RESUMEN
Mutant α-adducin and endogenous ouabain levels exert a causal role in hypertension by affecting renal Na-K ATPase. In addition, mutant ß-adducin is involved in glomerular damage through nephrin down-regulation. Recently, the salt-inducible kinase 1 (SIK1) has been shown to exert a permissive role on mutant α-adducin effects on renal Na-K ATPase activity involved in blood pressure (BP) regulation and a SIK1 rs3746951 polymorphism has been associated with changes in vascular Na-K ATPase activity and BP. Here, we addressed the role of SIK1 on nephrin and glomerular functional modifications induced by mutant ß-adducin and ouabain, by using congenic substrains of the Milan rats expressing either mutant α- or ß-adducin, alone or in combination, ouabain hypertensive rats (OHR) and hypertensive patients. SIK1 co-localized and co-immunoprecipitated with nephrin from glomerular podocytes and associated with caveolar nephrin signaling. In cultured podocytes, nephrin-gene silencing decreased SIK1 expression. In mutant ß-adducin congenic rats and in OHR, the podocyte damage was associated with decreased nephrin and SIK1 expression. Conversely, when the effects of ß-adducin on podocytes were blocked by the presence of mutant α-adducin, nephrin and SIK1 expressions were restored. Ouabain effects were also reproduced in cultured podocytes. In hypertensive patients, nephrinuria, but not albuminuria, was higher in carriers of mutant SIK1 rs3746951 than in wild-type, implying a more direct effect of SIK1 on glomerular damage. These results demonstrate that, through nephrin, SIK1 is involved in the glomerular effects of mutant adducin and ouabain and a direct effect of SIK1 is also likely to occur in humans.
RESUMEN
Rats of the Milan hypertensive strain (MHS) are resistant to both hypertensive and diabetic renal disease. Genetically determined hypertrophy of intrarenal arteries has been suggested as the putative mechanism preventing transmission of systemic hypertension to the glomerular microcirculation or diabetes-induced loss of autoregulation, which lead to glomerular hypertension and consequent podocyte injury and proteinuria. This study aimed to investigate glomerular barrier function and structure in ageing and diabetic MHS rats under basal conditions and after injection of 2.5 g of bovine serum albumin (BSA) causing increased workload and possibly removing haemodynamic protection by inducing renal cortical vasodilatation. Genetically related rats of the Milan normotensive strain (MNS) served as a proteinuric counterpart. No change in renal function or structure was detected in diabetic MHS rats, whereas MNS rats developed diabetic nephropathy superimposed on that occurring spontaneously in this strain. Diabetic, but not non-diabetic, MHS rats showed significantly reduced synaptopodin and nephrin expression, though to a lesser extent than non-diabetic and diabetic MNS rats, together with unchanged podocyte number, density and structure and no proteinuria. Agrin expression was significantly altered in diabetic versus non-diabetic MHS animals, whereas collagen I was expressed only in diabetic MHS rats and collagen IV content did not change significantly between the two groups. Upon BSA injection, proteinuria increased markedly and abundant BSA was detected only in kidneys from diabetic MHS rats. BSA injection was associated with changes in intrarenal arteries suggesting vasodilatation, without any influx of inflammatory cells. These data indicate that while MNS rats show marked changes in the glomerular filtration barrier with either age or diabetes, glomerulosclerosis-resistant MHS rats develop only minor diabetes-induced podocyte (and extracellular matrix) alterations, which are not associated with proteinuria unless they are unmasked by an increased workload or removal of the haemodynamic protection.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Nefropatías Diabéticas/fisiopatología , Glomérulos Renales/fisiopatología , Envejecimiento/patología , Envejecimiento/fisiología , Animales , Glucemia/metabolismo , Creatinina/sangre , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Progresión de la Enfermedad , Predisposición Genética a la Enfermedad , Hemoglobina Glucada/metabolismo , Glomérulos Renales/patología , Masculino , Podocitos/fisiología , Proteinuria/fisiopatología , Ratas , Ratas Mutantes , Arteria Renal/fisiopatología , Albúmina Sérica Bovina , Especificidad de la Especie , Aumento de PesoRESUMEN
