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Background: Renal dysfunction is an important independent prognostic factor in heart failure (HF). Cardiac resynchronization therapy (CRT) improves functional status and left ventricular (LV) function in HF patients with ventricular dyssynchrony, but the impact of CRT on renal function is less defined. We hypothesized that CRT would improve glomerular filtration rate as estimated by the abbreviated Modification of Diet in Renal Disease equation (eGFR).Methods and Results: The Multicenter InSync Randomized Clinical Evaluation (MIRACLE) study evaluated CRT in HF patients with NYHA Class III-IV, ejection fraction or=130 ms. Patients were evaluated before and 6 months after randomization to control (n = 225) or CRT (n = 228). Patients were categorized according to their baseline eGFR: >or=90 (category A), 60 Conclusions: CRT increased eGFR and reduced blood urea nitrogen in HF patients with moderately 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. [ABSTRACT FROM AUTHOR]

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.

The natriuretic peptides (NPs) B-type NP (BNP) and urodilatin (URO) exert renal protective properties via the particulate guanylyl cyclase A receptor (pGC-A). As a potential renal-enhancing strategy, we engineered a novel designer peptide that we call CRRL269. CRRL269 was investigated in human cell lines and in normal canines to define potential cardiorenal enhancing actions. The mechanism of its cardiorenal selective properties was also investigated. In vitro NP receptor activity was quantified with guanosine 3',5'-cyclic monophosphate generation. In vivo effects were determined in normal canine acute infusion studies. We observed that CRRL269 demonstrated enhanced pGC-A activity in renal compared with nonrenal cell lines. CRRL269 exerted enhanced resistance to neprilysin compared with URO. Importantly, CRRL269 exhibited significant and greater increases in urinary sodium excretion and diuresis, with less blood pressure reduction, than BNP or URO in normal canines. CRRL269 retained potent renin-angiotensin-aldosterone system (RAAS) suppressing properties shared by URO and BNP. Also, CRRL269 exerted less arterial relaxation and higher cAMP cardiomyocytes generation than BNP. CRRL269 possessed superior renal and pGC-A activating properties compared with BNP or URO in vitro. CRRL269 exerted enhanced renal actions while suppressing RAAS in vivo and with less hypotension compared with URO or BNP. Together, our study suggests that CRRL269 is a promising innovative renal-enhancing drug, with favorable protective actions targeting cardiorenal disease states through the pGC-A receptor.

Background: Heart failure (HF) is a disease of neurohumoral dysfunction and current pharmacological therapies for HF have not improved mortality rates, thus requiring additional new strategies. Waon therapy for HF patients may be a complementary strategy with peripheral vasodilation via nitric oxide. We hypothesized that Waon therapy would improve neurohumoral factors, such as natriuretic peptides (NP) and the renin-angiotensin-aldosterone system (RAAS) in HF., Methods and results: Plasma samples were collected from patients enrolled in the WAON-CHF Study (Waon therapy (n=77) or control (n=73)) before and after the treatment. B-type NP (BNP), C-type NP (CNP), and aldosterone (Aldo) levels were measured by respective specific radioimmunoassays. Although clinical parameters significantly improved in the Waon group compared with the control group, BNP, Aldo, and CNP levels were not statistically different between groups. On subanalysis with patient variables, BNP levels were improved in the Waon group treated with angiotensin-converting enzyme inhibitor/angiotensin-receptor blocker or spironolactone. In addition, Aldo levels were improved in the Waon group patients with diabetes mellitus, hypertension, and inotrope use, and CNP levels were improved in Waon group patients with estimated glomerular filtration rate <60 mL/min/1.73 m 2 . These changes were not observed in the control group., Conclusions: Waon therapy may accelerate the favorable actions of RAAS modulators in HF. (WAON-CHF Study: UMIN000006705).

Background: Elevated plasma fibroblast growth factor 23 (FGF23) is a prognostic marker in chronic kidney disease. Recently, FGF23 was reported to also be a predictive factor in chronic congestive heart failure (HF). To date however, plasma levels in acute decompensated HF (ADHF) have not been reported and myocardial production and distribution of FGF23 in HF is poorly defined. We aimed to determine plasma levels and myocardial production of FGF23 in ADHF., Methods: Plasma FGF23, N-terminal pro B-type natriuretic peptide (NT-proBNP) and estimated glomerular filtration rate (eGFR) were assessed in 21 ADHF patients and 19 controls. Myocardial gene expression and distribution of FGF23 was determined on left ventricular samples from HF patients and normal controls., Results: Plasma FGF23 was markedly higher in ADHF patients compared with controls (1498 ± 1238 versus 66 ± 27 RU/mL, P < 0.0001). There were no correlations between FGF23 and eGFR, NT-proBNP, ejection fraction or age. ADHF subjects with eGFR >60 mL/min/1.73 m(2) had FGF23 levels of 1526 ± 1601 RU/mL versus 55 ± 20 RU/mL in controls (P = 0.007). Quantified myocardial FGF23 gene expression was similar between HF patients and controls. Myocardial FGF23 immunostaining was similar between HF patients and controls, with equal distribution throughout cardiomyocytes., Conclusion: Patients with ADHF had markedly elevated plasma FGF23 levels. Myocardial FGF23 gene expression was present in HF at a similar level as normal controls, and immunohistochemistry showed similar cellular distribution of FGF23 in HF and controls, suggesting that the myocardium does not contribute to the elevated circulating FGF23 in HF., (© The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Background: Cyclic guanosine monophosphate-protein kinase G-phosphodiesterase 5 signaling may be disturbed in heart failure (HF) with preserved ejection fraction, contributing to cardiac remodeling and dysfunction. The purpose of this study was to manipulate cyclic guanosine monophosphate signaling using the dipeptidyl-peptidase 4 inhibitor saxagliptin and phosphodiesterase 5 inhibitor tadalafil. We hypothesized that preservation of cyclic guanosine monophosphate cGMP signaling would attenuate pathological cardiac remodeling and improve left ventricular (LV) function., Methods and Results: We assessed LV hypertrophy and function at the organ and cellular level in aortic-banded pigs. Concentric hypertrophy was equal in all groups, but LV collagen deposition was increased in only HF animals. Prevention of fibrotic remodeling by saxagliptin and tadalafil was correlated with neuropeptide Y plasma levels. Saxagliptin better preserved integrated LV systolic and diastolic function by maintaining normal LV chamber volumes and contractility (end-systolic pressure-volume relationship, preload recruitable SW) while preventing changes to early/late diastolic longitudinal strain rate. Function was similar to the HF group in tadalafil-treated animals including increased LV contractility, reduced chamber volume, and decreased longitudinal, circumferential, and radial mechanics. Saxagliptin and tadalafil prevented a negative cardiomyocyte shortening-frequency relationship observed in HF animals. Saxagliptin increased phosphodiesterase 5 activity while tadalafil increased cyclic guanosine monophosphate levels; however, neither drug increased downstream PKG activity. Early mitochondrial dysfunction, evident as decreased calcium-retention capacity and Complex II-dependent respiratory control, was present in both HF and tadalafil-treated animals., Conclusions: Both saxagliptin and tadalafil prevented increased LV collagen deposition in a manner related to the attenuation of increased plasma neuropeptide Y levels. Saxagliptin appears superior for treating heart failure with preserved ejection fraction, considering its comprehensive effects on integrated LV systolic and diastolic function., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background: We recently reported that normal aldosterone levels are associated with cardiovascular, renal, and metabolic disease in a sample of the US general community (Visit 1). For the current analyses we used the same cohort in a new 4-year follow-up study (Visit 2)., Methods and Results: We measured aldosterone at Visit 1 and analyzed its predictive role for new diseases at Visit 2 (n=1140). We measured aldosterone at Visit 2 and investigated its associations with disease at Visit 2 (n=1368). We analyzed aldosterone continuously and we also dichotomized the variable as whether subjects were in the third tertile versus second and first tertiles. As continuous variable at Visit 1, aldosterone predicted new onset hypertension (HTN) (OR=1.36, CI=1.13-1.63, P=0.001), central obesity (OR=1.36, CI=1.07-1.73, P=0.011), and use of lipid-lowering drugs (OR=1.25, CI=1.05-1.48, P=0.012) at Visit 2, after adjustment for age, sex, and body mass index. When in the third tertile (8.5-88.6 ng/dL), aldosterone predicted type 2 diabetes (T2DM, OR=1.96, CI=1.03-3.70, P=0.039). At Visit 2, aldosterone remained associated with HTN, obesity, and chronic kidney disease (CKD), as reported for Visit 1. However, aldosterone was not associated with heart failure (HF) at Visit 1 and 2, nor was aldosterone a predictor of HF between visits., Conclusions: Aldosterone predicts new HTN, central obesity, T2DM, and use of lipid-lowering drugs in the general community and remains associated with HTN, obesity, and CKD over 4 years. Aldosterone is not associated nor predicts HF. Further studies are warranted to evaluate aldosterone as therapeutic target in the general community., (© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

