Urinary cGMP Excretion Research Articles

The Cardiorenal Effects of Chronic Phosphodiesterase-V Inhibition in Preclinical Diastolic Dysfunction (Stage B Heart Failure)

ObjectiveTo determine whether chronic phosphodiesterase-V (PDEV) inhibition with tadalafil will improve urinary sodium excretion, glomerular filtration rate (GFR), plasma cyclic guanosine 3’, 5’-monophosphate (cGMP), and urinary cGMP excretion in response to volume expansion (VE) in patients with preclinical diastolic dysfunction (PDD) or stage B heart failure. BackgroundPDD is defined as abnormal diastolic function with normal systolic function, without clinical heart failure. PDD is predictive of development of heart failure and all-cause mortality. Impaired renal function and attenuated cGMP response to VE are hallmarks of PDD. MethodsA double-blind, placebo-controlled, proof-of-concept study was conducted to compare 12 weeks of tadalafil 20 mg daily (n = 14) vs placebo (n = 7). Subjects underwent 2 study visits 12 weeks apart. Renal, neurohormonal and echocardiographic assessments were performed before and after intravascular VE (normal saline 0.25 mL/kg/min for 1 hour). ResultsBaseline characteristics were similar. There was no increase in GFR, plasma cGMP or urinary cGMP excretion in response to VE in either group at visit 1. At visit 2, tadalafil did not result in significant change in GFR but increased plasma cGMP and urinary cGMP excretion at baseline. In response to VE, tadalafil resulted in increased urine flow, urinary sodium excretion, GFR (7.00 [-1.0, 26.3] vs -9.00 [-24.5, 2.0] mL/min/1.73m2; P = 0.02) and plasma cGMP (0.50 [-0.1, 0.7] vs -0.25 [-0.6, -0.1] pmol/mL; P = 0.02). It did not improve urinary cGMP excretion after VE. ConclusionIn PDD, chronic PDEV inhibition with tadalafil improved renal response to VE through increased urine flow, urinary sodium excretion, GFR, and plasma cGMP. Further studies are required to determine whether this enhanced renal response can mitigate progression to clinical heart failure.

Cardiorenal Effects of Long-Term Phosphodiesterase V Inhibition in Pre-Heart Failure.

BackgroundPhosphodiesterase V (PDEV) is upregulated in heart failure, leading to increased degradation of cGMP and impaired natriuresis. PDEV inhibition improves the renal response to B‐type natriuretic peptide in animal models. We tested the hypothesis that long‐term PDEV inhibition would improve renal function and cardiorenal response after short‐term volume load in subjects with pre–heart failure.Methods and ResultsA total of 20 subjects with pre–heart failure (defined as an ejection fraction ≤45% without previous diagnosis of heart failure) and renal impairment were randomized in a 2:1 manner to tadalafil or placebo. Baseline echocardiography and renal clearance study were performed, followed by a short‐term saline load and repeated echocardiography and renal clearance study. Subjects then received either tadalafil at a goal dose of 20 mg daily or placebo, and the study day was repeated after 12 weeks. Long‐term tadalafil did not improve glomerular filtration rate (median increase of 2.0 mL/min in the tadalafil group versus 13.5 mL/min in the placebo group; P=0.54). There was no difference in urinary sodium or cGMP excretion with PDEV inhibition following short‐term saline loading.ConclusionsGlomerular filtration rate and urinary sodium/cGMP excretion were not significantly different after 12 weeks of tadalafil compared with placebo. These results do not support the use of PDEV inhibition to improve renal response in patients with pre–heart failure.RegistrationURL: https://www.clinicaltrials.gov; Unique identifier: NCT01970176.

Blockade of the natriuretic peptide clearance receptor attenuates proteinuria in a mouse model of focal segmental glomerulosclerosis.

Glomerular podocytes play a key role in proteinuric diseases. Accumulating evidence suggests that cGMP signaling has podocyte protective effects. The major source of cGMP generation in podocytes is natriuretic peptides. The natriuretic peptide clearance receptor (NPRC) binds and degrades natriuretic peptides. As a result, NPRC inhibits natriuretic peptide‐induced cGMP generation. To enhance cGMP generation in podocytes, we blocked natriuretic peptide clearance using the specific NPRC ligand ANP(4‐23). We then studied the effects of NPRC blockade in both cultured podocytes and in a mouse transgenic (TG) model of focal segmental glomerulosclerosis (FSGS) created in our laboratory. In this model, a single dose of the podocyte toxin puromycin aminonucleoside (PAN) causes robust albuminuria in TG mice, but only mild disease in non‐TG animals. We found that natriuretic peptides protected cultured podocytes from PAN‐induced apoptosis, and that ANP(4‐23) enhanced natriuretic peptide‐induced cGMP generation in vivo. PAN‐induced heavy proteinuria in vehicle‐treated TG mice, and this increase in albuminuria was reduced by treatment with ANP(4‐23). Treatment with ANP(4‐23) also reduced the number of mice with glomerular injury and enhanced urinary cGMP excretion, but these differences were not statistically significant. Systolic BP was similar in vehicle and ANP(4‐23)‐treated mice. These data suggest that: 1. Pharmacologic blockade of NPRC may be useful for treating glomerular diseases such as FSGS, and 2. Treatment outcomes might be improved by optimizing NPRC blockade to inhibit natriuretic peptide clearance more effectively.

