RESUMO
The management of sodium intake is clinically important in many disease states including heart failure, kidney disease, and hypertension. Tenapanor is an inhibitor of the sodium-proton (Na(+)/H(+)) exchanger NHE3, which plays a prominent role in sodium handling in the gastrointestinal tract and kidney. When administered orally to rats, tenapanor acted exclusively in the gastrointestinal tract to inhibit sodium uptake. We showed that the systemic availability of tenapanor was negligible through plasma pharmacokinetic studies, as well as autoradiography and mass balance studies performed with (14)C-tenapanor. In humans, tenapanor reduced urinary sodium excretion by 20 to 50 mmol/day and led to an increase of similar magnitude in stool sodium. In salt-fed nephrectomized rats exhibiting hypervolemia, cardiac hypertrophy, and arterial stiffening, tenapanor reduced extracellular fluid volume, left ventricular hypertrophy, albuminuria, and blood pressure in a dose-dependent fashion. We observed these effects whether tenapanor was administered prophylactically or after disease was established. In addition, the combination of tenapanor and the blood pressure medication enalapril improved cardiac diastolic dysfunction and arterial pulse wave velocity relative to enalapril monotherapy in this animal model. Tenapanor prevented increases in glomerular area and urinary KIM-1, a marker of renal injury. The results suggest that therapeutic alteration of sodium transport in the gastrointestinal tract instead of the kidney--the target of current drugs--could lead to improved sodium management in renal disease.
Assuntos
Mucosa Intestinal/metabolismo , Rim/patologia , Miocárdio/patologia , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Sódio/metabolismo , Albuminúria/complicações , Albuminúria/tratamento farmacológico , Albuminúria/fisiopatologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Pressão Sanguínea/efeitos dos fármacos , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Eletrólitos/urina , Enalapril/farmacologia , Enalapril/uso terapêutico , Fezes , Voluntários Saudáveis , Humanos , Hipertrofia , Intestinos/efeitos dos fármacos , Isoquinolinas/administração & dosagem , Isoquinolinas/farmacocinética , Isoquinolinas/farmacologia , Isoquinolinas/uso terapêutico , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Miocárdio/metabolismo , Nefrectomia , Ratos , Ratos Sprague-Dawley , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/fisiopatologia , Cloreto de Sódio na Dieta/administração & dosagem , Cloreto de Sódio na Dieta/farmacologia , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/metabolismo , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêuticoRESUMO
INTRODUCTION: Bioimpedance spectroscopy (BIS) has been used in human and large animal research to assess body fluid compartment volumes (BFC) such as total body water (TBW), extracellular fluid volume (ECFV), and intracellular fluid volume (ICFV). To date, the application of BIS for determination of BFC in small research animals has been limited. METHODS: We sought to evaluate the sensitivity and consistency of BIS for the determination of BFC in male SD rats. Thus, in separate series of experiments, we a) compared BFC values determined using BIS to BFC values obtained using radioisotope indicator dilution methods; b) examined day-to-day intra- and inter-rat BFC variability in small (267.8±5.4 g) and large (372.6±5.6 g) rats (n=8/group) as compared to empirical normative mammalian values; c) evaluated the sensitivity of BIS to detect time-dependent responses to repeated administration of a potent diuretic; and d) compared empirically generated BFC data to predicted osmotically-induced ECFV and ICFV shifts in response to i.v. administration of hypotonic (0.3%), isotonic (0.9%) or hypertonic (3.0%) saline (n=6/concentration). RESULTS: BFC values generated using radioisotope dilution agreed with those generated using BIS. BIS reliably detected differences between small and large rats (p<0.001), and was associated with low (<3.5%) day-to-day, intra-animal coefficient of variation (%=Standard Deviation/mean). BIS detected small reductions (~10%) in ECFV induced by as few as 2 days of the loop diuretic, furosemide, relative to vehicle treatment (70.8±1.5 ml vs. 84.0±1.5 ml; respectively, p<0.05). BIS rapidly detected shifts between ECFV and ICFV in response to osmotic saline challenge, and these responses were similar to physiologically predicted responses. DISCUSSION: The current studies support using BIS as a means of sensitively and reliably performing repeated measurements of BFC in rats of a) differing sizes, b) in response to therapeutic agents known to influence renal sodium handling and c) in response to osmotic challenge.