C-type natriuretic peptide (CNP) is an endothelium-derived peptide that is released as a protective mechanism in response cardiovascular injury or disease. However, no studies have investigated circulating CNP, identifying clinical factors that may influence CNP and its relationship to cardiovascular disease in the general population. We studied 1841 randomly selected subjects from Olmsted County, MN (mean age, 63±11 years; 48% men). Plasma CNP was measured by a well-established radioimmunoassay and echocardiography, clinical characterization, and detailed medical record review were performed. We report that CNP circulates at various concentrations (median, 13 pg/mL), was unaffected by sex, was weakly associated by age, and that highest quartile of CNP identified a high-risk phenotype. Subjects with CNP in the highest quartile were associated with increased risk of myocardial infarction (multivariable-adjusted hazard ratio, 1.51; 95% confidence interval, 1.09-2.09; P=0.01) but not heart failure, cerebrovascular accidents, or death during a follow-up of 12 years. Addition of the highest quartile of CNP to clinical variables led to a modest increase in the integrated discrimination improvement for risk of myocardial infarction. In a large community-based cohort, elevated circulating CNP identified a high-risk phenotype that included cardiovascular comorbidities and left ventricular dysfunction, and provided evidence that highest concentrations of CNP potentially has prognostic value in predicting future risk of myocardial infarction. Together, these data from the general population highlight the potential value of CNP and support the need for additional studies to evaluate whether mechanisms regulating CNP could improve outcomes., (© 2015 American Heart Association, Inc.)

Background: In epidemiologic studies, obesity has been associated with reduced natriuretic peptide (NP) concentrations. Reduced NP production could impair the ability of obese individuals to respond to salt loads, increasing the risk of hypertension and other disorders. We hypothesized that weight loss enhances NP production before and after salt loading., Methods and Results: We enrolled 15 obese individuals (mean BMI 45±5.4 kg/m(2)) undergoing gastric bypass surgery. Before and 6 months after surgery, subjects were admitted to the clinical research center and administered a large-volume intravenous saline challenge. Echocardiography and serial blood sampling were performed. From the pre-operative visit to 6 months after surgery, subjects had a mean BMI decrease of 27%. At the 6-month visit, N-terminal pro-atrial NP (Nt-proANP) levels were 40% higher before, during, and after the saline infusion, compared with levels measured at the same time points during the pre-operative visit (P<0.001). The rise in Nt-pro-ANP induced by the saline infusion (≈50%) was similar both before and after surgery (saline, P<0.001; interaction, P=0.2). Similar results were obtained for BNP and Nt-proBNP; resting concentrations increased by 50% and 31%, respectively, after gastric bypass surgery. The increase in NP concentrations after surgery was accompanied by significant decreases in mean arterial pressure (P=0.004) and heart rate (P<0.001), and an increase in mitral annular diastolic velocity (P=0.02)., Conclusion: In obese individuals, weight loss is associated with a substantial increase in the "setpoint" of circulating NP concentrations. Higher NP concentrations could contribute to an enhanced ability to handle salt loads after weight loss., (© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background: We have reported that pro-B-type natriuretic peptide (BNP)-1-108 circulates and is processed to mature BNP1-32 in human blood. Building on these findings, we sought to determine whether proBNP1-108 processed forms in normal circulation are biologically active and stimulate cGMP, and whether proBNP1-108 processing and activity are altered in human heart failure (HF) compared with normal. Because BNP1-32 is deficient whereas proBNP1-108 is abundant in HF, we hypothesize that proBNP1-108 processing and degradation are impaired in HF patients ex vivo., Methods and Results: We measured circulating molecular forms, including BNP1-32, proBNP1-108, and N-terminal-proBNP, and all were significantly higher in patients with HF when compared with that in normals. Fresh serum samples from normals or patients with HF were incubated with or without exogenous nonglycosylated proBNP1-108 tagged with 6 C-terminal Histidines to facilitate peptide isolation. His-tag proBNP1-108 was efficiently processed into BNP1-32/3-32 at 5 minutes in normal serum, persisted for 15 minutes, then disappeared. Delayed processing of proBNP1-108 was observed in HF samples, and the degradation pattern differed depending on left ventricular function. The 5-minute processed forms from both normal and HF serums were active and generated cGMP via guanylyl cyclase-A receptors; however, the 180-minute samples were not active. The proBNP1-108 processing enzyme corin and BNP-degrading enzyme dipeptidyl peptidase-4 were reduced in HF versus normal, perhaps contributing to differential BNP metabolism in HF., Conclusions: Exogenous proBNP1-108 is processed into active BNP1-32 and ultimately degraded in normal circulation. The processing and degradation of BNP molecular forms were altered but complete in HF, which may contribute to the pathophysiology of HF., (© 2014 American Heart Association, Inc.)

We sought to investigate the role of aldosterone as a mediator of disease and its relationship with the counter-regulatory natriuretic peptide (NP) system. We measured plasma aldosterone (n=1674; aged≥45 years old) in a random sample of the general population from Olmsted County, MN. In a multivariate logistic regression model, aldosterone analyzed as a continuous variable was associated with hypertension (odds ratio [OR]=1.75; 95% confidence interval [CI]=1.57-1.96; P<0.0001), obesity (OR=1.34; 95% CI=1.21-1.48; P<0.0001), chronic kidney disease (OR=1.39; 95% CI=1.22-1.60; P<0.0001), central obesity (OR=1.47; 95% CI=1.32-1.63; P<0.0001), metabolic syndrome (OR=1.41; 95% CI=1.26-1.58; P<0.0001), high triglycerides (OR=1.23; 95% CI=1.11-1.36; P<0.0001), concentric left ventricular hypertrophy (OR=1.22; 95% CI=1.09-1.38; P=0.0007), and atrial fibrillation (OR=1.24; 95% CI=1.01-1.53; P=0.04), after adjusting for age and sex. The associations with hypertension, central obesity, metabolic syndrome, triglycerides, and concentric left ventricular hypertrophy remained significant after further adjustment for body mass index, NPs, and renal function. Furthermore, aldosterone in the highest tertile correlated with lower NP levels and increased mortality. Importantly, most of these associations remained significant even after excluding subjects with aldosterone levels above the normal range. In conclusion, we report that aldosterone is associated with hypertension, chronic kidney disease, obesity, metabolic syndrome, concentric left ventricular hypertrophy, and lower NPs in the general community. Our data suggest that aldosterone, even within the normal range, may be a biomarker of cardiorenal and metabolic disease. Further studies are warranted to evaluate a therapeutic and preventive strategy to delay the onset and progression of disease, using mineralocorticoid antagonists or chronic NP administration in high-risk subjects identified by plasma aldosterone., (© 2014 American Heart Association, Inc.)