Experimental and clinical evidence indicates that Endogenous Ouabain (EO) and Adducin polymorphism play a pathogenetic role in hypertension and related organ complications. These effects occur through a complex interaction of genetic molecular mechanisms regulating renal sodium reabsorption and vascular function. The activation of a Na-K ATPase-Src-EGFr-ERK signaling pathway within the restricted membrane subdomains of caveolae by Ouabain has been associated to hypertension and cardiac remodeling. Rostafuroxin (PST 2238) is a novel anti-hypertensive compound able to selectively antagonize EO/Ouabain and Adducin hypertensive effect and Ouabain-induced cardiac hypertrophy in rats. Studies have been conducted in vivo and in a cell-free system to prove that Rostafuroxin exerts its antihypertensive and antihypertrophic effects by antagonizing the Src-dependent signaling triggered by Ouabain. At the vascular level, Rostafuroxin antagonizes the Ouabain-mediated increase of myogenic vascular tone. This peculiar and novel mechanism of action, together with a good tolerability and efficacy both in animal models and hypertensive patients, make Rostafuroxin the prototype of a new class of antihypertensive compounds able to antagonize EO/ Ouabain and Adducin molecular effects.
Asunto(s)
Androstanoles/farmacología , Antihipertensivos/farmacología , Caveolas/metabolismo , Hipertensión/tratamiento farmacológico , Ouabaína/metabolismo , Animales , Proteínas de Unión a Calmodulina/antagonistas & inhibidores , Proteínas de Unión a Calmodulina/metabolismo , Caveolas/efectos de los fármacos , Humanos , Ratas , Transducción de SeñalRESUMEN
The evidence that high levels of endogenous ouabain (EO), a closely related isomer of ouabain, are implicated in human hypertension and cardiac hypertrophy and failure stimulated the pharmacological research for developing novel anti-hypertensive agents active as ouabain antagonists. The pathogenetic mechanisms through which increased EO levels affect cardiovascular system involve the modulation of Na-K ATPase, the key enzyme responsible for renal tubular sodium reabsorption and the activation of signalling transduction pathways implicated in growth-related gene transcription. By studying both genetic and experimental rat models of hypertension and comparing them with humans, our group has demonstrated that elevated levels of circulating EO and the genetic polymorphism of the cytoskeletal protein adducin associate with hypertension and high renal Na-K pump activity. Ouabain itself induces hypertension and up-regulates renal Na-K pump when chronically infused at low doses into rats (OS). In renal cultured cells, either incubated for several days with nanomolar concentrations of ouabain or transfected with the hypertensive adducin genetic variant, the Na-K pump results enhanced. Moreover, both EO and adducin polymorphism affect cardiac complications associated to hypertension, the former through the activation of a signalling transduction pathway. As a consequence, a compound able to interact with the cellular and molecular alterations, sustained by EO or mutated adducin, may represent the suitable treatment for those patients in whom these mechanisms are at work. A new antihypertensive compound, PST 2238, that selectively antagonises the pressor effect and the alteration of renal Na-K pump, sustained both by ouabain and adducin polymorphism, is described. A selective ability of PST 2238 to antagonise the ouabain-induced organ hypertrophy is also documented. The specificity of PST 2238 mechanism of action is supported by the absence of interactions with receptors or hormones involved in blood pressure regulation and by the lack of diuretic activity and diuretic-associated side effects. It is concluded that this compound could be useful for the treatment of those forms of essential hypertension in which renal Na handling alterations and cardiac complications are associated with either increased EO levels and/or adducin polymorphism.