Assuntos
Compartimentos de Líquidos Corporais/fisiologia , Água Corporal/fisiologia , Impedância Elétrica , Líquido Extracelular/fisiologia , Animais , Composição Corporal/efeitos dos fármacos , Composição Corporal/fisiologia , Compartimentos de Líquidos Corporais/química , Compartimentos de Líquidos Corporais/efeitos dos fármacos , Água Corporal/efeitos dos fármacos , Líquido Extracelular/efeitos dos fármacos , Injeções Intravenosas , Masculino , Técnica de Diluição de Radioisótopos , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Cloreto de Sódio/administração & dosagemRESUMO
Contractile reserve decreases with advancing age and chronic isoproterenol (ISO) administration is a well-characterized model of cardiac hypertrophy known to impair cardiovascular function. This study evaluated whether nonsenescent, mature adult rats are more susceptible to detrimental effects of chronic ISO administration than younger adult rats. Rats received daily injections of ISO (0.1 mg/kg sc) or vehicle for 3 wk. ISO induced a greater impairment in contractile reserve [maximum of left ventricular pressure development (Δ+dP/dt(max))] in mature adult ISO-treated (MA-ISO) than in young adult ISO-treated rats (YA-ISO) in response to infusions of mechanistically distinct inotropes (digoxin, milrinone; 20-200 µl·kg(-1)·min(-1)), while basal and agonist-induced changes in heart rate and systolic arterial pressure (SAP) were not different across groups. ISO decreased expression of the calcium handling protein, sarco(endo)plasmic reticulum Ca(2+)-ATPase-2a, in MA-ISO compared with YA, YA-ISO, and MA rats. Chronic ISO also induced greater increases in cardiac hypertrophy [left ventricular (LV) index: 33 ± 3 vs. 22 ± 5%] and caspase-3 activity (34 vs. 5%) in MA-ISO relative to YA-ISO rats. Moreover, ß-myosin heavy chain (ß-MHC) and atrial natriuretic factor (ANF) mRNA expression was significantly elevated in MA-ISO. These results demonstrate that adult rats develop greater impairments in systolic performance than younger rats when exposed to chronic catecholamine excess. Reduced contractile reserve may result from calcium dysregulation, increased caspase-3 activity, or increased ß-MHC and ANF expression. Although several studies report age-related declines in systolic performance in older and senescent animals, the present study demonstrates that catecholamine excess induces reductions in systolic performance significantly earlier in life.
Assuntos
Envelhecimento/fisiologia , Cardiotônicos/farmacologia , Catecolaminas/farmacologia , Isoproterenol/farmacologia , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Animais , Digoxina/farmacologia , Esquema de Medicação , Isoproterenol/administração & dosagem , Milrinona/farmacologia , RatosRESUMO
Stress signaling in the myocardium results in enhanced expression of fetal ß-myosin heavy chain (ß-MyHC) and reduced expression of adult α-myosin heavy chain (α-MyHC), with the net outcome of diminished myofibrillar ATPase activity and impaired contractility. Pharmacological approaches aimed at preventing this myosin isoform "switch" could provide therapeutic benefit to patients with heart failure. Myosin isoform protein expression is typically quantified using gel electrophoresis methods, which are time-consuming and prone to variability. Here we describe a facile, reversed-phase high-performance liquid chromatography (HPLC) method for rapidly determining the relative amounts of full-length α- and ß-MyHC in rat hearts. The assay was validated using cardiac tissues from rats in which a key transcriptional regulator of MyHC expression, the thyroid hormone receptor, was pharmacologically manipulated. This novel assay should facilitate drug discovery efforts focused on the MyHC axis.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Cadeias Pesadas de Miosina/análise , Animais , Eletroforese em Gel de Poliacrilamida/métodos , Masculino , Miocárdio/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Hepatic and circulating endothelin-1 (ET-1) are increased in patients with cirrhosis and in cirrhotic animals. However, the distinct roles of ET receptor subtypes ETA and ETB in cirrhosis and portal hypertension (PHT) have not been clearly elucidated. Thus, we studied the effects of selective ET-1 antagonists (ETA-ant or ETB-ant) and nonselective ET-1 antagonist (ETA/B-ant) on hepatic hemodynamics in cirrhotic rats. Liver fibrosis and PHT were induced by complete bile duct ligation (BDL) in rats. Two weeks after BDL or sham surgery, hemodynamic responses were measured during intraportal infusion of incremental doses of the following ET-ants: (i) BQ-123, (ii) BQ-788, and (iii) bosentan. After equilibration with vehicle, doses of ET-ants were infused for 30 min periods, and steady-state systemic and hepatic hemodynamics, portal venous pressure (PVP), and hepatic blood flow (HBF) were measured. BDL induced significant PHT and elevated concentrations of plasma ET-1 compared with sham. ETA-ant decreased PVP of cirrhotic rats but had no effect on sham, whereas ETB-ant increased PVP in sham but had no effect in BDL. Nonselective ETA/B-ant decreased PVP of BDL similarly to ETA-ant. Both ETA-ant and ETB-ant decreased local HBF, whereas a nonselective antagonist did not change HBF in sham; however no significant changes were observed in HBF of BDL rats with any of the antagonists. These findings suggest ETA activation contributes to PHT in cirrhotic rats, whereas ETB-mediated portal depressor effects are attenuated in cirrhotic rats compared with noncirrhotic rats.