We analyzed the phenotype associated with the atrial natriuretic peptide (ANP) genetic variant rs5065 in a random community-based sample. We also assessed and compared the biological action of 2 concentrations (10(-10) mol/L, 10(-8) mol/L) of ANP and ANP-RR, the protein variant encoded by the minor allele of rs5065, on activation of the guanylyl cyclase (GC)-A and GC-B receptors, production of the second messenger 3',5'-cGMP in endothelial cells, and endothelial permeability. rs5065 genotypes were determined in a cross-sectional adult cohort from Olmsted County, MN (n=1623). Genotype frequencies for rs5065 were 75%, 24%, and 1% for TT, TC, and CC, respectively. Multivariate analysis showed that the C allele was associated with increased risk of cerebrovascular accident (hazard ratio, 1.43; 95% confidence interval, 1.09-1.86; P=0.009) and higher prevalence of myocardial infarction (odds ratio, 1.82; 95% confidence interval, 1.07-3.09; P=0.026). ANP-RR 10(-8) mol/L activated the GC-A receptor (83.07±8.31 versus no treatment 0.18±0.04 per 6 wells; P=0.006), whereas ANP-RR 10(-10) mol/L did not. Neither 10(-8) mol/L nor 10(-10) mol/L ANP-RR activated GC-B receptor (P=0.10, P=0.35). ANP 10(-8) mol/L and ANP-RR 10(-8) mol/L stimulated 3',5'-cGMP production in endothelial cells similarly (P=0.58). Both concentrations of ANP-RR significantly enhanced human aortic endothelial cell permeability (69 versus 29 relative fluorescence units [RFUs], P=0.012; 58 versus 39 RFUs, P=0.015) compared with ANP. The minor allele of rs5065 was associated with increased cardiovascular risk. ANP-RR activated the GC-A receptor, increased 3',5'-cGMP in endothelial cells, and when compared with ANP, augmented endothelial cell permeability.

Background: High-sensitivity cardiac troponin (hs-cTn) assays are now available that can detect measurable troponin in significantly more individuals in the general population than conventional assays. The clinical use of these hs-cTn assays depends on the development of proper reference values. Therefore, our objective was to define hs-cTnI reference values and determinants in the general community, in a healthy reference cohort, and in subsets with diseases., Materials and Methods: A well-characterized community-based cohort of 2042 study participants underwent clinical assessment and echocardiographic evaluation. Baseline hs-cTnI measurements were obtained in 1843 individuals. A healthy reference cohort (n = 565) without cardiac, renal, or echocardiographic abnormalities was identified., Results: Measurable hs-cTnI was identified in 1716 (93%) of the community-based study cohort and 499 (88%) of the healthy reference cohort. Parameters that significantly contributed to higher hs-cTnI concentrations in the healthy reference cohort included age, male sex, systolic blood pressure, and left ventricular mass. Glomerular filtration rate and body mass index were not independently associated with hs-cTnI in the healthy reference cohort. Individuals with diastolic and systolic dysfunction, hypertension, and coronary artery disease (but not impaired renal function) had significantly higher hs-cTnI values than the healthy reference cohort., Conclusions: We assessed an hs-cTnI assay with the aid of echocardiographic imaging in a large, well-characterized community-based cohort. hs-cTnI is remarkably sensitive in the general population, and there are important sex and age differences among healthy reference individuals. These results have important implications for defining hs-cTnI reference values and identifying disease.

Objectives: Using a novel, specific assay for proBNP(1-108), this study tested the hypotheses that proBNP(1-108) is secreted by both nonfailing and failing human hearts and that proBNP(1-108) secretion is increased in failing hearts., Background: The prohormone of B-type natriuretic peptide (proBNP(1-108)) is a 108-amino acid peptide produced primarily by the heart and cleaved into biologically active BNP(1-32) and the biologically inactive NT-proBNP(1-76). It is unknown to what extent increased cardiac proBNP1-108 secretion compared to reduced peripheral processing is responsible for elevated proBNP(1-108) levels in patients with heart failure (HF) compared to subjects without HF., Methods: The transcardiac gradient of proBNP(1-108) was determined by collecting arterial blood and blood from the coronary sinus (CS). Samples from subjects without overt heart disease (n = 9) were collected during cardiac catheterization after coronary artery disease had been excluded. Samples from HF patients (n = 21) were collected during implantation of a biventricular pacemaker. ProBNP(1-108) was measured with a new assay. Values are medians (25th/75th percentiles)., Results: The gradient of proBNP(1-108) across the nonfailing hearts was 8 (2/20) ng/l (aorta: 15 [1/25] ng/l; CS: 24 [8/41] ng/l; p = 0.018). The transcardiac gradient of proBNP(1-108) in the failing hearts was 326 (96/482) ng/l (arterial: 381 [201/586] ng/l; CS: 709 [408/1,087] ng/l; p<0.001). The transcardiac gradient was greater in failing than nonfailing hearts (p = 0.001)., Conclusions: ProBNP(1-108) is secreted by nonfailing and failing human hearts, but more so in the latter. It remains to be established where peripheral processing of proBNP(1-108) occurs and how this is affected by disease., (Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objectives: This study was conducted to determine whether urinary excretion of C-type natriuretic peptide (CNP) is elevated in acute decompensated heart failure (ADHF) and whether elevated levels predict adverse outcomes., Background: Urinary CNP has been detected in patients with heart failure, but its clinical significance and prognostic utility, compared to established kidney injury biomarkers, in ADHF is unknown., Methods: We measured 24-h urinary excretion and concurrent plasma concentrations of CNP22, CNP53, and NT-CNP53 in 58 ADHF patients and 20 healthy control subjects. Urinary kidney injury molecule (KIM)-1 and neutrophil gelatinase–associated lipocalin (NGAL) and plasma N-terminal pro-B type natriuretic peptide (NT-proBNP) were also measured. Mortality and all-cause rehospitalization/death were assessed over a follow-up of 1.5 ± 0.9 years., Results: ADHF patients had higher urinary excretion of all 3 CNP molecular forms than did controls. Plasma CNP22 and CNP53 were elevated in ADHF but showed limited correlation with urinary excretion, suggesting that mainly renal-derived CNP appears in urine. Plasma NT-proBNP and urinary KIM-1 were also elevated in ADHF (p < 0.0001); urinary NGAL was similar to that in controls. At 6 months, event-free survival values in ADHF patients were 86% for mortality and 59% for all-cause rehospitalization/death. On Cox regression analysis, urinary NT-CNP53 was the only predictor of mortality (hazard ratio: 1.7; 95% confidence interval: 1.1 to 2.4; p = 0.01) and all-cause rehospitalization/death (hazard ratio: 1.8; 95% confidence interval: 1.3 to 2.4; p = 0.0004), even after adjustment. Integrated discrimination analysis suggested that urinary NT-CNP53 combined with plasma NT-proBNP improved the prediction of adverse outcomes., Conclusions: The findings from this study support the clinical utility of urinary CNP molecular forms. In ADHF, urinary NT-CNP53 correlated with prognosis, better predicted outcomes than did urinary NGAL and KIM-1, and improved the prognostic value of plasma NT-proBNP.

Renal aging is characterized by structural changes in the kidney including fibrosis, which contributes to the increased risk of kidney and cardiac failure in the elderly. Studies involving healthy kidney donors demonstrated subclinical age-related nephropathy on renal biopsy that was not detected by standard diagnostic tests. Thus there is a high-priority need for novel noninvasive biomarkers to detect the presence of preclinical age-associated renal structural and functional changes. C-type natriuretic peptide (CNP) possesses renoprotective properties and is present in the kidney; however, its modulation during aging remains undefined. We assessed circulating and urinary CNP in a Fischer rat model of experimental aging and also determined renal structural and functional adaptations to the aging process. Histological and electron microscopic analysis demonstrated significant renal fibrosis, glomerular basement membrane thickening, and mesangial matrix expansion with aging. While plasma CNP levels progressively declined with aging, urinary CNP excretion increased, along with the ratio of urinary to plasma CNP, which preceded significant elevations in proteinuria and blood pressure. Also, CNP immunoreactivity was increased in the distal and proximal tubules in both the aging rat and aging human kidneys. Our findings provide evidence that urinary CNP and its ratio to plasma CNP may represent a novel biomarker for early age-mediated renal structural alterations, particularly fibrosis. Thus urinary CNP could potentially aid in identifying subjects with preclinical structural changes before the onset of symptoms and disease, allowing for the initiation of strategies designed to prevent the progression of chronic kidney disease particularly in the aging population.

