Huntley, Brenda K.

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.

Introduction: Human and experimental studies have suggested that a decline in renal function after myocardial infarction (MI), possibly due to the activation of adverse molecular pathways, contributes to heart failure (HF) progression and poor outcomes. The apoptosis and the transforming growth factor-beta (TGF-beta) pathways have been implicated in post-MI injury, however it is unclear if these 2 key pathways are activated post-MI in the kidney and may contribute to renal fibrosis and dysfunction. Hypothesis: We hypothesized that MI activates widespread renal apoptotic and TGF-beta related genes and is associated with renal fibrosis and dysfunction. Methods: Wistar rats were randomized into two groups: Sham-operated (S; n=10) and MI (produced by left coronary artery ligation; n=10). Cardiorenal structure and function were assessed at 3 weeks and included mean arterial pressure (MAP), EF, GFR, renal blood flow (RBF), sodium (Na) excretion and renal fibrosis by picrosirius red staining. Microarray analysis (Affymetrix GeneChip Rat Genome) was performed on kidney cortex (KC) and medulla (KM) and focused on apoptotic and TGF-beta related genes. Data presented as mean±SE,*p<0.05. Results: Experimental MI was characterized by a significant decrease in EF (S: 79±1, MI: 49±4 %*) and LV hypertrophy (S: 2.06±0.03, MI: 2.57±0.07 mg/g*), while MAP did not differ between groups. GFR and Na excretion were similar between the groups, however there was a significant decline in post-MI RBF (S: 14±1, MI: 8±1 ml/min*). Notably, after MI the KC (S: 1.3±0.3, MI: 3.4±0.8 %*) and KM (S: 1.0±0.3, MI: 11±2.8 %*) exhibited an increase in fibrosis. Microarray analysis revealed that 138 apoptotic and 15 TGF-beta pathway genes in the KC and 122 apoptotic and 10 TGF-beta pathway genes in the KM were significantly altered (1.5 fold, p<0.05) in the MI group. Conclusions: These findings demonstrate that MI results in adverse renal fibrotic remodeling together with extensive modulation of apoptotic and TGF-beta pathways of which may contribute to post-MI renal dysfunction. This study suggests that therapies should target detrimental molecular pathways in the kidney as well the heart to prevent the progression of post-MI cardiorenal failure.

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

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

Background : Studies in human myocardial infarction (MI) suggest that even in the absence of heart failure (HF) alterations in renal function may occur and contribute to poor outcomes. After MI a decline in renal function may be seen acutely by mechanisms which are unclear. The long term consequences of MI upon renal function and structure remain poorly defined. We hypothesized that even without pre-existing renal disease, renal functional and structural changes would be present following MI. Methods : Cardiorenal function and structure were assessed in Wistar rats, Sham (S; n=10) and MI groups (n=9) 3 weeks after MI. GFR was determined by inulin clearance. Blood was obtained for PRA and aldosterone. Hearts and kidneys were harvested for histological analysis. Cardiac function was assessed by echo. Genome-wide microarray analysis was performed on kidney cortex (KC) and medulla (KM) (Affymetrix GeneChip® Rat Genome 230 2.0). Results : EF decreased after MI (S:62.8±2.3, MI:42.8±6.5 %, p<0.01) and LVEDd increased (p<0.005) PRA and aldosterone activation were absent. Blood pressure (BP) was not different between groups. There was no HF as sodium and water excretion was maintained. GFR tended to decrease (S:2.9±0.3, MI:2.4±0.2 ml/min, NS). Picrosirius Red staining for collagen in the KC and KM after MI showed greater fibrosis especially in the RM (KC S:1.1±0.2, MI:3.5±0.6 %, p<0.001 and KM S:1±0.2, MI:18.8±6 %, p<0.005). Microarray analysis revealed that 303 genes significantly changed in KM and 407 genes in the KC after MI (1.5 fold, P<0.05). Gene dysregulation was related to cell proliferation, metabolic processes and cell communication (Z value>2). Conclusion : We conclude that experimental MI results in renal structural remodeling characterized by renal cortical and medullary fibrosis with a mild reduction in GFR and extensive modulation of molecular pathways related to renal growth and metabolism. This investigation provides evidence for a heart-kidney connection after MI by mechanisms which remain to be defined. We also conclude that therapies for MI targeting the heart also should be evaluated for properties of renoprotection.