Asunto(s)
Androstanoles/uso terapéutico , Antihipertensivos/uso terapéutico , Hipertensión/tratamiento farmacológico , Ouabaína/antagonistas & inhibidores , Androstanoles/farmacología , Androstanoles/toxicidad , Animales , Antihipertensivos/farmacología , Antihipertensivos/toxicidad , Humanos , Ratas , Ratas Endogámicas SHR , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacosRESUMEN
A primary impairment of the kidney sodium excretion has been documented both in hypertensive patients (EH) and genetic animal models (Milan hypertensive rat [MHS]) carrying mutations of the cytoskeletal protein adducin and/or increased plasma levels of endogenous ouabain (EO). Ouabain (OU) itself induces hypertension in rats and both OU and mutated adducin activate the renal Na/K-ATPase function both in vivo and in cultured renal cells (NRK). A new antihypertensive agent, PST 2238, able to selectively interact with these alterations has been developed. PST lowers blood pressure (BP) by normalizing the expression and activity of the renal Na-K pump selectively in those rat models carrying the adducin mutation (MHS) and/or increased EO levels (OS) at oral doses of 0.1-10 micro g/kg. In NRK cells either transfected with mutated adducin or incubated with 10(-9) M OU, PST normalizes the Na-K pump activity. Recently, an association between EO and cardiac complications has been observed in both EH and rat models consistent with a prohypertrophic activity of OU. OS rats showed a 10% increase of left ventricle and kidney weights as compared with controls, and PST 2238 (1 micro g/kg OS) prevented both ventricle and renal hypertrophy. This effect was associated with the ability of PST to antagonize the OU-dependent activation of growth-related genes, in the membrane subdomains of caveolae. In conclusion, PST is a new antihypertensive agent that may prevent cardiovascular complications associated with hypertension through the selective modulation of the Na-K pump function.
Asunto(s)
Androstanoles/farmacología , Antihipertensivos/farmacología , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacos , Animales , Presión Sanguínea/efectos de los fármacos , Células Cultivadas , Humanos , Riñón/enzimología , Microsomas/enzimología , Ratas , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , TransfecciónRESUMEN
An ouabain-like factor has been implicated repeatedly in salt-sensitive hypertension as a natriuretic agent. However, the response of plasma ouabain-like factor to acute and chronic variation of body sodium is unclear. We studied 138 patients with essential hypertension who underwent an acute volume expansion/contraction maneuver (2 days) and 20 patients who entered a blind randomized crossover design involving chronically controlled sodium intake and depletion (170 to 70 mmol/d; 2 weeks each period). In both studies, plasma levels of ouabain-like factor were higher during sodium depletion (acute: 338.8+/-17.4 and 402.7+/-22.8 pmol/L for baseline and low sodium, respectively, P<0.01; chronic: 320.4+/-32.0 versus 481.0+/-48.1 pmol/L, P=0.01). No significant change in plasma ouabain-like factor was observed after a 2-hour saline infusion (333.4+/-23.9 pmol/L) or controlled sodium (402.1+/-34.9 pmol/L). When patients were divided into salt-sensitive or salt-resistant groups, no differences in plasma ouabain-like factor were observed in the 2 groups at baseline or in response to the 2 protocols: salt resistant (n=69, 340.1+/-25.9 pmol/L) versus salt sensitive (n=69, 337.4+/-23.6 pmol/L) and chronic salt resistant (n=11, 336.0+/-53.2) versus salt sensitive (n=9, 301.1+/-331.4 pmol/L). However, circulating ouabain-like factor was increased by sodium depletion in both groups. These results demonstrate that circulating ouabain-like factor is raised specifically by maneuvers that promote the loss of body sodium. Acute expansion of body fluids with isotonic saline is not a stimulus to plasma ouabain-like factor. Moreover, basal levels of plasma ouabain-like factor do not differ among patients with salt-sensitive or salt-resistant hypertension. Taken together, these new results suggest that ouabain-like factor is involved in the adaptation of humans to sodium depletion and argue against the hypothesis that ouabain-like factor is a natriuretic hormone.