Objectives: We sought to define the cardiometabolic phenotype associated with rs5068, a genetic variant of the atrial natriuretic peptide (ANP) gene., Background: The ANP and B-type natriuretic peptide play an important role in cardiorenal homeostasis but also exert metabolic actions., Methods: We genotyped 1,608 randomly selected residents from Olmsted County, Minnesota. Subjects were well-characterized., Results: Genotype frequencies were: AA 89.9%, AG 9.7%, and GG 0.4%; all subsequent analyses were AA versus AG+GG. The G allele was associated with increased plasma levels of N-terminal pro-atrial natriuretic peptide (p = 0.002), after adjustment for age and sex. The minor allele was also associated with lower body mass index (BMI) (p = 0.006), prevalence of obesity (p = 0.002), waist circumference (p = 0.021), lower levels of C-reactive protein (p = 0.027), and higher values of high-density lipoprotein cholesterol (p = 0.019). The AG+GG group had a lower systolic blood pressure (p = 0.011) and lower prevalence of myocardial infarction (p = 0.042). The minor allele was associated with a lower prevalence of metabolic syndrome (p = 0.025). The associations between the G allele and high-density lipoprotein cholesterol, C-reactive protein values, myocardial infarction, and metabolic syndrome were not significant, after adjusting for BMI; the associations with systolic blood pressure, BMI, obesity, and waist circumference remained significant even after adjusting for N-terminal pro-atrial natriuretic peptide., Conclusions: In a random sample of the general U.S. population, the minor allele of rs5068 is associated with a favorable cardiometabolic profile. These findings suggest that rs5068 or genetic loci in linkage disequilibrium might affect susceptibility for cardiometabolic diseases and support the possible protective role of natriuretic peptides by their favorable effects on metabolic function. Replication studies are needed to confirm our findings., (Copyright © 2011 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Objective: To assess in a US general adult population the effect of the functional single-nucleotide polymorphism rs198389 in the promoter region of the gene of brain-type natriuretic peptide (BNP) on 3 commonly used BNP assays, clinical phenotype, disease prevalence, overall survival, and diagnostic test characteristics of BNP as a biomarker., Patients and Methods: We genotyped for rs198389 in a random sample of the general population (aged ≥ 45 years; n = 1970; enrolled between June 1, 1997, and September 30, 2000) from Olmsted County, Minnesota. Patients were characterized biochemically, clinically, echocardiographically, and regarding BNP molecular forms (2 assays for BNP and 1 assay for amino-terminal proBNP). Median follow-up was 9 years., Results: Genotype frequencies were in Hardy-Weinberg equilibrium (P = .98): TT genotype, n = 645 (32.7%); TC genotype, n = 983 (49.9%); and CC genotype, n = 342 (17.4%). The C allele independently predicted higher BNP forms (P<.001 for all assays). Genotypes did not differ with regard to clinical and echocardiographic phenotype or overall survival. When previously reported genotype-unadjusted cut points for the detection of left ventricular ejection fraction less than or equal to 40% (n = 37 [1.9%]) and less than or equal to 50% (n = 116 [6.0%]) were used, sensitivity generally increased with the number of C alleles, whereas specificity decreased, both on average by more than 10% for the TT vs CC genotype., Conclusion: The C allele of rs198389 is common in the general US population and is associated with higher concentrations of BNP molecular forms but not with cardiovascular phenotype or survival. The C allele confounds the test characteristics of commonly used assays.

Background: B-type natriuretic peptide (BNP), which is activated in heart failure (HF), is processed to an active form by corin. The corin gene is expressed in the human heart and kidney, but corin protein expression in the heart, kidney, and circulation, along with whether proBNP is processed by circulating corin, remains unknown., Methods: We examined corin protein expression by immunostaining and Western blot in human heart and kidney, and we assessed the circulating corin concentration by ELISA. We examined histidine-tagged (His-tag) proBNP(1-108) processing in serum and plasma by immunoprecipitation and Western blot and sequenced the processed form., Results: Normal human heart and kidney displayed the presence of corin, especially in cells around the vasculature. Both corin and proBNP(1-108) were present in the plasma of healthy human subjects, with circulating corin significantly higher in men than women (P < 0.0001) and a positive correlation of corin to age (P = 0.0497, r = 0.27). In fresh normal plasma and serum, His-tag proBNP(1-108) was processed to a lower molecular weight form confirmed to be BNP. Processed BNP was higher in men than women (P = 0.041) and was positively correlated to plasma corin concentrations (P = 0.041, r = 0.65)., Conclusions: Our results support the concept that proBNP(1-108) may be processed outside of the heart in the circulation where the proprotein convertase is present. Moreover, sex may impact this process, since corin concentrations are higher in men. These findings may have important physiologic and pathophysiologic implications for the proBNP/corin system in the human.

Atrial fibrillation is a common arrhythmia that is hereditary in a small subgroup of patients. In a family with 11 clinically affected members, we mapped an atrial fibrillation locus to chromosome 1p36-p35 and identified a heterozygous frameshift mutation in the gene encoding atrial natriuretic peptide. Circulating chimeric atrial natriuretic peptide (ANP) was detected in high concentration in subjects with the mutation, and shortened atrial action potentials were seen in an isolated heart model, creating a possible substrate for atrial fibrillation. This report implicates perturbation of the atrial natriuretic peptide-cyclic guanosine monophosphate (cGMP) pathway in cardiac electrical instability., (2008 Massachusetts Medical Society)

Recent studies support the speculation that different molecular forms of the cardiac hormone BNP with differential biological activity may circulate in heart failure and be detected by conventional assays. In the current study we determined the ability of 3 widely used conventional assays to detect these different forms thought to circulate in heart failure. We also evaluated the ability of pro-BNP (1-108), N-terminal peptide (NT)-pro-BNP (1-76), and BNP 3-32, the latter a cleavage product of BNP 1-32 by dipeptidyl peptidase IV, on an equimolar basis to activate cGMP in cultured cardiac fibroblasts and cardiomyocytes compared with the biologically active mature BNP 1-32. Specifically, we observed that the Roche NT-pro-BNP assay detected both NT-pro-BNP 1-76 and pro-BNP 1-108 and that Biosite Triage and Shionogi detected both mature BNP 1-32 and the shortened BNP 3-32. Moreover, in cultured cardiac fibroblasts and cardiomyocytes, BNP 1-32 (10(-6) mol/L) activated cGMP. BNP 3-32 demonstrated a similar cGMP activating property in both cardiac cell types. In contrast, the cGMP response to pro-BNP 1-108 and NT-pro-BNP 1-76 was not significantly greater than no treatment alone. We conclude that widely used commercial assays for NT-pro-BNP 1-76 and BNP 1-32 cannot differentiate among pro-, processed, or degraded forms and, thus, may not thoroughly identify circulating BNP forms in heart failure patients. These findings also demonstrate differential cGMP activating properties of BNP forms and, importantly, that pro-BNP 1-108 and NT-pro-BNP 1-76 have reduced cGMP activity in vitro that may have biological relevance to human heart failure.

We evaluated relationships among two circulating molecular forms of brain natriuretic peptide (BNP32 and NT-proBNP), severity of hypertension (HTN), and cardiac hypertrophy in subjects with mild, moderate, and severe HTN. We prospectively studied 78 patients (43 males; mean age 51.4 +/- 11 yr) with essential HTN and 28 age- and sex-matched controls. BNP32 and NT-proBNP were measured by radioimmunoassay. In grade 1 HTN, BNP32 was not elevated and NT-proBNP was reduced (P = 0.030) compared with controls. However, log-transformed values of BNP32 and NT-proBNP were both increased with severity of HTN from grade 1 to 3 (P <0.0001 and P = 0.003, respectively). By multivariate analysis, log BNP32 was independently predicted by age (beta = 0.210, P = 0.026) and HTN grade (beta = 0.274, P = 0.004), whereas log NT-proBNP was independently predicted by sex (beta = 0.235, P = 0.012) and HTN grade (beta = 0.218, P = 0.0023). Two forms of BNP were measured in normal subjects and patients with essential HTN. In grade 1 HTN, BNP32 was unchanged and NT-proBNP was significantly reduced compared with controls. As severity increased in humans with grade 1 to 3 HTN, both BNP32 and NT-proBNP levels were increased while not being affected by the presence of left ventricular hypertrophy. The lack of activation of BNP32 together with the reduction of NT-proBNP in grade 1 HTN may represent an impaired response of the BNP system in the early phase of HTN. The later activation of both forms of BNP may be a late compensatory effect, because it correlates with severity of HTN rather than cardiac hypertrophy/remodeling.