Background: Decreased renal function is associated with increased cardiovascular morbidity and mortality by mechanisms that remain unclear. We hypothesized that even mild renal insufficiency produced by unilateral nephrectomy results in changes in ventricular structure and gene expression in the absence of hypertension or volume overload underscoring a kidney-heart connection in the control of myocardial structure. Methods: Cardiorenal function and structure were assessed in Wistar rats [sham (S; n=10) and unilateral nephrectomized (UNX; n=9)] 4 weeks after UNX. GFR was determined by inulin clearance and renal blood flow (RBF) by PAH. Blood was obtained for BNP, PRA, and aldosterone. Hearts and kidneys were harvested for histological analysis. Cardiac function was assessed by echo. Genome-wide microarray analysis was performed on left ventricular myocardium (Affymetrix GeneChip® Rat Genome 230 2.0). Results: Glomerular hypertrophy was observed in UNX (S:1±0.04, Nx:1.6±0.1 u3x106, p<0.001). GFR tended to decrease with a reduction in RBF (S:8±1, Nx:5±1 ml/min, p<0.005). Sodium and water excretions were not different between groups with no activation of PRA, aldosterone or BNP. LVEF and LV end-systolic and end-diastolic diameters were normal. Blood pressure (BP) was not different between groups. Importantly, Picrosirius Red staining in the ventricular myocardium of UNX compared to S revealed greater fibrosis (S:2.4±0.1, Nx:4.2±0.4 %, p<0.001). Further, microarray analysis of ventricular myocardium revealed that 278 genes significantly changed with UNX (1.5 fold, P<0.05) compared to S in genes related to cell growth, bone remodeling and muscle contraction (Z value>2). Conclusion: We conclude that even mild renal insufficiency produced by unilateral nephrectomy initiates myocardial gene responses and fibrosis independent of alterations in the circulating RAAS, BNP, BP, volume overload or systolic dysfunction. These studies support a kidney - heart connection in early renal dysfunction resulting in molecular ventricular remodeling and fibrosis. These studies in experimental mild renal dysfunction produced by removal of one kidney reveal a renal mediated mechanism for myocardial remodeling.

BACKGROUND: C-type natriuretic peptide (CNP) is a 22-amino-acid peptide produced mainly in the endothelium with potent cardiac unloading and modest blood pressure lowering actions, but minimal renal actions. Based on our previous knowledge, we recently fused a 6 aa sequence from BNP to the C-terminus and a 5 aa sequence from ANP to the N-terminus of CNP. This novel hybrid peptide, CBA-NP, has cardiac unloading actions and mild hypotensive effects similar to CNP. Importantly however, the N and C terminus alterations resulted in potent renal excretory actions. here we test the hypothesis that the 3 aa GSM 15–17 in the disulfide-ring mediate the vascular and hypotensive actions. We therefore mutated GSM 15–17 to REA 15–17 , which we named ABC-NP and compared its in vivo and in vitro actions to CBA-NP. METHODS: We determined the cardiorenal and humoral actions of intravenous bolus administration of CBA-NP (n=5) and ABC-NP (n=5) at 25 microgram/Kg in 2 separate group of normal anesthetized dogs. We also assessed the cGMP response of both peptides in human aortic endothelial cells (HAEC), human cardiac fibroblast (HCF) and isolated canine glomeruli. * p<0.05 RESULTS: IV bolus administration of CBA-NP and ABC-NP resulted in diuresis* and natriuresis*. There was a significant decrease in mean arterial blood (MAP) pressure with CBA-NP (126±6 to113±7 mmHg*) but no change with ABC-NP(126±8 to126±8 mmHg) . In addition, the reduction in pulmonary capillary wedge pressure (PCWP) and right atrial pressure (RAP) was significantly greater with CBA-NP as compared to ABC-NP. cGMP generation in HAEC and HCF was minimal with ABC-NP and was significantly higher with CBA-NP*. In contrast, cGMP generation was similar in isolated glomeruli between the two peptides. CONCLUSION: Our studies demonstrates that mutation of three amino acid (aa) residues within the CNP ring of CBA-NP from GSM 15–17 to REA alters the vascular but not the renal excretory properties. Hence by this strategic mutation within the ring of CBA-NP, we have designed a renal specific peptide ABC-NP resulting in new sequence specific functional information which can be used to design organ specific therapeutic peptides with unique properties tailored for a specific disease state.

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

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