire NP system and global gene profiles including fibrotic and inflammatory pathways in experimental HF. Hypothesis: Components of the NP, pro-fibrotic and inflammatory system will be up-regulated in the atrium in HF. Methods: Experimental canine HF was induced by rapid right ventricular pacing at 240 bpm for 10 days. We examined hemodynamics, echo parameters, circulating neurohumoral factors, and global gene profiles using RT-PCR microarrays related to the NP system, fibrosis, growth factors, inflammation and cardiac hypertrophy in left atrium (LA) from normal and HF canines (n=4 of each). Results: Experimental HF was characterized by decreased % EF and LV radial strain, and increased circulating ANP, cGMP and aldosterone levels. HF LA weight significantly increased with fibrosis and interstitial edema compared to normals. Gene profile analyses showed increases in profibrotic cytokines and extracellular matrix related genes in LA. Increased ANP expression but also increased ANP degrading enzyme IDE and decreased proANP processing enzyme corin were also observed. Genes related to inflammatory cytokines such as TNF-alpha, members of the IL-6 family, MCP-1, and cell adhesion molecules were up-regulated in LA compared to normals. Conclusions: Experimental HF was characterized by on-going atrial fibrosis with increased circulating ANP levels. Although ANP gene expression increased in failing LA, impaired proANP processing and accelerated ANP degradation may impair any anti-fibrotic ANP effects. Further, atrium may be a source of inflammatory cytokines and pro-fibrotic growth factors which may advance organ damage in HF. Our studies suggest NP treatment, perhaps in a form less susceptible to IDE degradation, may be a novel therapeutic for HF.

Introduction: ProANP is a 108 amino acid (AA) prohormone which is produced in the atria and processed to the mature 28 AA ANP which activates the guanylyl cyclase receptor-A (GC-A) and its second messenger cGMP. ProANP is found in the human circulation but its bioavailability is undefined. Hypothesis: ProANP represents a novel cardiac peptide with more sustained natriuretic actions than either carperitide (ANP) or nesiritide (BNP). ProANP itself can activate GC-A in vitro and the proANP can be processed in human serum from normals and in patients with heart failure (HF). Methods: We examined the in vivo cardiorenal actions of proANP compared to ANP, BNP, or placebo in normal canines (667 pmol/kg, n=5/group). We determined cGMP activation of proANP, ANP and BNP in GC-A expressing HEK293 cells. ProANP processing and degradation was also defined in serum from normal subjects (n=13) and patients with HF (n=14) ex vivo. Results: ProANP had significantly greater diuretic and natriuretic properties with increased renal blood flow and sustained renal tubular actions compared to ANP or BNP in canines. ProANP also resulted in greater and more prolonged cardiac unloading than ANP, and much less hypotensive effects than BNP. The plasma half-life of proANP was 4.5 times longer than ANP and 3.8 times longer than BNP. ProANP itself stimulated cGMP generation in GC-A expressing HEK cells which was equal to ANP. ProANP was processed to ANP in fresh serum from normals as well as HF patients ex vivo and the processed peptide activated cGMP in GC-A expressing HEK cells. Conclusions: ProANP represents a novel GC-A activator with a longer half-life and greater natriuresis and diuresis than ANP and BNP and less hypotensive effects than BNP. The bioactivity of exogenous proANP may be through direct stimulation of GC-A, as well as through processing into ANP, as proANP is processed in the circulation of both normal and HF. As proANP circulates in the human circulation, proANP may represent a key circulating hormone in cardiorenal and blood pressure homeostasis as well as a potential innovative therapeutic beyond ANP (carperitide) or BNP (nesiritide) for cardiorenal diseases including HF.

Abstract Introduction The rapid increase of patients of heart failure (HF) is a major health burden worldwide. Most importantly is the need to develop innovative new drugs for treatment of HF, such as sacubitril/valsartan which in part functions by enhancing the natriuretic peptides (NPs). We engineered NPA7 as a novel 30 amino acid bispecific designer peptide which activates the particulate guanylyl cyclase A receptor (pGC-A)/cGMP and for which the NPs both ANP and BNP are ligands and the Mas-receptor (MasR)/cAMP pathways for Angiotensin 1–7 (Ang1–7) is the endogenous ligand. We previously reported that acute intravenous (IV) administration of NPA7 shows cardiorenal protective and renin-aldosterone suppressing actions that go beyond the native peptides, BNP or Ang 1–7, which may have therapeutic potential for HF. Purpose To support the clinical development of NPA7 as a potential therapy in HF which promotes NP and MasR pathways, we investigated the actions and stability of subcutaneous (SQ) administration of NPA7 in normal canines. We also defined NPA7's peptide stability and metabolites in canine plasma. Methods Plasma and urinary cGMP, cardiorenal and renin-aldosterone responses to SQ injection (10μg/kg) were determined over 4 hours in normal canines (n=5) in vivo. Ex vivo, we established stability of NPA7 and key metabolites in canine serum using liquid chromatography-mass spectrometry (LC-MS). Data are expressed as mean ± SEM. * P<0.05 vs. BL. Results In vivo, SQ NPA7 resulted in a sustained increase at 2 hours in plasma (BL: 10±3; 120 min: 30±6* pmol/ml) and urinary (BL: 1033±198; 120 min: 5792±857* pmol/min) cGMP, GFR (BL: 29±6; 120 min: 70±12* ml/min) and sodium excretion (BL: 18±10; 120 min: 144±33* ueq/min). We observed a gradual reduction in BP at 60 min (BL: 109±4; 60 min: 99±7* mmHg) with a sustained decrease in PCWP at 4 hours (BL: 5±0.9; 240 min: 3.1±0.6* mmHg). SQ NPA7 also suppressed plasma renin and aldosterone up to 3 hours after SQ injection. LC-MS revealed that NPA7 was highly stable with both the pGC-A and MasR activating moieties intact ex vivo in canine serum with a disappearance time of 2 hours. We also identified 2 major NPA7 metabolites NPA71–27 and NPA71–28. Conclusions SQ NPA7 possesses cGMP activating, cardiac unloading, diuretic, natriuretic, and renin-aldosterone suppressing actions in normal canines. NPA7 is also highly stable in serum. These studies support SQ administration as an effective delivery strategy for NPA7, a first-in-class innovative bispecific dual pGC-A/MasR activator now in preclinical development for HF.