Objectives: This study sought to characterize factors influencing amino-terminal pro-B-type natriuretic peptide (NT-proBNP) and to evaluate the ability of NT-proBNP to detect left ventricular (LV) dysfunction in a large community sample., Background: Secretion of BNP increases in cardiac disease, making BNP an attractive biomarker. Amino-terminal proBNP, a fragment of the BNP prohormone, is a new biomarker. We evaluated factors influencing NT-proBNP in normal patients and compared the ability of NT-proBNP and BNP to detect LV dysfunction in a large community sample., Methods: Amino-terminal pro-BNP was determined in plasma samples of a previously reported and clinically and echocardiographically characterized random sample (n = 1,869, age > or =45 years) of Olmsted County, Minnesota., Results: In normal patients (n = 746), female gender and older age were the strongest independent predictors of higher NT-proBNP. Test characteristics for detecting an LV ejection fraction < or =40% or < or =50% were determined in the total sample with receiver operating characteristic curves. Amino-terminal pro-BNP had significantly higher areas under the curve for detecting an LV ejection fraction < or =40% or < or =50% than BNP in the total population and in several male and age subgroups, whereas areas were equivalent in female subgroups. Age- and gender-adjusted cutpoints improved test characteristics of NT-proBNP. Both assays detected patients with systolic and/or moderate to severe diastolic dysfunction to a similar degree, which was less robust than the detection of LV systolic dysfunction alone., Conclusions: Amino-terminal pro-BNP in normal patients is affected primarily by gender and age, which should be considered when interpreting values. Importantly, in the entire population sample NT-proBNP performed at least equivalently to BNP in detecting LV dysfunction and was superior in some subgroups in detecting LV systolic dysfunction.

C-terminal brain (B-type) natriuretic peptide (BNP)-32 is a widely used clinical biomarker for the diagnosis, prognosis, and treatment of heart failure (HF). The 32-aa peptide is synthesized primarily in the atrial and ventricular myocardium and constitutes the mature biologically active form of immature BNP (pro-BNP). There has been mounting evidence that suggests BNP circulates in different structural forms that impact HF diagnosis and in vivo activity. Herein, we have developed and used an immunoaffinity purification assay to isolate endogenous BNP-32 from New York Heart Association class IV patient plasma for subsequent analysis by nano-liquid chromatography (LC) electrospray ionization Fourier transform ion cyclotron resonance (FT-ICR) MS. We have introduced stable isotope-labeled BNP-32 to the assayed plasma to enable quantification of endogenous levels of BNP-32. Unlike the chemically nonspecific point-of-care tests (POCTs) and RIAs used worldwide to quantify BNP-32 from plasma, FT-ICR-MS (unprecedented mass measurement accuracy) coupled with LC (retention time) affords extraordinary molecular specificity, and when combined with the use of internal standards is able to confidently identify and quantify BNP-32. The significance of this work is despite exceedingly high circulating levels of BNP-32 in the New York Heart Association class IV patients as determined by POCTs (>290 fmol/ml) nano-LC-electrospray ionization-FT-ICR-MS data did not reveal any endogenous BNP-32. These results provide molecularly specific evidence for the absence of circulating BNP-32 in advanced-stage HF patients and suggest the existence of altered forms of BNP that are contributing to the POCT values.

Objective: Hypovolemia after aneurysmal subarachnoid hemorrhage (SAH) may be mediated by natriuretic peptides and can further impair cerebral perfusion in dysautoregulated and vasospastic arterial territories. Dendroaspis natriuretic peptide (DNP), derived from the venom of Dendroaspis augusticeps, the Green Mamba snake, has recently been discovered in human plasma and atrial myocardium. There is no information regarding the presence or putative role of this peptide in patients with aneurysmal SAH., Methods: A sensitive and specific DNP radioimmunoassay was performed on venous blood samples obtained on post-SAH Days 1, 3, and 7 from 10 consecutive SAH patients (cases) and randomly from 9 healthy volunteers (controls). Clinical and laboratory data, including daily serum sodium concentration and fluid balance, were collected prospectively up to 7 days after the ictus., Results: Increase in plasma DNP levels occurred in five (63%) of eight patients who had DNP levels measured on Days 1 and 3 (mean increase, 29%). An increase in DNP level was significantly associated with development of a negative fluid balance (P = 0.003) and hyponatremia (P = 0.008). Three (75%) of the four patients who developed cerebral vasospasm during this study experienced an increase in DNP levels from Days 1 to 3., Conclusion: The present study is the first to find a significant association between elevated levels of DNP, a new member of the natriuretic peptide family, and the development of diuresis and natriuresis in patients with aneurysmal SAH. Our findings warrant further investigation by means of a large-scale, prospective, case-control study.

Background: The renal actions of brain natriuretic peptide (BNP) in congestive heart failure (CHF) are associated with increased diuresis and natriuresis, preserved glomerular filtration rate (GFR), and lack of activation of the renin-angiotensin-aldosterone system (RAAS). In contrast, diuretic-induced natriuresis may be associated with reduced GFR and RAAS activation. The objective of this study was to test the hypothesis that exogenous BNP enhances the renal diuretic and natriuretic actions of furosemide (Fs) and retards the activation of aldosterone in a model of CHF., Methods and Results: CHF was produced in 2 groups of dogs by ventricular pacing. One group received continuous (90-minute) intravenous Fs (1 mg x kg(-1) x h(-1)). A second group (Fs+BNP) received 45-minute intravenous coinfusion of Fs (1 mg x kg(-1) x h(-1)) and low-dose (2 pmol x kg(-1) x min(-1)) BNP followed by 45-minute coinfusion of Fs (1 mg x kg(-1) x h(-1)) and high-dose (10 pmol x kg(-1) x min(-1)) BNP. Fs increased urinary flow, but the effect of Fs+BNP was greater. Similarly, urinary sodium excretion was higher in the Fs+BNP group. Although GFR tended to decrease in the Fs group, it increased in the Fs+BNP group (35+/-3 to 56+/-4*) (* indicates P<0.05 versus baseline) (P<0.0001 between groups). Plasma aldosterone increased with Fs (41+/-10 to 100+/-11* ng/dL) but was attenuated in the Fs+BNP group (44+/-11 to 54+/-9 ng/dL low-dose and to 47+/-7 ng/dL high-dose) (P=0.0007 between groups)., Conclusions: Fs+BNP has more profound diuretic and natriuretic responses than Fs alone and also increases GFR without activation of aldosterone. Coadministration of BNP and loop diuretic is effective in maximizing natriuresis and diuresis while preserving renal function and inhibiting activation of aldosterone.

Background: Plasma C-terminal atrial natriuretic peptide (C-ANP), N-terminal ANP (N-ANP), and brain natriuretic peptide (BNP) have diagnostic utility in detecting left ventricular dysfunction. Their relative value in monitoring symptom status during the chronic treatment of congestive heart failure (CHF) remains undefined., Methods and Results: Ninety-eight subjects with CHF were evaluated. Baseline natriuretic peptides were measured by radioimmunoassay, left ventricular ejection fraction (LVEF) was estimated with echocardiography, and New York Heart Association (NYHA) class was determined independently by attending heart failure specialists. Forty-one subjects were restudied during a 6- to 12-month follow-up period after optimizing therapy. At baseline, all natriuretic peptides and LVEF correlated positively with NYHA class (P <.005). Plasma BNP, however, correlated best with NYHA class. At follow-up, only changes of BNP correlated to changes of NYHA class (P =.04). BNP decreased (-45% +/- 12%, N = 14, P =.002) in subjects whose NYHA class improved whereas BNP remained unchanged (-1% +/- 10%, N = 25, P =.95) in those whose NYHA class was stable., Conclusions: This investigation demonstrates the superiority of plasma BNP as compared to ANP and LVEF in objectively assessing NYHA class during the chronic treatment of CHF. Given that clinical assessment of CHF is subjective, plasma BNP is a useful objective biomarker in monitoring human CHF in the outpatient setting.