Abstract 15217: Combination Phosphodiesterase V Inhibition and Low Dose B-type Natriuretic Peptide Did Not Enhance Renal Function in Acute Heart Failure

Introduction: Cardiorenal dysfunction with impaired cyclic guanosine monophosphate (cGMP) response to volume load is a hallmark of heart failure. Phosphodiesterase V (PDEV) is known to be upregulated and may explain the dysfunction of renal response. Hypothesis: We tested the hypothesis that PDEV inhibition in combination with low dose intravenous (IV) B-type natriuretic peptide (BNP) would improve renal function and potentiate urinary sodium and cGMP excretion in patients with acute heart failure. Methods: Randomized open label study in 67 patients admitted to the hospital with acute heart failure. These patients were randomized to standard care, low dose IV BNP (0.005μg/kg/min), or combination low dose BNP/PDEV inhibition with sildenafil (25 mg q12 hrs) for 48 hours. Plasma and urine studies were obtained at baseline, 24 hours, and 48 hours after study drug initiation to assess renal function. The primary endpoint was the percent change in estimated glomerular filtration rate (eGFR) and blood urea nitrogen (BUN) from baseline to 48 hours. Changes from baseline were summarized with median and quartiles and groups were compared using two-sample t-test. [ClinicalTrials.gov Identifier: NCT00972569] Results: Baseline characteristics were similar between groups. Median age was 78 years and median ejection fraction 39%. Treatment with BNP and BNP/PDEV inhibitor significantly increased plasma cGMP at 24 hr (% increase of 25.6 (8.9, 13.1) and 60.8 (32.3, 103.8) for BNP and BNP/PDEV vs % decrease of 13.5 (-29.1, 14.2) for placebo, p=0.001). BNP levels were significantly higher in both groups at 48 hr compared with placebo. However, there was no significant change in eGFR, BUN, or urinary sodium/cGMP excretion between groups. Hypotension was more common in the BNP/PDEV inhibitor group. Conclusions: Low dose IV BNP and combination BNP/PDEV inhibition increased plasma cGMP in patients with acute heart failure but did not improve renal function or urinary sodium/cGMP excretion. Our study does not support the use of low dose IV BNP with or without PDEV inhibition to enhance renal function in patients admitted with acute heart failure.

ANP-stimulated Na+ secretion in the collecting duct prevents Na+ retention in the renal adaptation to acid load.

We have recently reported that type A intercalated cells of the collecting duct secrete Na+ by a mechanism coupling the basolateral type 1 Na+-K+-2Cl- cotransporter with apical type 2 H+-K+-ATPase (HKA2) functioning under its Na+/K+ exchange mode. The first aim of the present study was to evaluate whether this secretory pathway is a target of atrial natriuretic peptide (ANP). Despite hyperaldosteronemia, metabolic acidosis is not associated with Na+ retention. The second aim of the present study was to evaluate whether ANP-induced stimulation of Na+ secretion by type A intercalated cells might account for mineralocorticoid escape during metabolic acidosis. In Xenopus oocytes expressing HKA2, cGMP, the second messenger of ANP, increased the membrane expression, activity, and Na+-transporting rate of HKA2. Feeding mice with a NH4Cl-enriched diet increased urinary excretion of aldosterone and induced a transient Na+ retention that reversed within 3 days. At that time, expression of ANP mRNA in the collecting duct and urinary excretion of cGMP were increased. Reversion of Na+ retention was prevented by treatment with an inhibitor of ANP receptors and was absent in HKA2-null mice. In conclusion, paracrine stimulation of HKA2 by ANP is responsible for the escape of the Na+-retaining effect of aldosterone during metabolic acidosis.

A Human Study to Evaluate Safety, Tolerability, and Cyclic GMP Activating Properties of Cenderitide in Subjects With Stable Chronic Heart Failure.

Cenderitide is a novel designer natriuretic peptide (NP) composed of C‐type natriuretic peptide (CNP) fused to the C‐terminus of Dendroaspis natriuretic peptide (DNP). Cenderitide was engineered to coactivate the two NP receptors, particulate guanylyl cyclase (pGC)‐A and ‐B. The rationale for its design was to achieve the renal‐enhancing and antifibrotic properties of dual receptor activation, but without clinically significant hypotension. Here we report the first clinical trial on the safety, tolerability, and cyclic guanosine monophosphate (cGMP) activating properties of Cenderitide in subjects with stable heart failure (HF). Four‐hour infusion of Cenderitide was safe, well‐tolerated, and significantly increased plasma cGMP levels and urinary cGMP excretion without adverse effects with no change in blood pressure. Thus, Cenderitide has a favorable safety profile and expected pharmacological effects in stable human HF. Our results support further investigations of Cenderitide in HF as a potential future cGMP‐enhancing therapeutic strategy.