Assuntos
Antagonistas dos Receptores de Endotelina , Hemodinâmica/efeitos dos fármacos , Cirrose Hepática/fisiopatologia , Fígado/irrigação sanguínea , Animais , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Bosentana , Modelos Animais de Doenças , Endotelina-1/sangue , Endotelina-1/genética , Expressão Gênica/genética , Hemodinâmica/fisiologia , Células Estreladas do Fígado/metabolismo , Hipertensão Portal/etiologia , Hipertensão Portal/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Cirrose Hepática/sangue , Cirrose Hepática/complicações , Cirrose Hepática/metabolismo , Masculino , Oligopeptídeos/administração & dosagem , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/administração & dosagem , Peptídeos Cíclicos/farmacologia , Piperidinas/administração & dosagem , Piperidinas/farmacologia , Pressão na Veia Porta/efeitos dos fármacos , Pressão na Veia Porta/fisiologia , Ratos , Ratos Sprague-Dawley , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Sanguíneo Regional/fisiologia , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacologiaRESUMO
Vascular injury triggers dedifferentiation and cytoskeletal remodeling of smooth muscle cells (SMCs), culminating in vessel occlusion. Serum response factor (SRF) and its coactivator, myocardin, play a central role in the control of smooth muscle phenotypes by regulating the expression of cytoskeletal genes. We show that SRF and myocardin regulate a cardiovascular-specific microRNA (miRNA) cluster encoding miR-143 and miR-145. To assess the functions of these miRNAs in vivo, we systematically deleted them singly and in combination in mice. Mice lacking both miR-143 and miR-145 are viable and do not display overt abnormalities in smooth muscle differentiation, although they show a significant reduction in blood pressure due to reduced vascular tone. Remarkably, however, neointima formation in response to vascular injury is profoundly impeded in mice lacking these miRNAs, due to disarray of actin stress fibers and diminished migratory activity of SMCs. These abnormalities reflect the regulation of a cadre of modulators of SRF activity and actin dynamics by miR-143 and miR-145. Thus, miR-143 and miR-145 act as integral components of the regulatory network whereby SRF controls cytoskeletal remodeling and phenotypic switching of SMCs during vascular disease.
Assuntos
Citoesqueleto/metabolismo , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Miócitos de Músculo Liso/metabolismo , Actinas/metabolismo , Animais , Sequência de Bases , Lesões das Artérias Carótidas/metabolismo , Células Cultivadas , Elementos Facilitadores Genéticos/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Dados de Sequência Molecular , Mutação , Miócitos Cardíacos/metabolismo , Miócitos de Músculo Liso/patologia , Proteínas Nucleares/metabolismo , Ratos , Alinhamento de Sequência , Transativadores/metabolismoRESUMO
Atrial cardiomyocytes, neurons, and endocrine tissues secrete neurotransmitters and peptide hormones via large dense-core vesicles (LDCVs). We describe a new member of the Ras family of G-proteins, named RRP17, which is expressed specifically in cardiomyocytes, neurons, and the pancreas. RRP17 interacts with Ca(2+)-activated protein for secretion-1 (CAPS1), one of only a few proteins known to be associated exclusively with LDCV exocytosis. Ectopic expression of RRP17 in cardiomyocytes enhances secretion of atrial natriuretic peptide (ANP), a regulator of blood pressure and natriuresis. Conversely, genetic deletion of RRP17 in mice results in dysmorphic LDCVs, impaired ANP secretion, and hypertension. These findings identify RRP17 as a component of the cellular machinery involved in regulated secretion within the heart and potential mediator of the endocrine influence of the heart on other tissues.
Assuntos
Fator Natriurético Atrial/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica , Proteínas ras/metabolismo , Sequência de Aminoácidos , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Átrios do Coração/metabolismo , Humanos , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Pâncreas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
In response to pathological stresses such as hypertension or myocardial infarction, the heart undergoes a remodeling process that is associated with myocyte hypertrophy, myocyte death, and fibrosis. Histone deacetylase 5 (HDAC5) is a transcriptional repressor of cardiac remodeling that is subject to phosphorylation-dependent neutralization in response to stress signaling. Recent studies have suggested a role for protein kinase C (PKC) and its downstream effector, protein kinase D1 (PKD1), in the control of HDAC5 phosphorylation. While PKCs are well-documented regulators of cardiac signaling, the function of PKD1 in heart muscle remains unclear. Here, we demonstrate that PKD1 catalytic activity is stimulated in cardiac myocytes by diverse hypertrophic agonists that signal through G protein-coupled receptors (GPCRs) and Rho GTPases. PKD1 activation in cardiomyocytes occurs through PKC-dependent and -independent mechanisms. In vivo, cardiac PKD1 is activated in multiple rodent models of pathological cardiac remodeling. PKD1 activation correlates with phosphorylation-dependent nuclear export of HDAC5, and reduction of endogenous PKD1 expression with small interfering RNA suppresses HDAC5 shuttling and associated cardiomyocyte growth. Conversely, ectopic overexpression of constitutively active PKD1 in mouse heart leads to dilated cardiomyopathy. These findings support a role for PKD1 in the control of pathological remodeling of the heart via its ability to phosphorylate and neutralize HDAC5.
