Citations

Introduction: While left ventricular assist devise (LVAD) support improves the prognosis of end-stage heart failure (HF) patients, the long-term outcomes remains suboptimal. Improved outcomes may occur with organ protection including reverse myocardial remodeling. Cenderitide (CDNP) is a Mayo engineered designer natriuretic peptide (NP) that co-activates particulate guanylyl cyclase A and B receptors and functions via the second messenger cGMP. We reported a NP deficient state in the heart with LVAD support which lays the rationale for NP replacement therapy with Cenderitide which has cardiorenal protective actions in experimental HF. We sought to determine if Cenderitide co-therapy would be safe and activate cGMP in patients with LVAD support. Hypothesis: Acute single dose of subcutaneous (SQ) Cenderitide will be safe and activate cGMP in patients with LVAD support. Methods: Stable HF patients with LVAD support (Heartmate II, all males) were enrolled in a cross-over design comparing SQ placebo and low (Low-C, 5 ug/kg, n=8) or high dose (High-C, 10 ug/kg, n=8) Cenderitide (Capricor Therapeutics) administration. Vital signs, plasma C(D)NP, cGMP, Aldosterone (Aldo), and NT-proBNP levels were measured before or after drug administration over 24 hours. All parameters in Cenderitide group were adjusted by those in placebo. Results: Both Low-C and High-C group demonstrated significantly increased plasma C(D)NP and cGMP levels for 2 hours after drug injection and those in High-C group showed more prolonged and higher increases than Low-C group. Mean arterial pressure (MAP) in Low-C did not show any changes but in High-C group tended to be lower for 18 hours but this was not significant. Plasma Aldo and NT-proBNP levels in Low-C group did not show any change but those in High-C group tended to be lower after drug injection s similar to the change in MAP. No adverse events were observed in the study. Conclusions: SQ Cenderitide was successfully absorbed, activated cGMP and well tolerated in patients with LVAD support. High dose Cenderitide may have cardiac unloading effects suggested by decreases in NT-proBNP. Further clinical trial for efficacy is warranted to clarify cardiorenal protective effects of Cenderitide in the LVAD population to improve outcomes.

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

Introduction: As importantly underscored by the SPRINT Trial, innovative blood pressure (BP) lowering and multiorgan protective drugs are needed for treating hypertension (HTN). We engineered a Mayo Clinic designer atrial natriuretic peptide (ANP)-based drug, ZD100, which possesses a unique 12 amino acid (AA) carboxy-terminus (CT) extension while maintaining the N-terminus (NT) of ANP. ZD100 is more potent than ANP in reducing BP, inducing natriuresis and inhibiting aldosterone via pGC-A and its second messenger cGMP. To date, the in vivo actions of ZD100 in a model of essential HTN are undefined. Further, in attempt to enhance and/or minimize pGC-A activation, the importance of specific AAs in the CT and NT of ZD100 is unknown and is critical to define. Hypothesis: We hypothesize that ZD100 is a potent anti-HTN drug activating pGC-A/cGMP system and that specific AA variations in the CT and/or NT of ZD100 will modify the ability of activating pGC-A. Methods: Spontaneously hypertensive rats (SHRs) received intravenous (IV) ZD100 or vehicle and BP, plasma and urinary cGMP and the natriuretic effects were determined. We then engineered 15 strategic ZD100 variants (Vs) consisting of mutated peptides in the CT or NT or both. We stimulated HEK293 cells overexpressing pGC-A with ZD100 and the 15 Vs (10-8 M) and measured intracellular cGMP generation. Results: In SHRs, acute IV ZD100 reduced BP, increased plasma and urinary cGMP generation and induced natriuresis demonstrating efficacy in HTN. In vitro, we found two more potent pGC-A activators (+100% cGMP activation) only among the CT-only mutated ZD100 Vs. Importantly, key mutations of either the CT or NT resulted in 6 loss-of-function Vs identifying importance of peptide-receptor dependency. Conclusions: We demonstrate for the first time in a genetic model of HTN that ZD100 reduces BP, activates systemic and renal pGC-A/cGMP and induces natriuresis. Enhanced or reduced pGC-A activation with targeted AA mutations in the CT and NT of ZD100 provides key structure function insights into ZD100 which may lead to next generation ZD100 therapeutic peptides. These findings support ZD100 as a novel peptide therapeutic strategy for HTN and will help guide the selection of best-in-class ZD100 analogues.

Background: The primary role of natriuretic peptide receptor 3 (NPR3) is clearance of natriuretic peptides. Genetic variation in NPR3 has been associated with blood pressure regulation and obesity. Despite its importance, variation in NPR3 characterizing coding single nucleotide polymorphisms (SNPs) has not been addressed by resequencing the gene. Objective: We set out to identify and functionally characterize common variation in NPR3 in 3 ethnic groups. DNA samples from such subjects are studied because common genetic variation needs to be defined initially in “normal” individuals prior to studying drug effect and response. Also, rare variants identified by sequencing are enriched in complex disease traits. Methods and Results: DNA samples from 96 European-American, 96 African-American, and 96 Han Chinese-American healthy subjects were used to resequence NPR3. There were 105 polymorphisms identified of which 50 were novel, including 8 nonsynonymous (ns) SNPs of which 7 were novel. Expression constructs for the nsSNPs were created and HEK-293 cells were transfected with constructs for wild type (WT) and the variants. Recombinant proteins were analyzed by quantitative Western blot analysis. A significant reduction in NPR3 immunoreactive protein (60% or less of WT) was observed for the following variants Cys3, Arg146, Val499, and Ser477+Asp521. The Arg146 novel polymorphism resulted in the least protein expression (20% of WT). After treatment of WT and Arg146 cells with proteasome (MG132) and autophagy (3MA) inhibtors, it was determined that autophagy and not proteasomes plays an important role in the degradation of the Arg 146 variant protein. Modeling based on the NPR3 x-ray crystallography structure confirmed that the Arg146 variant were not compatible with WT conformation and could result in protein misfolding or instability. Conclusions: Multiple novel polymorphisms of NPR3 were identified in 3 ethnic groups. There were 4 variant receptors that displayed a significant decrease in NPR3 protein quantity, Arg146 being the variant with the least expression. Degradation of the variant Arg146 was mediated predominantly by autophagy. This SNP could have a significant effect on the metabolism of natriuretic peptides with clinical implications.

