Martín, Fernando L.

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.

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.

Background: Chronic renal insufficiency (CRI) is a strong predictor of increased mortality in the presence of heart failure (HF) and its prevalence has been increasing in the US. In addition, CRI is a major contributing factor to the progression of HF as it is associated with chronic volume overload, accelerated atherosclerosis and hypertension (HTN). The first aim of the current study was to evaluate the prevalence of CRI in an adult general population. The second aim was to address whether calculated glomerular filtration rate (cGFR) adds value to the cardiac biomarkers BNP and NT-proBNP in detecting early cardiac structural and functional impairments in the general population. Methods: From 1997 to 1999 medical history, physical examination, echocardiography, BNP (Biosite) and NT-proBNP (Roche) and serum creatinine (SCr) were obtained in 1,982 randomly selected residents of Olmsted County, MN >= 45 years (range 45 – 96 years). GFR was calculated using the Cockroft Gault equation. CRI was defined as a GFR < 60 ml/min. Cardiac structure and function were evaluated by in-depth echocardiographic examination. Results: The prevalence of cardiovascular co-morbidities was coronary atherosclerosis 12%, HTN 29.5% and diabetes mellitus 7.5%. The prevalence of CRI was 22.8% when cGFR was used for its detection. Importantly, by Chi-Square model, cGFR significantly increased the ability of BNP in detecting left ventricular hypertrophy (LVH) (p=0.004), increased relative wall thickness (p=0.0036), diastolic dysfunction (DDF) (p=0.0001) and HTN (p=0.0002). Similarly, when combined with NT-proBNP, cGFR increased the predictive power of detecting DDF (p=0.0001) and HTN (p=0.0366). Conclusion: This study reports a higher than predicted prevalence of CRI in this general US population equal to 23% and closely associated with the prevalence of HTN (29.5%). Importantly, this study also demonstrates that cGFR has additive predictive power when used in combination with two cardiac biomarkers of cardiac dysfunction and remodeling, BNP and NT-proBNP. These studies underscore the increasing burden of CRI in the general population and the utility of assessing cardiorenal function by combining renal and cardiac biomarkers in assessment of cardiac structure and function.

Decreased renal function is associated with increased CV morbidity and mortality. The changes mild renal impairment may cause structurally/molecularly in the heart remain unclear. We hypothesized that mild renal dysfunction produced by unilateral nephrectomy(UNX)results in changes in ventricular structure and gene expression in the absence of hypertension or vol overload. Wistar rats were used[sham (S; n=10) and UNX; n=9]. At 4 weeks, GFR was determined by inulin clearance and RBF by PAH. Hearts and kidneys were harvested for histological analysis. Cardiac function assessed by echo. Genomewide microarray analysis was performed on LV myocardium (Affymetrix). Glomerular hypertrophy was observed in UNX. GFR tended to decrease with a reduction in RBF (S:8±1, Nx:5±1 ml/min, p<0.005). Blood pressure (BP), Na and water excretions were maintained without activation of PRA, aldo or BNP. LVEF and LV diameters were normal. Microarray analysis of LV revealed that 278 genes changed with UNX (1.5 fold, P<0.05) related to cell growth, bone remodeling and muscle contraction (Z value>2). Importantly, Pic Red staining in the LV of UNX revealed greater fibrosis (S:2±0.1, Nx:4±0.4 %, p<0.001). We conclude that mild renal insufficiency initiates myocardial gene responses and fibrosis independent of alterations in the circulating RAAS, BNP, BP, vol overload or systolic dysfunction.