Abstract 12893: Design and cGMP-Generating Actions of a Novel Selective and Non-Selective Particulate Guanylyl Cyclase B Activator Targeting Cardiorenal Fibrotic Remodeling

Introduction: Cardiorenal fibrotic remodeling (CFR), often in the setting of heart failure (HF) and kidney disease (CKD), is a major cause for increased risk in mortality, for which there are no effective drugs. C-type natriuretic peptide (CNP) is a 22 amino acid (AA) ligand for the particulate guanylyl cyclase receptor-B (GC-B) and exerts anti-fibrotic actions, with little effect on blood pressure (BP) or renal function, via its second messenger cGMP. To overcome CNP’s limited efficacy with its short half-life, we designed 2 novel CNP-based peptides (C53 and CU32). C53 consists of CNP plus a 31 AA N-terminus extension, while CU32 is the CNP ring fused with the N- and C-termini of urodilatin (GC-A agonist). Here we investigated the cGMP generating actions of C53 and CU32, with the goal of advancing novel GC-B/cGMP therapeutics for CFR. Hypotheses: We hypothesized that C53 and CU32 would have cGMP activating actions in vivo, however only C53 would generate cGMP production selectively via GC-B in vitro. We also hypothesize that the addition of the C-termini to CU32 would form a non-selective GC-B/cGMP agonist with renal and BP lowering actions. Methods: HEK293 cells expressing human GC-B or -A and human fibroblasts (hFs), where GC-B is abundant, were stimulated with C53 or CU32 (10-8M) for 10 min. Two groups of normal rats (n=8) received a 75 min equimolar IV infusion of C53 or CU32. Change in mean arterial pressure (ΔMAP), sodium (Na) excretion and plasma and urinary cGMP were assessed. *p<0.05. Results: C53 and CU32 activated cGMP in GC-B cells (C53: 75±9, CU32: 24±2 pmol/well*), while only CU32 activated cGMP in GC-A cells (C53: 0±0, CU32: 50±3 pmol/well*). In hFs, both C53 and CU32 generated cGMP. In vivo, plasma cGMP (C53: 91±4, CU32: 94±6 pmol/ml) and urinary cGMP excretion (C53: 177±28, CU32: 227±28 pmol/min) were similar with both peptides. Yet, CU32 induced a greater reduction in ΔMAP (C53: -7±1, CU32: -16±3 mmHg*) and increased Na excretion (C53: 1.4±0.2, CU32: 2.4±0.2 uEq/m*) compared to C53. Conclusions: We report the engineering of a selective (C53) and non-selective (CU32) GC-B agonist that has favorable cGMP generating action in hFs and in vivo. Thus, C53 and CU32 may represent novel GC-B therapeutics that could be tailored to specific phases of HF or CKD to prevent CFR.

April 2016 at a glance. Focus on cardiac remodeling, biomarkers and treatment.