Assuntos
Regulação da Expressão Gênica , Miócitos Cardíacos/metabolismo , Proteínas Quinases/fisiologia , Transdução de Sinais , Estresse Fisiológico/metabolismo , Animais , Animais Recém-Nascidos , Células COS , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Domínio Catalítico , Células Cultivadas , Chlorocebus aethiops , Ativação Enzimática , Ventrículos do Coração/citologia , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Miócitos Cardíacos/patologia , Proteína Quinase C , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WF , Ratos Sprague-DawleyRESUMO
L-Arginine inhibits thick ascending limb (THAL) NaCl absorption by activating endothelial NO synthase (eNOS) and increasing NO production. Inhibition of renal NO production combined with a high-salt diet produces hypertension, and the THAL has been implicated in salt-sensitive hypertension. We hypothesized that a high-salt diet enhances the inhibitory action of L-arginine on NaCl absorption by THALs because of increased eNOS expression and NO production. To test this, we used isolated THALs from rats on a normal-salt (NS) or high-salt diet (HS) for 7 to 10 days. L-Arginine (1 mmol/L) decreased chloride absorption by 56+/-10% in THALs from rats on a HS diet, but only 29+/-3% in THALs from rats on a NS diet. eNOS expression in isolated THALs from rats on a HS diet was increased by 3.9-fold compared with NS (P<0.03). However, L-arginine increased NO levels to the same extent in THALs from both groups, as measured with DAF-2 DA or a NO-sensitive electrode. To determine whether a HS diet increases the sensitivity of the THAL to NO, we tested the effects of the NO donor spermine NONOate on chloride absorption. In THALs from rats on a HS diet, 1 and 5 micromol/L spermine NONOate reduced chloride absorption by 35+/-5% and 58+/-6%, respectively. In contrast, these same concentrations of spermine NONOate reduced chloride absorption by 4+/-4% (P<0.03 versus HS diet) and 43+/-9% in THALs from rats on a NS diet. We conclude that a HS diet enhances the effect of NO in the THAL. L-Arginine-stimulated NO production was not enhanced by a HS diet, despite increased eNOS protein.
Assuntos
Alça do Néfron/enzimologia , Alça do Néfron/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/biossíntese , Cloreto de Sódio/farmacologia , Animais , Arginina/farmacologia , Cloretos/metabolismo , Técnicas de Cultura , Dieta , Transporte de Íons , Alça do Néfron/anatomia & histologia , Masculino , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: The thick ascending limb of the loop of Henle (THAL) plays an important role in the maintenance of salt, water, and acid-base balance. While techniques for gene transfer of renal vascular cells and some tubular segments have been described, in vivo transduction of THALs has not been successful. We hypothesized that in vivo injection of adenoviral vectors into the renal medulla would result in efficient transduction of THALs. METHODS: We injected recombinant adenoviruses containing the reporter gene, green fluorescent protein (GFP), driven by either the cytomegalovirus promoter (Ad-CMVGFP) or the promoter for the Na/K/2 Cl cotransporter (Ad-NKCC2GFP), which is THAL-specific, into the outer medullary interstitium of Sprague-Dawley rat kidneys. Kidneys were removed at various times after viral injection and analyzed for GFP expression. RESULTS: Western blots revealed strong GFP expression in the outer medulla (which is composed primarily of THALs) 5 days after Ad-CMVGFP injection. We quantified THAL transduction efficiency by scoring the number of fluorescent tubules in THALs suspensions, which showed that at least 77 +/- 3% of THAL expressed GFP. To specifically transduce THALs, we injected Ad-NKCC2GFP into the medullary interstitium. As determined by Western blot, GFP expression was only detected in the outer medulla. Immunohistochemistry and confocal microscopy showed that GFP was localized to tubular cells positive for Tamm-Horsfall protein. Thus, GFP fluorescence was only detected in THALs, not in cortical, inner medulla or vascular cells. Time-course studies showed that GFP expression in THALs was measurable from 4 to 14 days, peaked at 7 days, and had returned to background levels by 21 days. CONCLUSION: This method facilitates highly efficient, THAL-specific transduction. While application of this technique for gene therapy in humans is unlikely due to the transient gene expression observed and the impossibility for repeated injections of adenoviral vectors, this method provides a valuable tool for investigators studying regulation and mechanisms of THAL ion transport and its relationship to whole-kidney physiology and pathophysiology.