Rationale: Acute kidney injury (AKI) has a high prevalence and mortality in critically ill patients. It is also a powerful risk factor for heart failure incidence driven by hemodynamic changes and neurohormonal activation. However, no drugs have been approved by the Food and Drug Administration. Endogenous pGC-A (particulate guanylyl cyclase A receptor) activators were reported to preserve renal function and improve mortality in AKI patients, although hypotension accompanied by pGC-A activators have limited their therapeutic potential. Objective: We investigated the therapeutic potential of a nonhypotensive pGC-A activator/designer natriuretic peptide, CRRL269, in a short-term, large animal model of ischemia-induced AKI and also investigated the potential of uCNP (urinary C-type natriuretic peptide) as a biomarker for AKI. Methods and Results: We first showed that CRRL269 stimulated cGMP generation, suppressed plasma angiotensin II, and reduced cardiac filling pressures without lowering blood pressure in the AKI canine model. We also demonstrated that CRRL269 preserved glomerular filtration rate, increased renal blood flow, and promoted diuresis and natriuresis. Further, CRRL269 reduced kidney injury and apoptosis as evidenced by ex vivo histology and tissue apoptosis analysis. We also showed, compared with native pGC-A activators, that CRRL269 is a more potent inhibitor of apoptosis in renal cells and induced less decreases in intracellular Ca 2+ concentration in vascular smooth muscle cells. The renal antiapoptotic effects were at least mediated by cGMP/PKG pathway. Further, CRRL269 inhibited proapoptotic genes expression using a polymerase chain reaction gene array. Additionally, we demonstrated that AKI increased uCNP levels. Conclusions: Our study supports developing CRRL269 as a novel renocardiac protective agent for AKI treatment.

Despite optimal current therapies, cardiovascular disease remains the leading cause for death worldwide. Importantly, advances in peptide engineering have accelerated the development of innovative therapeutics for diverse human disease states. Additionally, the advancement of bispecific therapeutics targeting >1 signaling pathway represents a highly innovative strategy for the treatment of cardiovascular disease. We, therefore, engineered a novel, designer peptide, which simultaneously targets the pGC-A (particulate guanylyl cyclase A) receptor and the MasR (Mas receptor), potentially representing an attractive cardiorenoprotective therapeutic for cardiovascular disease. We engineered a novel, bispecific receptor activator, NPA7, that represents the fusion of a 22-amino acid sequence of BNP (B-type natriuretic peptide; an endogenous ligand of pGC-A) with Ang 1–7 (angiotensin 1–7)—the 7-amino acid endogenous activator of MasR. We assessed NPA7’s dual receptor activating actions in vitro (second messenger production and receptor interaction). Further, we performed an intravenous peptide infusion comparison study in normal canines to study its biological actions in vivo, including in the presence of an MasR antagonist. Our in vivo and in vitro studies demonstrate the successful synthesis of NPA7 as a bispecific receptor activator targeting pGC-A and MasR. In normal canines, NPA7 possesses enhanced natriuretic, diuretic, systemic, and renal vasorelaxing and cardiac unloading properties. Importantly, NPA7’s actions are superior to that of the individual native pGC-A or MasR ligands. These studies advance NPA7 as a novel, bispecific designer peptide with potential cardiorenal therapeutic benefit for the treatment of cardiovascular disease, such as hypertension and heart failure.

Methods developed previously for studying the effect of cerebroventricular injection or ventriculocisternal perfusion of test substances are unsatisfactory because the test substance is not confined to the central compartment. Most likely the test substance enters the peripheral circulation via the arachnoid villi. The purpose of this paper is to describe a method for perfusing the cerebroventricular system of conscious dogs without passage of test substances to the peripheral circulation. With the method described, the mean (+/- SE) cerebroventricular pressure in conscious dogs was 7.4 +/- 0.8 cmH2O (n = 16), and the mean (+/- SE) production of cerebrospinal fluid (CSF) was 25 +/- 0.3 microliter/min (n = 16). Endogenously occurring migrating myoelectric complexes (MMCs) of the small intestine were recorded in dogs before catheters were implanted in the left and right lateral ventricles and the fourth ventricle and after catheter implantation during cerebroventricular perfusion with artificial CSF alone or with CSF containing sulfated (S-CCK-OP) or nonsulfated cholecystokinin octapeptide (NS-CCK-OP). Only cerebroventricular perfusion with S-CCK-OP (1.2 pmol.kg-1.min-1; n = 20) replaced spontaneously occurring MMCs with a fed-like pattern of myoelectric activity. The results suggest that replacement of the fasting pattern of myoelectric activity with a fed-like pattern in the fasted dog was mediated by CCK-A receptors located in one or more brain nuclei surrounding the third ventricle.