Mohamed et al. show the long-term effects of volume overload induced by aortacaval shunt in mice followed by serial echocardiography until the development of reduced left ventricular ejection fraction (LVEF) and heart failure (HF). Transition to HF was characterized by reduced sarcomeric titin phosphorylation, a cause of increased sarcomeric stiffness, activation of calcium/calmodulin-dependent protein kinase II, decreased protein kinase B (Akt) phosphorylation, high oxidative stress, and increased apoptosis. These changes were worsened in Akt-deficient mice showing the critical role of this kinase.1 Tenascin-C is a large glycoprotein which appears in the extracellular matrix following tissue injury or tumor formation. Yokokawa et al. have measured it in 123 patients with dilated cardiomyopathy undergoing endomyocardial biopsy. Patients with high myocardial tenascin had worse cardiac remodeling and poorer outcomes. Diabetes and HF severity were independent determinants of its content.2 Suematsu et al. have compared valsartan and LCZ696 with a control group in streptozotocin induced diabetic mice. Compared with control and with valsartan groups, administration of LCZ696 improved LVEF and reduced LV ANP mRNA, the LV fibrotic area and TGF-β levels.3 Atrial flutter-related tachycardiomyopathy is a specific form of cardiac remodeling. Brembilla-Perot et al. report the 6 months follow-up of 1269 patients who underwent radiofrequency ablation for atrial flutter. A marked improvement in LVEF was shown in 56% of the patients who had a reduced LVEF before ablation. An ischemic etiology and prescription of antiarrhythmic drugs were associated with a lower likelihood of LVEF improvement. Patients who improved their LVEF had similar outcomes as those with a normal LVEF at baseline whereas those with a persistent low LVEF post-ablation had an almost 3-fold higher mortality rate.4 Ovchinnikova et al. have measured circulating microRNAs in 137 patients with acute HF, 20 with chronic HF, 8 with dyspnea caused by COPD exacerbation, and 41 healthy controls. Plasma levels of miRNAs were decreased in the patients with acute HF compared with all the other groups with 15 of the 226 miRNAs measured that showed a larger than a 4-fold difference. Among them, 12 were different also in the validation cohort and a prognostic value was shown for 7 of them so that their further drop within 48 hours after admission was associated with an increased risk of 180-day mortality.5 Another study shows that liver function exams were abnormal in up to 40% of the patients with acute HF enrolled in ASCEND-HF (Acute Study of Clinical Effectiveness of Nesiritide in Decompensated Heart Failure) and increased serum bilirubin had prognostic value also at multivariable analysis.6 Sleep-disordered breathing (SDB) is found in over half of the patients with HF and is associated with reduced quality of life and poor prognosis. Pearse and Cowie have reviewed the classification, pathophysiology, prognosis and treatment options for patients with HF and SDB. The results of the most recent trials, including SERVE-HF, are discussed.7 The effects of chronic subcutaneous (SQ) BNP administration were evaluated by McKie et al. in a double-blind, placebo controlled study in patients with asymptomatic LV dysfunction who underwent plasma volume expansion at baseline and after 12 weeks. Compared with placebo, chronic SQ, twice daily, BNP administration was associated with an increase in urinary sodium and cGMP excretion and in urine flow with a tendency to a different glomerular filtration rate response to volume expansion and to a lower LV mass.8

Cenderitide: structural requirements for the creation of a novel dual particulate guanylyl cyclase receptor agonist with renal-enhancing in vivo and ex vivo actions.

Cenderitide is a novel dual natriuretic peptide (NP) receptor chimeric peptide activator, which targets the particulate guanylyl cyclase B (pGC-B) receptor and pGC-A unlike native NPs. Cenderitide was engineered to retain the anti-fibrotic properties of C-type natriuretic peptide (CNP)/pGC-B with renal-enhancing actions facilitated by fusion to the carboxyl terminus of Dendroaspis NP (DNP), a pGC-A agonist, to CNP. Here, we address significance of the DNP carboxyl terminus in dual pGC receptor activation and actions of cenderitide compared with CNP on renal function and cyclic guanosine monophosphate (cGMP) in vivo and ex vivo in normal canines. In vitro, only cenderitide and not CNP or three CNP-based variants was a potent dual pGC-A/pGC-B activator of cGMP production (from 5 to 237 pmol/mL) in human embryonic kidney (HEK) 293 cells overexpressing human pGC-A while in pGC-B overexpressing cells cenderitide increased cGMP production (from 4 to 321 pmol/mL) while the three CNP-based variants were weak agonists. Based upon our finding that the DNP carboxyl terminus is a key structural requirement for dual pGC-A/pGC-B activation, we defined in vivo the renal-enhancing actions of cenderitide compared with CNP. Cenderitide increased urinary cGMP excretion (from 989 to 5977 pmol/mL), net generation of renal cGMP (821-4124 pmol/min), natriuresis (12-242 μEq/min), and glomerular filtration rate (GFR) (37-51 mL/min) while CNP did not. We then demonstrated the transformation of CNP ex vivo into a renal cGMP-activating peptide which increased cGMP in freshly isolated glomeruli eight-fold greater than CNP. The current study establishes that dual pGC-A and pGC-B activation with CNP requires the specific carboxyl terminus of DNP. In normal canines in vivo and in glomeruli ex vivo, the carboxyl terminus of DNP transforms CNP into a natriuretic and GFR-enhancing peptide. Future studies of cenderitide are warranted in cardiorenal disease states to explore its efficacy in overall cardiorenal homeostasis.

Abstract 12236: A Novel Particulate Guanylyl Cyclase B Agonist With Therapeutic Potential Targeting Fibrosis via cGMP Generation for Cardiorenal Disease