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

Background: C-type natriuretic peptide (CNP) is of endothelial cell origin and represents the most potent anti-fibrotic peptide of the natriuretic peptide family and is important for extracellular matrix homeostasis including mediating bone growth in the young. Cardiorenal aging is associated with altered cardiac and renal structure and function that may contribute to the development of heart and kidney failure, particularly in the elderly. The impact of aging on circulating CNP and associated changes in cardiorenal structure and function are undefined. Hypothesis: We hypothesized that a decrease in endogenous plasma CNP occurs with aging and is associated with an increase in cardiorenal fibrosis and altered left ventricular (LV) and renal structure and function. Methods: Studies were performed in 2, 11 and 20 month old male Fischer rats (n = 8 –10/group). Echo was used to assess LV structure and function. Blood pressure (BP), urinary protein excretion, plasma B-type natriuretic peptide (BNP) and CNP were measured. Kidneys and LV were harvested for gross and histology analysis. Mean±SE, *p<0.05. Results: Aging from 2 to 20 months was associated with progressive reductions in plasma CNP (30±3 to 21±1* to 9±1*pg/ml). Significant and progressive cardiac and renal fibrosis were observed with aging (LV: 9±0.3 to 15±0.7* to 21±1*% and kidney: 1±0.3 to 8±2* to 17±1* %). In contrast to CNP, plasma BNP was modestly increased from the 2 to 20 month old group (21±2 to 26±1*pg/ml). The reduction in CNP and increase in BNP, the latter a biomarker for LV dysfunction, were associated with decreased EF (88±1 to 80±1*%) and increased LVEDd (6.61±0.09 to 7.48±0.09*mm). BP and urinary protein excretion were mildly elevated with aging (91±1 to 101±3* mmHg and 8±0.3 to 20±3* mg/day, respectively). Conclusion: We report for the first time that aging is associated with a progressive decline in circulating CNP and progressive cardiorenal fibrosis, systolic dysfunction and proteinuria. Further studies are warranted to explore the hypothesis that a mechanism of cardiorenal aging with altered cardiorenal structure and function may include a decrease in the bioavailability of CNP.

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

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

Abstract The roles of sex hormones such as estradiol, testosterone, and sex hormone-binding globulin (SHBG) in the etiology of lung and colorectal cancers in women, among the most common cancers after breast cancer, are unclear. This Mendelian randomization (MR) study evaluated such potential causal associations in women of European ancestry. We used summary statistics data from genome-wide association studies (GWASs) on sex hormones and from the Trøndelag Health (HUNT) Study and large consortia on cancers. There was suggestive evidence of genetically predicted 1-standard deviation increase in total testosterone levels being associated with a lower risk of lung non-adenocarcinoma (hazard ratio (HR) 0.60, 95% CI 0.37–0.98) in the HUNT Study. However, this was not confirmed by using data from a larger consortium. In general, we did not find convincing evidence to support a causal role of sex hormones on risk of lung and colorectal cancers in women of European ancestry.

Abstract Background Variations in climate have been associated with a greater risk of surgical site infections, urinary tract infections, and changes in the skin microbiome; however, limited data exist on the impact of climate on inflatable penile prosthesis (IPP) infections. Aim We sought to evaluate the impact of climate on the risk of IPP infections in a large international, multicenter cohort. Methods We performed a multi-institutional, retrospective study of patients undergoing IPP surgery. We then evaluated whether the month or season, during which surgery was performed, affected device infections. Implant infections were defined as infections requiring device explantation. A univariate logistic regression analysis was undertaken. Outcomes Our primary outcome was implant infection. Results A total of 5289 patients with a mean age of 62.2 ± 10.8 years received IPP placement. There was a fairly even distribution of implants performed in each season. A total of 103 (1.9%) infections were recorded. There were 32 (31.1%) IPP infections in patients who underwent surgery in the summer, followed by 28 (27.2%) in the winter, 26 (25.2%) in the spring, and 17 (16.5%) in the fall. No statistically significant differences were recorded in terms of season (P = .19) and month (P = .29). The mean daily temperature (P = .43), dew point (P = .43), and humidity (P = .92) at the time of IPP placement was not associated with infection. Clinical Implications These findings provide reassurance to prosthetic urologists that infection reduction strategies do not need to be tailored to local climate. Strengths and Limitations Climate data were not directly recorded for each hospital, but rather based on the monthly averages in the city where the surgery was performed. Conclusion The climate at time of IPP placement and time of year of surgery is not associated with IPP infection risk.