Objectives: To determine levels of natriuretic peptides (NPs) in patients with end-stage renal disease (ESRD) and to examine the relationship of these cardiovascular peptides to left ventricular hypertrophy (LVH) and to cardiac mortality., Patients and Methods: One hundred twelve dialysis patients without clinical evidence of congestive heart failure underwent plasma measurement of NP concentrations and echocardiographic investigation for left ventricular mass index (LVMI)., Results: Plasma atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) concentrations correlated positively with LVMI and inversely with left ventricular ejection fraction, whereas C-type NP and Dendroaspis NP levels did not correlate with LVMI. In dialysis patients with LVH (LVMI >125 g/m2), plasma ANP and BNP concentrations were increased compared with those in dialysis patients without LVH (both P<001). In a subset of 15 dialysis patients without LVH or other concomitant diseases, plasma BNP concentrations were not significantly increased compared with those in 35 controls (mean +/- SD, 20.1+/-13.4 vs 13.5+/-9.6 pg/mL; P=.06), demonstrating that the BNP concentration was not increased by renal dysfunction alone. Furthermore, the BNP level was significantly higher in the 16 patients who died from cardiovascular causes compared with survivors (mean +/- SD, 129+/-13 vs 57+/-7 pg/mL; P<.003) and was significantly associated with greater risk of cardiovascular death in Cox regression analysis (P<.001), as was the ANP level (P=.002)., Conclusions: Elevation of the plasma BNP concentration is more specifically related to LVH compared with the other NP levels in patients with ESRD independent of congestive heart failure. Thus, BNP serves as an important plasma biomarker for ventricular hypertrophy in dialysis patients with ESRD.

The objective of this study was to determine the effect of N(G)-monomethyl-L-arginine (L-NMMA) infusion on plasma renin activity (PRA) in the presence or absence of the renal nerves in normotensive Wistar-Kyoto (WKY) rats and Okamoto spontaneously hypertensive rats (SHR). All rats were unilaterally nephrectomized two weeks before the acute experiment. On the day of the experiment, acute renal denervation (Dnx) of the remaining kidney was performed in one group of WKY rats (Dnx-WKY; n= 10) and one group of SHRs (Dnx-SHR: n=7). The renal nerves were left intact in a group of WKY rats (Inn-WKY; n=8) and SHRs (Inn-SHR; n=9). After a control clearance period, L-NMMA was administered i.v. (15 mg/kg bolus followed by 500 microg/kg/min infusion) and another clearance period of 20 min was taken. In all experimental groups L-NMMA infusion resulted in a significant natriuresis. L-NMMA infusion increased fractional excretion of sodium (FE(Na)) to a greater extent in the Inn-SHR than in the Inn-WKY (delta FE(Na) = 5.23+/-0.87% vs delta FE(Na) = 2.87+/-0.73% respectively; P=0.05), PRA did not change in the SHR with the infusion of L-NMMA. However, in the Inn-WKY group, the natriuresis of L-NMMA infusion was associated with a tendency for lower PRA levels as compared to a group of time control Inn-WKY rats. In Dnx-WKY, the natriuresis of L-NMMA infusion (delta FE(Na) = 4.60+/-0.52%) was associated with a significantly lower level of PRA (4.26+/-1.18 ng AI/ml/hr) as compared to a group of time control Dnx-WKY rats (9.83+/-1.32 ng AI/ml/hr; P<0.05). In the Dnx-SHR, the natriuretic response to L-NMMA infusion was significantly attenuated by renal denervation (delta FE(Na) = 2.36+/-0.34%) and PRA was unchanged. In conclusion, the natriuretic effect of systemic inhibition of nitric oxide (NO) synthesis was associated with decreased PRA in the Dnx-WKY suggesting that a potential interaction exists between NO and the renal nerves in the modulation of PRA in the normotensive WKY rat. Whereas, the natriuretic effect of L-NMMA infusion in the SHR in the presence and absence of the renal nerves, were independent of changes in PRA.

Background: The recently discovered vasodilating and positive inotropic peptide, adrenomedullin (ADM), has strong natriuretic actions. ADM-induced natriuresis is caused by an increase in glomerular filtration rate and a decrease in distal tubular sodium reabsorption. Although ADM is activated in human and experimental heart failure, the role of ADM in the kidney in heart failure remains undefined., Methods and Results: The present study was performed to determine the renal hemodynamic and urinary excretory actions of exogenously administered ADM in a canine model of acute heart failure produced by rapid ventricular pacing. Experimental acute heart failure was characterized by a decrease in cardiac output and an increase in pulmonary capillary wedge pressure with an increase in plasma ADM concentration. Intrarenal infusion of ADM (1 and 25 ng/kg/min) induced an increase in urinary sodium excretion in the normal control dogs (change in urinary sodium excretion [Delta UNaV], +94.5 microEq/min during 1 ng/kg/min ADM infusion and +128.1 microEq/min during 25 ng/kg/min ADM infusion). In the acute heart failure dogs, intrarenal ADM infusion resulted in an attenuated increase in urinary sodium excretion (Delta UNaV, +44.8 microEq/min during 1 ng/kg/min ADM infusion and +51.8 microEq/min during 25 ng/kg/min ADM infusion). Both glomerular and tubular actions of ADM were attenuated in the acute heart failure group compared with responses in the normal control group., Conclusion: The present study shows that the renal natriuretic responses to ADM are markedly attenuated in experimental acute heart failure. This study provides insight into humoral mechanisms that may promote sodium retention in heart failure via a renal hyporesponsiveness to natriuretic actions of ADM.

Object: Natriuresis is a common systemic manifestation of aneurysmal subarachnoid hemorrhage (SAH). Natriuresis and its accompanying hypovolemia may be a major contributing factor in the pathophysiology of symptomatic cerebral vasospasm., Methods: The authors studied 14 consecutive patients with aneurysmal SAH and compared levels of adrenomedullin (ADM), a novel endogenous natriuretic peptide that possesses additional profound vasodilatory properties, with the natriuretic peptide system by using radioimmunoassay. The mean ADM values on admission were 24.8 pg/ml, a twofold increase over control values, but no correlation was found with atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-natriuretic peptide (CNP) from the natriuretic peptide system. At Day 5 post-SAH, ADM levels were significantly elevated in patients with vasospasm documented angiographically or on transcranial Doppler studies as compared with those who suffered no vasospasm (mean 61.9 pg/ml compared with 15.3 pg/ml, p < 0.01)., Conclusions: The authors conclude that an elevation of ADM in plasma may indicate a physiological regulatory attempt to induce cerebral vasodilation. The regulation of ADM is uncoupled from ANP, BNP, and CNP.