Introduction: Fibrosis is a hallmark of cardiorenal diseases, including heart failure, for which there are few effective therapies. C-type natriuretic peptide (CNP) is a cardiorenal-derived peptide whose mature active form, CNP-22, possesses potent anti-fibrotic actions through the activation of guanylyl cyclase receptor B (GC-B) and its second messenger, cGMP. However, CNP-22’s therapeutic potential is limited due to its short half-life. We recently identified a 53 amino acid (AA) peptide, CNP-53, whose structure consists of CNP-22 with an additional 31 AA on the N-terminus. Herein, we investigated the cGMP generating actions of CNP-53 (compared to CNP-22) in vivo and in vitro, with the goal of advancing a potential anti-fibrotic strategy for cardiorenal disease. Hypotheses: We hypothesized that CNP-53 would elevate circulating CNP levels as well as have greater cGMP activating actions compared to CNP-22 in vivo. We also hypothesized that CNP-53 would stimulate GC-B specific cGMP production in vitro. Methods: In vivo, two groups of anesthetized normal rats (n=8) received a 75-minute intravenous infusion of an equimolar dose of CNP-53 or CNP-22. Mean arterial pressure (MAP), plasma CNP and plasma and urinary cGMP were assessed. In vitro, HEK 293 cells over-expressing GC-A and GC-B and human fibroblasts (hFs), where GC-B is abundant, were stimulated with CNP-53 or CNP-22 for 10 minutes at a dose of (10-8M). Data are mean ± SE, *p<0.05. Results: In vivo, plasma CNP (CNP-53: 311±66, CNP-22: 153±16 pg/ml*) and cGMP (CNP-53: 21±2, CNP-22: 11±1 pmol/ml*) and urinary cGMP excretion (CNP-53: 75±4, CNP-22: 38±1 pmol/min*) were greater with CNP-53 infusion than CNP-22. MAP was similar between the 2 groups (CNP-53: 85±2, CNP-22: 88±4 mmHg). In vitro, both CNP-53 and CNP-22 activated cGMP in GC-B HEK cells and in hFs, with no activation of cGMP in GC-A HEK cells. Conclusions: These findings demonstrate that CNP-53 is superior to CNP-22 in activating cGMP in vivo, without inducing hypotension. Additionally, CNP-53 is a GC-B specific cGMP activating peptide that potently increases cGMP in hFs. Thus, CNP-53 may represent a novel first in class GC-B agonist with therapeutic potential targeting fibrosis and preventing the progression of cardiorenal disease.

Improvements in Left Atrial Appendage Velocities Following Transcatheter Aortic Valve Replacement

Background: The natriuretic peptide family consists of atrial and b-type natriuretic peptide (ANP, BNP) and C-type natriuretic peptide (CNP). ANP and BNP are mainly produced in the heart and possess potent blood pressure lowering as well as natriuretic and diuretic actions through guanylyl cyclase receptor A (GC-A) and cGMP activation. Meanwhile, CNP is an endothelium and renal derived hormone of which its mature biological active form, CNP22, has the most robust cardiorenal anti-remodeling properties via GC-B activation and generation of cGMP compared to GC-A. Recently we reported that heart failure (HF) patients have increased plasma and markedly elevated urinary levels of a larger CNP molecular form, CNP53, which also is biologically active and generates cGMP through the GC-B receptor. Notably, preliminary studies have demonstrated that CNP53 is more biologically active and has renal enhancing actions in vivo, compared to CNP22. However, it remains unclear if CNP53’s biological and renal actions differ from that of BNP in vivo. Objectives: To define the cardiorenal and cGMP generating actions of CNP53, GC-B agonist, compared to BNP, a GC-A agonist, in vivo. Hypothesis: We hypothesize that CNP53 has cardiorenal and cGMP actions in vivo, but will exhibit less hypotension than BNP. Methods: Vehicle (V), an equimolar dose of BNP or CNP53 (Phoenix Pharmaceuticals, CA) was infused intravenously into anesthetized normal rats. Mean arterial blood pressure (MAP), glomerular filtration rate (GFR) and urinary sodium (Na) excretion were assessed. Plasma and urinary cGMP was determined by radioimmunoassay. Data are presented as Mean6SE. *p!0.05 between all groups. Results: During CNP53 and BNP infusion, plasma cGMP (V: 961, CNP53: 9164, BNP: 190637*, pmol/ml) and urinary cGMP excretion (V: 3366, CNP53: 176628, BNP: 5096124*, pmol/min) significantly increased compared to vehicle. Moreover, GFR (V: 4.860.3, CNP53: 5.760.2, BNP: 7.260.5*, ml/min/kg) and urinary Na excretion (V: 0.860.1, CNP53: 1.460.2, BNP: 2.460.1*,uEq/min) also significantly increased with CNP53 and BNP compared to vehicle. Notably, BNP had the greatest drop in MAP compared to vehicle and CNP53 (DMAP V: 2.06 3.0, CNP53: -4.062.7, BNP: -11.363.8*, mmHg). Conclusion: We report CNP53 exerts favorable cardiorenal and cGMP actions without excessive hypotension as seen with BNP. These findings support an emerging concept that CNP53 may be an alternative therapeutic strategy for HF, especially in the setting of hypotension, and this warrants further investigation.