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. We hypothesized that even without preexisting renal disease, renal functional, structural and molecular changes would be present following MI. Wistar rats were used, Sham(S;n=10) and MI(n=9). 3 weeks after MI, GFR was determined by inulin clearance. Blood was obtained for PRA and aldosterone. Cardiac function was assessed by echo. Genomewide microarray analysis was performed on kidney cortex (KC) and medulla (KM) (Affymetrix). EF decreased after MI (S:63±2, MI:43±6%, p<0.01) and LVEDd increased. PRA, aldosterone and blood pressure (BP) did not change. There was no HF as Na and water excretion was maintained. GFR tended to decrease. Pic Red staining in the KC and KM after MI showed greater fibrosis (KC S:1±0.2, MI:3±0.6%, p<0.001 and KM S:1±0.2, MI:19±6%, p<0.005). Microarray analysis showed that 303 genes changed in KM and 407 genes in KC after MI (1.5 fold, P<0.05) related to structure morphogenesis and metabolic processes(Z value>2). We conclude that experimental MI results in renal structural remodeling characterized by cortical and medullary fibrosis with a mild reduction in GFR and extensive modulation of molecular pathways related to renal growth and morphogenesis underscoring a heart‐kidney connection after MI.

Decreased renal function is associated with increased CV morbidity and mortality. However, the impact of mild renal dysfunction upon the heart remains to be fully elucidated. It is thought that a mild reduction in renal mass is not deleterious to the heart, yet exploration of the impact of unilateral nephrectomy (Nx) on myocardial gene responses is undefined. We hypothesized that even when ventricular function and structure are grossly preserved after Nx, such a reduction in renal mass and function would result in significant alterations in the cardiac genome. Wistar rats, Sham(S)(n=10), Nx(n=9). 4 weeks after Nx we assessed proteinuria, UNaV, UcGMP and BNP. Cardiac function was assessed by echo. GFR was determined by inulin clearance and renal blood flow (RBF) by PAH. Blood was obtained for BNP, aldosterone and cGMP. Kidneys and hearts were harvested. Gene microarrays were conducted of left ventricle (LV) myocardium (Affymetrix GeneChip® Rat Genome 230 2.0). GFR strongly tended to decrease and RBF was decreased in Nx (S:8±1, Nx:5±1 ml/min, p<0.005). Glomerular volume was increased in Nx (S:1±0.04, Nx:1.6±0.1 u 3 ×10 6 , p<0.001) while UNaV and volume were not different. There was no proteinuria in Nx. EF, LV end systolic and diastolic diameters were normal. Blood pressure (BP) was unchanged. There was no activation of the systemic RAAS or BNP. Importantly, 278 cardiac genes changed their expression after Nx (1.5 fold, P<0.05). 11% were increased and 89% decreased. Pathways involved included cell adhesion, growth and proliferation. We conclude that Nx, independently of alterations in circulating RAAS or BNP, BP or myocardial structure and function, results in widespread changes in myocardial gene expression. Further long‐term studies are needed to define the impact of these early gene responses to myocardial structure and function.

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

Background— Alterations at the level of the coronary circulation with aging may play an important role in the evolution of age-associated changes in left ventricular (LV) fibrosis and function. However these age-associated changes in the coronary vasculature remain poorly defined primarily due to the lack of high resolution imaging technologies. The current study was designed to utilize cardiac micro–computed tomography (micro-CT) technology as a novel imaging strategy, to define the 3-dimensional coronary circulation in the young and aged heart and its relationship to LV fibrosis and function. Methods and Results— Young (2 months old; n=10) and aged (20 months old; n=10) Fischer rats underwent cardiac micro-CT imaging as well as echocardiography, blood pressure, and fibrosis analysis. Importantly, when indexed to LV mass, which increased with age, the total and intramyocardial vessel volumes were lower, whereas the epicardial vessel volume, with and without indexing to LV mass, was significantly higher in the aged hearts compared with the young hearts. Moreover, the aged hearts had a significantly lower percentage of intramyocardial vessel volume and a significantly higher percentage of epicardial vessel volume, when normalized to the total vessel volume, compared with the young hearts. Further, the aged hearts had significant LV fibrosis and mild LV dysfunction compared with the young hearts. Conclusions— This micro-CT imaging study reports the reduction in normalized intramyocardial vessel volume within the aged heart, in association with increased epicardial vessel volume, in the setting of increased LV fibrosis, and mild LV dysfunction.