Asunto(s)
Digoxina , Hipertensión/sangre , Hipertensión/metabolismo , Saponinas/sangre , Sodio/metabolismo , Adulto , Presión Sanguínea , Cardenólidos , Estudios Cruzados , Femenino , Humanos , Hipertensión/fisiopatología , Masculino , Renina/sangreRESUMEN
We sought to evaluate the relationships among circulating levels of an endogenous ouabain-like factor (EO) and systemic hemodynamics and left ventricular (LV) geometry in patients with recently diagnosed essential hypertension. We selected 92 never-treated patients with essential hypertension. Blood samples were drawn for estimation of plasma EO (radioimmunoassay) and subjects underwent echocardiographic examination to evaluate LV end-systolic and end-diastolic wall thickness and internal dimensions. LV volumes, stroke volume, cardiac output, total peripheral resistance, LV mass, and relative wall thickness were calculated, and all except the last parameter were indexed by body surface area. LV mass also was indexed by height. On the basis of the values of LV mass index (body surface area or height) and relative wall thickness, subjects were divided into groups with either normal geometry, concentric remodeling, concentric hypertrophy, or eccentric nondilated hypertrophy. In the study population as a whole, circulating EO levels were significantly and directly correlated with mean blood pressure (r = 0.21, P = .048), relative wall thickness (r = 0.34, P = .001), and total peripheral resistance index (r = 0.37, P = .0003). Plasma EO also was significantly and inversely correlated with LV end-diastolic volume index (r = -0.32, P = .002), stroke index (r = -0.34, P = .0009), and cardiac index (r = -0.35, P = .0007). In multiple regression analysis, plasma EO was an independent correlate of total peripheral resistance index, cardiac index, and relative wall thickness. Regardless of the indexation method used for LV mass, plasma EO was higher in patients with concentric remodeling than in those with either normal geometry or concentric hypertrophy. Plasma EO tended to be higher (indexation by body surface area) or was significantly higher (indexation by height) in subjects with concentric remodeling than in those with eccentric nondilated hypertrophy. Patients with concentric remodeling showed the highest total peripheral resistance index and the lowest cardiac index. Our data suggest that EO plays a role in regulating systemic hemodynamics and LV geometry in patients with essential hypertension.
Asunto(s)
Factores Biológicos/sangre , Digoxina , Ecocardiografía , Hemodinámica , Hipertensión/fisiopatología , Saponinas , Función Ventricular Izquierda , Adulto , Cardenólidos , Cardiomegalia/diagnóstico por imagen , Cardiomegalia/etiología , Femenino , Humanos , Hipertensión/complicaciones , Hipertensión/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Remodelación VentricularRESUMEN
A primary renal alteration due to a genetic polymorphism of the cytoskeletal protein adducin associated with an up-regulation of the renal Na-K pump and increased levels of ouabainlike factor (OLF) has been identified as a possible causes of hypertension in Milan rats (MHS). This adducin polymorphism has also been found to be associated with hypertension and the blood pressure changes related to renal Na handling in humans and increased OLF levels have been found in a relevant portion of hypertensive patients. Increased activity and expression of the Na-K pump has also been observed under the following 'in vitro' and 'in vivo' conditions: rat renal cells transfected with the 'hypertensive' variant of adducin, as compared with normal cells; normal rat renal cells incubated for 5 days with 10(-9) M ouabain and normal rats made hypertensive by a chronic infusion of low doses of ouabain (OS rats). An up-regulation of the Na-K pump seems therefore to be a common biochemical alteration induced both by an adducin polymorphism and/or chronic exposure to low concentrations of ouabain (or OLF). A new antihypertensive compound, PST 2238, that selectively antagonizes the pressor effect and the alteration of the renal Na-K pump induced both by an adducin polymorphism and OLF, is described. The ability of PST 2238 to lower blood pressure and normalize the Na-K pump both in MHS and OS rats suggests that this compound could be useful in the treatment of those forms of essential hypertension in which renal Na-handling alterations are associated with either adducin polymorphisms and/or increased OLF levels.