Therapeutic Insights for Heart Failure: In Vivo Comparison Between a Guanylyl Cyclase Receptor A and a Novel Guanylyl Cyclase Receptor B Activator

The natriuretic peptide family consists of atrial and b-type natriuretic peptide (ANP, BNP) and C-type natriuretic peptide (CNP). ANP and BNP are mainly produced in the heart and possess potent blood pressure lowering as well as natriuretic and diuretic actions through guanylyl cyclase receptor A (GC-A) and cGMP activation. Meanwhile, CNP is an endothelium and renal derived hormone of which its mature biological active form, CNP22, has the most robust cardiorenal anti-remodeling properties via GC-B activation and generation of cGMP compared to GC-A. Recently we reported that heart failure (HF) patients have increased plasma and markedly elevated urinary levels of a larger CNP molecular form, CNP53, which also is biologically active and generates cGMP through the GC-B receptor. Notably, preliminary studies have demonstrated that CNP53 is more biologically active and has renal enhancing actions in vivo, compared to CNP22. However, it remains unclear if CNP53’s biological and renal actions differ from that of BNP in vivo. To define the cardiorenal and cGMP generating actions of CNP53, GC-B agonist, compared to BNP, a GC-A agonist, in vivo. We hypothesize that CNP53 has cardiorenal and cGMP actions in vivo, but will exhibit less hypotension than BNP. Vehicle (V), an equimolar dose of BNP or CNP53 (Phoenix Pharmaceuticals, CA) was infused intravenously into anesthetized normal rats. Mean arterial blood pressure (MAP), glomerular filtration rate (GFR) and urinary sodium (Na) excretion were assessed. Plasma and urinary cGMP was determined by radioimmunoassay. Data are presented as Mean±SE. *p<0.05 between all groups. During CNP53 and BNP infusion, plasma cGMP (V: 9±1, CNP53: 91±4, BNP: 190±37*, pmol/ml) and urinary cGMP excretion (V: 33±6, CNP53: 176±28, BNP: 509±124*, pmol/min) significantly increased compared to vehicle. Moreover, GFR (V: 4.8±0.3, CNP53: 5.7±0.2, BNP: 7.2±0.5*, ml/min/kg) and urinary Na excretion (V: 0.8±0.1, CNP53: 1.4±0.2, BNP: 2.4±0.1*,uEq/min) also significantly increased with CNP53 and BNP compared to vehicle. Notably, BNP had the greatest drop in MAP compared to vehicle and CNP53 (ΔMAP V: 2.0± 3.0, CNP53: -4.0±2.7, BNP: -11.3±3.8*, mmHg). We report CNP53 exerts favorable cardiorenal and cGMP actions without excessive hypotension as seen with BNP. These findings support an emerging concept that CNP53 may be an alternative therapeutic strategy for HF, especially in the setting of hypotension, and this warrants further investigation.

Abstract 12951: Human C-Type Natriuretic Peptide-53: A Distinct Molecular Form With in vitro and in vivo Biological Actions Through cGMP Generation

Introduction: ANP and BNP are cardiac hormones that have a natriuretic and diuretic role in heart failure (HF) through guanylyl cyclase receptor A (GC-A) and cGMP generation. C-type natriuretic peptide (CNP) is part of this hormone family and is produced in the heart and endothelium. The biologically active form, CNP-22, is known to have potent anti-remodeling properties through the activation of GC-B and cGMP, but lacks natriuretic actions. Recently we have identified that another larger CNP form, CNP-53, is elevated in the plasma of acute decompensated HF patients, however it unclear if CNP-53 possesses biological actions. Hypothesis: We hypothesized that CNP-53 would be biologically active in vivo and in vitro, and would stimulate GC-B specific cGMP production. Methods: HEK 293 cells over-expressing GC-A and GC-B and human cardiac fibroblasts (hCFs), in which GC-B are in abundance, were stimulated with CNP-53 for 10 minutes at a dose of (10-8M). Two groups of anesthetized rats (n=8) received a 75-minute infusion of normal saline vehicle (V) or CNP-53 (1 ug/kg/min) and then mean arterial pressure (MAP), sodium excretion and urinary and plasma cGMP were assessed. Results: CNP-53 significantly activated cGMP in GC-B HEK cells (75±9 vs. 0.4±0.1 pmol/well, p&lt;0.05) and in hCFs (0.17±0.03 vs. 0.00±0.00 pmol/well, p&lt;0.05) compared to no treatment. In contrast CNP-53 failed to generate cGMP in GC-A HEK cells. In vivo, CNP-53 decreased MAP compared to vehicle (V: 101±2, CNP-53: 94±2 mmHg, p&lt;0.05). CNP-53 had a greater natriuretic effect compared vehicle (V: 0.86±0.11, CNP-53: 1.39±0.21 uEq/min, p&lt;0.05). Urinary cGMP excretion (V: 33±6, CNP-53: 177±28 pmol/min, p&lt;0.05) and plasma cGMP generation was also significantly higher with CNP-53 compared to vehicle (V: 9±2, CNP-53: 91±3 pmol/ml, p&lt;0.05). Conclusions: CNP-53, a higher molecular form of CNP that was reported to be increased human HF plasma, is bioactive with GC-B specific cGMP generation in vitro. Moreover, infusion of CNP-53 generates significant urinary and plasma cGMP and has natriuretic actions with minimal hypotensive effects in vivo. This study advances our understanding of CNP biology and provides new insights into potential CNP therapeutic opportunities targeting the progression of HF.

