Design, Synthesis, and Actions of an Innovative Bispecific Designer Peptide.

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.

CRRL269: a novel designer and renal-enhancing pGC-A peptide activator.

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.

Neurohumoral Modulation During Waon Therapy in Chronic Heart Failure　- Subanalysis of Waon-CHF Study.

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 m2. 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).

Saxagliptin and Tadalafil Differentially Alter Cyclic Guanosine Monophosphate (cGMP) Signaling and Left Ventricular Function in Aortic-Banded Mini-Swine.

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.

Elevation of circulating but not myocardial FGF23 in human acute decompensated heart failure.

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.

Aldosterone Predicts Cardiovascular, Renal, and Metabolic Disease in the General Community: A 4-Year Follow-Up.

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.

Circulating C-type natriuretic peptide and its relationship to cardiovascular disease in the general population.

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.

Weight loss, saline loading, and the natriuretic peptide system.

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.

Pro-B-type natriuretic peptide-1-108 processing and degradation in human heart failure.

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.

Circulating aldosterone and natriuretic peptides in the general community: relationship to cardiorenal and metabolic disease.

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.

Urinary C-type natriuretic peptide: a new heart failure biomarker.

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 gelatinase­associated 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.

Atrial natriuretic peptide genetic variant rs5065 and risk for cardiovascular disease in the general community: a 9-year follow-up study.

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.

Defining high-sensitivity cardiac troponin concentrations in the community.

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.

Secretion of prohormone of B-type natriuretic peptide, proBNP1-108, is increased in heart failure.

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.

Human hypertension is characterized by a lack of activation of the antihypertensive cardiac hormones ANP and BNP.

OBJECTIVES: This study sought to investigate plasma levels of circulating cardiac natriuretic peptides, atrial natriuretic peptide (ANP) and B-type or brain natriuretic peptide (BNP), in the general community, focusing on their relative differences in worsening human hypertension. BACKGROUND: Although ANP and BNP are well-characterized regulators of blood pressure in humans, little is known at the population level about their relationship with hypertension. The authors hypothesized that hypertension is associated with a lack of activation of these hormones or their molecular precursors. METHODS: The study cohort (N = 2,082, age >45 years) was derived from a random sample from Rochester, Minnesota, and each subject had a medical history, clinical examination, and assessment of different plasma forms of ANP and BNP. Patients were stratified by blood pressure. Multivariable linear regression was used to assess differences in natriuretic peptide levels in worsening hypertension. RESULTS: Compared to normotensive, BNP(1-32) and N-terminal proBNP(1-76) (NT-proBNP(1-76)) were significantly decreased in pre-hypertension (p < 0.05), with BNP(1-32) significantly decreased in stage 1 as well (p < 0.05). Although proBNP(1-108) remained unchanged, the processed form was significantly increased only in stage 2 hypertension (p < 0.05). ANP(1-28) remained unchanged, while NT-ANP(1-98) was reduced in pre-hypertension (p < 0.05). CONCLUSIONS: The authors demonstrated the existence of an impaired production and/or release of proBNP(1-108) along with a concomitant reduction of BNP(1-32) and NT-proBNP(1-76) in the early stages of hypertension, with a significant elevation only in stage 2 hypertension. Importantly, they simultaneously demonstrated a lack of compensatory ANP elevation in advanced hypertension.

Urinary C-type natriuretic peptide excretion: a potential novel biomarker for renal fibrosis during aging.

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.

A genetic variant of the atrial natriuretic peptide gene is associated with cardiometabolic protection in the general community.

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.

The effect of the brain-type natriuretic peptide single-nucleotide polymorphism rs198389 on test characteristics of common assays.

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.

Pro-B-type natriuretic peptide(1-108) circulates in the general community: plasma determinants and detection of left ventricular dysfunction.

OBJECTIVES: The purpose of this study was to investigate circulating pro-B-type natriuretic peptide (proBNP(1-108)) in the general community and evaluate its ability to detect left ventricular (LV) dysfunction. BACKGROUND: The current concept for cardiac endocrine function is that, in response to cardiac stress, the heart secretes B-type natriuretic peptide (BNP(1-32)) and amino-terminal pro-B-type natriuretic peptide (NT-proBNP(1-76)) after intracardiac cleavage of their molecular precursor, proBNP(1-108). We hypothesized that proBNP(1-108) circulates in normal human subjects and that it is a useful biomarker for LV dysfunction. METHODS: Our population-based study included a cohort of 1,939 adults (age ≥45 years) from Olmsted County, Minnesota, with 672 participants defined as healthy. Subjects underwent in-depth clinical characterization, detailed echocardiography, and measurement of proBNP(1-108). Independent factors associated with proBNP(1-108) and test characteristics for the detection of LV dysfunction were determined. RESULTS: ProBNP(1-108) in normal humans was strongly influenced by sex, age, heart rate, and body mass index. The median concentration was 20 ng/l with a mean proBNP(1-108) to NT-proBNP(1-76) ratio of 0.366, which decreased with heart failure stage. ProBNP(1-108) was a sensitive (78.8%) and specific (86.1%) biomarker for detecting LV systolic dysfunction, which was comparable to BNP(1-32), but less than NT-proBNP(1-76), in several subsets of the population. CONCLUSIONS: ProBNP(1-108) circulates in the majority of healthy humans in the general population and is a sensitive and specific biomarker for the detection of systolic dysfunction. The proBNP(1-108) to NT-proBNP(1-76) ratio may provide insights into altered proBNP(1-108) processing during heart failure progression. Thus, this highly specific assay for proBNP(1-108) provides important new insights into the biology of the BNP system.

Corin is present in the normal human heart, kidney, and blood, with pro-B-type natriuretic peptide processing in the circulation.

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.