Background: Endothelin-converting enzyme-1 (ECE-1) processes big endothelin-1 (ET-1) to ET-1, a peptide implicated in atheroma formation. ECE-1 exists in 2 isoforms (ECE-1alpha and ECE-1beta), the result of alternative splicing of a common gene. Neutral endopeptidase (NEP) is a genetically distinct metallopeptidase that degrades the natriuretic peptides. These peptides mediate antiproliferative and vasodilating actions. We sought to demonstrate the distribution of the 2 ECE-1 isoforms in experimental atherosclerosis, to determine the effects of chronic NEP-I on plasma cGMP concentrations, vascular wall ECE-1 activity, and ET-1 concentration, and to correlate these actions with atheroma formation. Our hypothesis was that chronic NEP-I, in association with augmented cGMP, would inhibit ECE-1 conversion of big ET-1 to active ET-1, thus reducing tissue ET-1 concentrations and associated atheroma formation., Methods and Results: Cholesterol-fed New Zealand White rabbits (n=8, 1% cholesterol diet) and NEP-I-treated cholesterol-fed New Zealand White rabbits (n=8; candoxatril, 30 mg/kg per day, Pfizer) were euthanized after 8 weeks of feeding. ECE-1alpha and ECE-1beta immunoreactivity was present in the aortas of both groups. Compared with control values, plasma cGMP concentrations were increased (2.8+/-0.6 versus 8.4+/-1.2 pmol/mL, P<0.05), ECE-1 activity was attenuated (68+/-3% versus 32+/-8%, P<0. 05), aortic tissue ET-1 concentrations were reduced (4.6+/-0.5 versus 3.2+/-0.3 pg/mg protein, P<0.05), and atheroma formation was attenuated (62+/-6% versus 34+/-5%, P<0.01) by NEP-I., Conclusions: These studies suggest that ECE-1 is present and functionally active in the vascular wall in atherosclerosis. Inhibition of ECE-1 by NEP-I represents a novel approach to interruption of the endothelin system in this cardiovascular disease state.

Background: Dendroaspis natriuretic peptide (DNP), recently isolated from the venom of the green Mamba snake Dendroaspis angusticeps, is a 38 amino acid peptide containing a 17 amino acid disulfide ring structure similar to that of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). DNP-like immunoreactivity (DNP-LI) was reported to be present in human plasma and atrial myocardium and to be elevated in human congestive heart failure. Although previously named DNP, it remains unknown if DNP is natriuretic or if is it present in canine plasma, urine, and atrial myocardium., Method: Studies were performed in vivo in anesthetized dogs (N = 6) using intravenous infusion of synthetic DNP at 10 and 50 ng/kg/min. Employing a sensitive and specific radioimmunoassay for DNP, the presence of DNP-like peptide was assessed in the canine plasma and urine before, during, and following the administration of exogenous synthetic DNP. Additionally, we performed immunohistochemical studies using the indirect immunoperoxidase method with polyclonal DNP antiserum in normal atrial myocardium (N = 10). Atrial concentrations of DNP-LI were also assessed., Results: We report that DNP is markedly natriuretic and diuretic, which, like ANP and BNP, is associated with the increase in urinary and plasma cGMP. DNP-like peptide is also detected in canine plasma, urine, and atrial myocardium., Conclusion: These studies establish that DNP is a potent natriuretic and diuretic peptide with tubular actions linked to cGMP and that DNP may play a physiological role in the regulation of sodium excretion.

Objective: To determine whether Dendroaspis natriuretic peptide (DNP), a novel peptide isolated from the venom of the Dendroaspis angusticeps snake that contains a 17-amino acid disulfide ring structure similar to that in atrial, brain, and C-type natriuretic peptides, is present in normal human plasma and myocardium and whether, like the other natriuretic peptides, DNP-like immunoreactivity (DNP-LI) is activated in human congestive heart failure (CHF)., Material and Methods: Circulating DNP-LI was assessed in 19 normal human subjects and 19 patients with CHF (New York Heart Association class III or IV) with a specific and sensitive radioimmunoassay for DNP with no cross-reactivity with the other natriuretic peptides. Immunohistochemical studies that used polyclonal rabbit anti-DNP antiserum were performed on human atrial myocardial tissue obtained from four patients with end-stage CHF who were undergoing cardiac transplantation and from three donor hearts at the time of transplantation., Results: We report that DNP-LI circulates in normal human plasma and is present in the normal atrial myocardium. In addition, DNP-LI is increased in the plasma of patients with CHF., Conclusion: This study demonstrates, for the first time, the presence of a DNP-like peptide in normal human plasma and in the atrial myocardium. Additionally, these studies demonstrate increased plasma DNP-LI in human CHF. These results support the possible existence of an additional new natriuretic peptide in humans, which may have a role in the neurohumoral activation that characterizes human CHF.

1. A family of structurally related but genetically distinct natriuretic peptides exist which include atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) of myocardial cell origin and C-type natriuretic peptide (CNP) of endothelial and renal epithelial cell origin. All three exert actions via cGMP, with ANP and BNP functioning via the natriuretic peptide A receptor and CNP via the natriuretic peptide B receptor. 2. Circulating and urinary natriuretic peptides were determined in response to acute intravascular volume overload (AVO). Additionally, their functional role in cardiorenal regulation during AVO was investigated by utilizing the natriuretic peptide receptor antagonist HS-142-1. Control (n=5) and study dogs (HS-142-1, n=9) underwent AVO with normal saline equal to 10% of body weight over 1 h. Both groups demonstrated similar significant increases in right atrial pressure, pulmonary capillary wedge pressure, pulmonary artery pressure and cardiac output. Circulating ANP paralleled increases in right atrial pressure and pulmonary capillary wedge pressure, with no changes in plasma BNP or CNP. At peak AVO, urinary CNP excretion was increased compared with baseline (7.0+/-4.2 versus 62+/-8.0 pg/min, P<0.05). 3. In the HS-142-1-treated group, plasma cGMP was decreased compared with the control group (9.6+/-1.1 to 5.0+/-1.2 pmol/ml, P<0.05). A significant attenuation of natriuresis (566+/-91 versus 1241+/-198 microEq/min, P<0.05) and diuresis (4.8+/-0.7 versus 10.1+/-2.0 ml/min, P<0.05) was also observed at peak AVO in the HS-142-1 treated group. 4. These findings support differential and selective responses of the three natriuretic peptides to AVO, in which plasma ANP and urinary CNP are markers for AVO. Secondly, these studies confirm the role of ANP and CNP but not BNP in the natriuretic and diuretic response to acute volume overload.

Endothelin-1 (ET-1) is a 21-amino-acid local and circulating factor whose plasma concentrations are increased in advanced atherosclerosis. ET-1 is cleaved from a prohormone (big ET-1) by endothelin-converting enzymes (ECEs) into the biologically active mature form which mediates vasoconstriction and cell proliferation. This study was designed to test by immunohistochemistry the hypothesis that ECE is present locally in the neointima of atherosclerotic vessels. Two groups of rabbits, control (n = 6) and cholesterol-fed (1% cholesterol diet for 8 weeks; n = 6) were sacrificed. Aortas were excised and divided for determination of tissue ET-1 concentration by RIA and immunohistochemical analysis of ECE. Vascular wall ET-1 was increased in the atherosclerotic aorta (6.1 +/- 0.8 vs. 9.8 +/- 0.9 pg/mg protein; p < 0.05), whereas circulating ET-1 concentrations were similar in the two groups (3.8 +/- 0.4 vs. 2.4 +/- 1.4 pg/ml). Immunostaining revealed the presence of ECE in endothelial and vascular smooth-muscle cells of the control group. Enhanced ECE immunoreactivity was present in atherosclerotic aortas, particularly in the neointimal macrophages and smooth-muscle cells. We conclude that local vascular wall, but not circulating ET-1, is increased in early atherosclerosis. In addition, ECE immunoreactivity is increased in early atherosclerosis and may therefore contribute to the generation of local ET-1 in early experimental atherosclerosis. These studies provide important insights into the regulation of ET-1 in early atherosclerosis, which may contribute to the elucidation of factors involved in the progression of atherosclerosis.

Adrenomedullin (ADM), originally discovered in human pheochromocytoma, is also of renal cell origin and has natriuretic and diuretic actions. The present study was designed to investigate the role of prostaglandins and renal nerves in the renal hemodynamic and natriuretic actions ofADM. ADM was administered intrarenally (1, 5 and 25 ng x kg(-1) x min(-1)) with and without prostaglandin inhibition (meclofenamate, 5 mg/kg intravenous bolus) in anesthetized normal mongrel dogs (n = 5, each). To elucidate the role of renal nerves, ADM was administered intrarenally to the denervated kidney in five dogs. ADM mediated a natriuretic action via increases in glomerular filtration rate and decreases in distal tubular sodium reabsorption, which was attenuated by renal denervation and completely abolished by prostaglandin inhibition. The renal vasodilatation induced by ADM was attenuated by meclofenamate, as well as by renal denervation, although not significantly. Additionally, renal nerves mediated hemodynamic effects of hypertension that were produced by intrarenal infusion of ADM. This study establishes an important mechanistic role for renal prostaglandins as a mediator of ADM-mediated natriuresis at the level of the glomerulus and terminal nephron.