Abstract 19094: Antihypertensive Properties of Subcutaneous M-ANP, a Novel Designer Atrial Natriuretic Peptide, in a Canine Model of Chronic Angiotensin-II Induced Hypertension

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&lt;0.05 Ang II Day 16 vs. baseline; *P&lt;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.

Chronic uninephrectomy in a large animal is characterized by preservation of cardiovascular homeostasis but with cardiorenal activation of the cGMP system together with atrial fibrosis

Purpose: While renal donation is safe and widely employed for the treatment of endstage chronic kidney disease, recent experimental and human studies suggest complex cardiovascular adaptation that may impact long-term cardiovascular homeostasis. Indeed, in a large animal model of acute uninephrectomy (UNX) we observed reduced CO and increased SVR. Here we define chronic cardiovascular, renal and humoral adaptation in a large animal model 12 weeks after UNX with the goal of understanding integrative adaptations so as to advance possible cardiorenal protective strategies in the setting of mild renal insufficiency and UNX commonly associated with renal donation. Methods: Dogs were assigned to UNX (n=6) or sham operation (n=6). Echocardiography was performed at baseline and after 12 weeks. At 12 weeks cardiovascular, renal and humoral functions were assessed invasively. Fibrosis was assessed by Picrosirius red staining, and microarray analyses were performed on tissue from the left atrium (LA) and left ventricle (LV). Data are presented as mean±SD, * indicates p<0.05. Results: UNX dogs had compensatory increased left kidney (LK) weight at 12 weeks (80±10 vs. 59±5g, *) as well as increased LK GFR (59±11 vs. 41±6 mL/min, *). The groups were equal regarding MAP, PAP, PCWP, CO and SVR, and diastolic and systolic functions were unaltered in both groups 12 weeks after surgery. Plasma ANP, BNP and aldosterone were not different after 12 weeks, however plasma cGMP was higher in the UNX dogs (6.2±1.2 vs. 4.6±1.0 pmol/mL, *). LK urinary cGMP excretion was increased in the UNX dogs (1132±325 vs. 667±121 pmol/min, *) and there was a trend for increased renal generation of cGMP (UNX 777±345 vs. sham 475±112 pmol/min, p=0.07). Left atrial fibrosis was higher in the UNX group (13.7±5.1 vs. 7.7±2.3%, *). The UNX group had > 2 fold significant changes in the expression of 10 genes in the LA and 7 genes in the LV, associated with cardiac remodeling. Conclusion: UNX in a large animal is characterized by compensatory renal hypertrophy and preserved cardiovascular homeostasis after 12 weeks. Importantly however, the cardiorenal protective cGMP system is activated which may reflect triggering of endogenous vasodilating pathways mediating the preserved cardiovascular and renal function. Furthermore, our model is characterized by subclinical atrial fibrosis and altered remodeling-related gene expressions in the LA and LV. These studies provide insights into the complex responses to UNX and possible cGMP therapeutic strategies for cardioprotection in the setting of reduced renal mass.

Concerted Action of ANP and Dopamine D1-Receptor to Regulate Sodium Homeostasis in Nephrotic Syndrome

The edema formation in nephrotic syndrome (NS) is associated with a blunted response to atrial natriuretic peptide (ANP). The natriuretic effects of ANP have been related to renal dopamine D1-receptors (D1R). We examined the interaction between ANP and renal D1R in rats with puromycin aminonucleoside-induced NS (PAN-NS). Urinary sodium, cyclic guanosine monophosphate (cGMP) excretion, and D1R protein expression and localization in renal tubules were evaluated in PAN-NS and control rats before and during volume expansion (VE). The effects of zaprinast (phosphodiesterase type 5 inhibitor), alone or in combination with Sch-23390 (D1R antagonist), were examined in both groups. The increased natriuresis and urinary cGMP excretion evoked by acute VE were blunted in PAN-NS despite increased levels of circulating ANP. This was accompanied in PAN-NS by a marked decrease of D1R expression in the renal tubules. Infusion of zaprinast in PAN-NS resulted in increased urinary excretion of cGMP and sodium to similar levels of control rats and increased expression of D1R in the plasma membrane of renal tubular cells. Combined administration of Sch-23390 and zaprinast prevented natriuresis and increased cGMP excretion induced by zaprinast alone. We conclude that D1R may play a major role in the ANP resistance observed in PAN-NS.

Sildenafil Treatment Prevents Glomerular Hypertension and Hyperfiltration in Rats with Renal Ablation

Background: Sildenafil treatment ameliorates progressive renal injury resulting from extensive renal ablation; however, modifications induced by sildenafil in the glomerular hemodynamic pathophysiology of the remnant kidney have not been investigated. Aim: To determine the effects of sildenafil in the glomerular microcirculation and their relation to histological damage in the renal ablation model. Methods: Micropuncture studies were performed 60 days after 5/6 nephrectomy in rats that received no treatment, sildenafil (5 mg/kg/day) and reserpine, hydralazine and hydrochlorothiazide to maintain the blood pressure within normal levels. Sham-operated rats untreated and treated with sildenafil served as controls. Results: As expected, renal ablation induced systemic and glomerular hypertension, hyperfiltration, proteinuria, glomerulosclerosis and tubulointerstitial inflammatory damage in the remnant kidney. Sildenafil treatment prevented single-nephron hyperfiltration and hypertension, suppressed renal arteriolar remodeling, ameliorated systemic hypertension and proteinuria, increased urinary excretion of cGMP and NO₂^–/NO₃^–, decreased oxidative stress and improved histological damage in the remnant kidney. Normalization blood pressure with reserpine, hydralazine and hydrochlorothiazide did not modify glomerular hemodynamics, proteinuria or histological changes induced by renal ablation. Conclusions: Beneficial effects of sildenafil in the remnant kidney are associated with a reduction in the arteriolar remodeling, renal inflammatory changes and prevention of changes in the glomerular microcirculation.

Ile-Pro-Pro and Val-Pro-Pro Tripeptide-Containing Milk Product has Acute Blood Pressure Lowering Effects in Mildly Hypertensive Subjects

Casein-derived tripeptides isoleucine-proline-proline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro) lower blood pressure (BP) in long-term clinical studies. Their acute effects on BP and vascular function, important for daily dosing scheme, were studied in a placebo-controlled double-blind crossover study using a single oral dose of a fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro as well as plant sterols. Twenty-five subjects with untreated mild hypertension received in random order 250 g of study product (25 mg peptides and 2 g plant sterols) or placebo. Ambulatory BP was monitored for 8 h post-dose and arterial stiffness measured by pulse wave analysis at 2, 4, and 8 h. Blood and urine samples were analyzed for markers of the renin-angiotensin system (RAS) and endothelial function. Baseline adjusted treatment effect for systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial BP was −2.1 mmHg (95% CI: −4.1 to −0.1, p = 0.045), −1.6 mmHg (95% CI: −3.1 to −0.1, p = 0.03), and −1,9 mmHg (95% CI: −3–3 to −0.4, p = 0.0093), respectively, in favor of the active treatment for 8 h post- dose. No significant differences between the treatments were seen in brachial or aortic augmentation index, pulse wave velocity, or markers of RAS. Urinary excretion of cGMP, the second messenger of endothelial nitric oxide, was higher in the active group vs. placebo (p = 0.01). The results indicate that a single dose of a fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro and plant sterols acutely lowers brachial SBP and DBP in mildly hypertensive subjects.

Multidrug Resistance Protein 4 (MRP4/ABCC4) Regulates cAMP Cellular Levels and Controls Human Leukemia Cell Proliferation and Differentiation

Increased intracellular cAMP concentration plays a well established role in leukemic cell maturation. We previously reported that U937 cells stimulated by H2 receptor agonists, despite a robust increase in cAMP, fail to mature because of rapid H2 receptor desensitization and phosphodiesterase (PDE) activation. Here we show that intracellular cAMP levels not only in U937 cells but also in other acute myeloid leukemia cell lines are also regulated by multidrug resistance-associated proteins (MRPs), particularly MRP4. U937, HL-60, and KG-1a cells, exposed to amthamine (H2-receptor agonist), augmented intracellular cAMP concentration with a concomitant increase in the efflux. Extrusion of cAMP was ATP-dependent and probenecid-sensitive, supporting that the transport was MRP-mediated. Cells exposed to amthamine and the PDE4 inhibitor showed enhanced cAMP extrusion, but this response was inhibited by MRP blockade. Amthamine stimulation, combined with PDE4 and MRP inhibition, induced maximal cell arrest proliferation. Knockdown strategy by shRNA revealed that this process was mediated by MRP4. Furthermore, blockade by probenecid or MRP4 knockdown showed that increased intracellular cAMP levels induce maturation in U937 cells. These findings confirm the key role of intracellular cAMP levels in leukemic cell maturation and provide the first evidence that MRP4 may represent a new potential target for leukemia differentiation therapy.

Abstract 5265: Alternatively Spliced B-Type Natriuretic Peptide (ASBNP) is a Renal Selective Natriuretic and Diuretic Peptide Without Hypotensive Properties

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&lt;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.