The natriuretic peptide (NP) and nitric oxide (NO) systems are activated in congestive heart failure (CHF), resulting in increased synthesis of cGMP, which serves as a second messenger for both humoral systems. These two regulatory systems play functional roles in the preservation of glomerular filtration rate (GFR) and sodium excretion in both acute and chronic CHF. A progressive decline in glomerular responsiveness to atrial natriuretic peptide (ANP) characterizes the terminal stage of chronic CHF despite elevation of plasma ANP. Phosphodiesterase isozymes (PDEs) are integral factors in determining cellular content and accumulation of cGMP, and up-regulation of PDE activity could participate in the glomerular resistance to ANP in severe CHF. To date, characterization of possible alteration of glomerular PDE isozyme activities in CHF is unknown, as is the in vitro glomerular response to the nitric oxide-soluble guanylyl cyclase pathway. We, therefore, first determined cGMP generation in response to particulate and soluble guanylyl cyclase activation by ANP and sodium nitroprusside (SNP) in isolated glomeruli from normal (N = 6) and CHF dogs (N = 5) in which CHF was induced by rapid ventricular pacing for 18 to 28 days. Secondly, we explored the presence of major PDE isozymes in glomeruli isolated from the control and CHF dogs. When ANP or SNP (10(-10) to 10(-4) M) were incubated with the suspension of isolated glomeruli, cGMP accumulation was lower by -72 to -96% with ANP and -42 to -77% with SNP in all glomerular medias obtained from CHF compared to controls. PDE hydrolyzing activity of both cAMP and cGMP were higher in the glomerular homogenates obtained from the kidneys of the CHF group (N = 5) compared to those of the control group (N = 5). We conclude that in severe chronic experimental CHF, glomerular cGMP accumulation decreases in response to both ANP and SNP, and CHF is characterized by enhanced cGMP- and cGMP-PDE activities that may participate in glomerular maladaptation to this cardiovascular syndrome.

Although angiotensin II (Ang II) has been implicated in the pathophysiology of congestive heart failure, its temporal and regional changes during the development and progression of the disease are poorly defined. Our objective was to assess circulating, renal, cardiac, and vascular Ang II in a canine model of rapid ventricular pacing-induced heart failure that evolves from early left ventricular dysfunction to overt congestive heart failure. Ang II was measured by radioimmunoassay with low cross-reactivity to other angiotensins. Control, early left ventricular dysfunction, and overt congestive heart failure dogs were studied. Early left ventricular dysfunction was characterized by impaired cardiac function, cardiac enlargement, preserved renal perfusion pressure, maintained urinary sodium excretion, and normal plasma renin activity. Overt congestive heart failure was characterized by further impaired cardiac function and cardiac enlargement, reduced renal perfusion pressure, urinary sodium retention, and increased plasma renin activity and plasma Ang II. In early left ventricular dysfunction dogs, renal cortical, renal medullary, ventricular, and aortic Ang II were unchanged, and atrial Ang II was decreased. In overt congestive heart failure dogs, Ang II was increased in the kidney and heart compared with normal dogs and in all tissues compared with early left ventricular dysfunction dogs. The greatest increase in tissue Ang II occurred in the renal medulla. We conclude that early increases in local renal, myocardial, and vascular Ang II do not occur in this model of early left ventricular dysfunction and may even be suppressed. In contrast, increased myocardial and particularly renal Ang II in association with increased circulating Ang II are hallmarks of overt experimental congestive heart failure. These studies provide new insights into the temporal and regional alterations in Ang II during the progression of experimental congestive heart failure.

Endothelin (ET) is a potent vasoconstrictor peptide which is elevated in plasma in congestive heart failure. Recent studies suggest an important role for angiotensin II (AII) in the activation of ET in cultured cardiomyocytes. Chronic thoracic inferior vena caval constriction (TIVCC) is a model of reduced cardiac output that mimics the neurohumoral activation observed in congestive heart failure. We hypothesized that activation of the renin-angiotensin system in TIVCC plays a role in the activation of ET and that the elevation of endogenous ET contributes to the systemic and renal vasoconstriction that characterizes this model of venous congestion. We studied conscious dogs after 7 d of TIVCC in the presence or absence of chronic angiotensin converting enzyme inhibition with enalapril. TIVCC resulted in marked activation of plasma AII and ET in plasma, right atrium, lung, and renal medulla which was further localized to cardiomyocytes, pulmonary, and renal epithelial cells. Chronic angiotensin converting enzyme inhibition abolished the increases in plasma AII and ET during TIVCC. Acute endothelin A receptor blockade with FR-139317 resulted in significant decreases in mean arterial pressure and systemic vascular resistance in TIVCC. We conclude that activation of the renin-angiotensin system contributes to the activation of circulating and local ET in TIVCC and that this activation plays an important role in the regulation of arterial pressure and systemic vascular resistance in this model of congestive failure.

C-type natriuretic peptide (CNP) is a newly described 22-amino acid peptide of endothelial cell origin, which has selective cardiovascular actions and is structurally related to atrial natriuretic peptide (ANP). Recent in vitro studies have demonstrated that an important regulatory pathway for the clearance of natriuretic peptides involves binding to a common clearance receptor [natriuretic peptide C receptor (NPR-C)]. Although CNP has also been identified as a ligand for NPR-C in binding assays, no studies have defined the in vivo interaction of CNP with NPR-C. CNP (10 ng.kg-1.min-1) followed by C-ANP-(4-23), a specific ligand for NPR-C blockade, was infused intravenously in two groups (both n = 7) of anesthetized dogs at two different doses (0.1 or 1.0 micrograms.kg-1.min-1) to permit calculation of total metabolic clearance rate (TMCR). C-ANP-(4-23) increased circulating CNP and reduced TMCR in both groups. Pulmonary metabolic clearance rate was negative at baseline, suggesting a net secretion of CNP across the lung, which was increased during CNP infusion and was abolished with NPR-C blockade. Renal and femoral metabolic clearance rates were positive at baseline and increased with CNP infusion. A decrease in cardiac output and cardiac filling pressures in response to CNP administration was potentiated by NPR-C blockade. We conclude that 1) circulating CNP achieved by CNP infusion is regulated by NPR-C in vivo, 2) the pulmonary circulation is a possible site of CNP secretion, 3) the renal and peripheral circulations are sites of CNP clearance, and 4) NPR-C blockade potentiates the selective cardiovascular actions of CNP.

Adrenomedullin (ADM) is a newly described 52-amino acid peptide originally isolated from extracts of human pheochromocytoma and, more recently, detected in human plasma. Based on the report that ADM mRNA and immunoreactivity are present in the kidney, the current study was designed to determine the renal distribution of ADM by immunohistochemistry and the renal biological actions of ADM. In the immunohistochemical studies, the present investigation demonstrated the localization of ADM in glomeruli, cortical distal tubules, and medullary collecting duct cells of the normal canine kidney. In the in vivo studies, ADM was administered (0.25 ng.kg-1.min-1 in group I and 1, 5, and 25 ng.kg-1.min-1 in group II) intrarenally in normal mongrel dogs with the contralateral kidney receiving only saline vehicle. Intrarenal infusion of ADM resulted in a marked diuretic and natriuretic response, whereas the contralateral kidney showed no renal effects. These significant natriuresis and diuresis in the ADM kidney were associated with increases in glomerular filtration rate and fractional sodium excretion and with a decrease in distal tubular sodium reabsorption. Intrarenal infusion of ADM also caused an increase in mean arterial blood pressure and a decrease in heart rate. Plasma concentrations of atrial natriuretic peptide, renin activity, aldosterone, and guanosine 3',5'-cyclic monophosphate were not changed during the infusion of ADM. The current study demonstrates that ADM is present in renal glomerular and tubular cells and is a potent natriuretic peptide that may play an important role in the regulation of sodium excretion.