Asunto(s)
Androstanoles/farmacología , Antihipertensivos/farmacología , Hipertensión/tratamiento farmacológico , Hipertensión/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Humanos , Técnicas In VitroRESUMEN
This review focuses on the most recent data published in the field of the sodium-potassium pump inhibitors regarding the hypothetical structure, the secretory stimuli and the pathophysiological implications for particular diseases, such as hypertension. On the basis of the findings published so far, we reconsider and discuss the 'natriuretic hypothesis' for explaining the role of the endogenous sodium-potassium ATPase inhibitor. We propose the ouabain-like factor as a modulator of the renal sodium-potassium pump, that can be considered as a new pharmacological target for hypertension therapy.
Asunto(s)
Factores Biológicos/fisiología , Digoxina , Natriuréticos/fisiología , Saponinas , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Cardenólidos , Humanos , Hipertensión Renal/tratamiento farmacológico , Hipertrofia Ventricular Izquierda/etiología , Factores de RiesgoAsunto(s)
Presión Sanguínea/genética , Presión Sanguínea/fisiología , Proteínas de Unión a Calmodulina/fisiología , Natriuresis/genética , Natriuresis/fisiología , Animales , Proteínas de Unión a Calmodulina/genética , Marcadores Genéticos , Humanos , Hipertensión/genética , Ratas , Ratas Endogámicas SHR/genéticaRESUMEN
Adducin point mutations are associated with genetic hypertension in Milan hypertensive strain (MHS) rats and in humans. In transfected cells, adducin affects actin cytoskeleton organization and increases the Na(+)-K(+)-pump rate. The present study has investigated whether rat and human adducin polymorphisms differently modulate rat renal Na(+)-K(+)-ATPase in vitro. We report the following. 1) Both rat and human adducins stimulate Na(+)-K(+)-ATPase activity, with apparent affinity in tens of nanomolar concentrations. 2) MHS and Milan normotensive strain (MNS) adducins raise the apparent ATP affinity for Na(+)-K(+)-ATPase. 3) The mechanism of action of adducin appears to involve a selective acceleration of the rate of the conformational change E(2) (K) --> E(1) (Na) or E(2)(K). ATP --> E(1)Na. ATP. 4) Apparent affinities for mutant rat and human adducins are significantly higher than those for wild types. 5) Recombinant human alpha- and beta-adducins stimulate Na(+)-K(+)-ATPase activity, as do the COOH-terminal tails, and the mutant proteins display higher affinities than the wild types. 6) The cytoskeletal protein ankyrin, which is known to bind to Na(+)-K(+)-ATPase, also stimulates enzyme activity, whereas BSA is without effect; the effects of adducin and ankyrin when acting together are not additive. 7) Pig kidney medulla microsomes appear to contain endogenous adducin; in contrast with purified pig kidney Na(+)-K(+)-ATPase, which does not contain adducin, added adducin stimulates the Na(+)-K(+)-ATPase activity of microsomes only about one-half as much as that of purified Na(+)-K(+)-ATPase. Our findings strongly imply the existence of a direct and specific interaction between adducin and Na(+)-K(+)-ATPase in vitro and also suggest the possibility of such an interaction in intact renal membranes.
Asunto(s)
Proteínas de Unión a Calmodulina/fisiología , Hipertensión/genética , Hipertensión/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/fisiología , Animales , Ancirinas/farmacología , Proteínas de Unión a Calmodulina/genética , Proteínas de Unión a Calmodulina/aislamiento & purificación , Proteínas de Unión a Calmodulina/farmacología , Eritrocitos/enzimología , Eritrocitos/metabolismo , Humanos , Riñón/enzimología , Riñón/metabolismo , Mutación/fisiología , Ratas , Ratas Endogámicas , Proteínas Recombinantes/farmacología , Albúmina Sérica Bovina/farmacología , ATPasa Intercambiadora de Sodio-Potasio/aislamiento & purificación , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Many patients with essential hypertension (EH) exhibit increased left ventricular mass. Similarly, elevated circulating levels of an endogenous ouabainlike factor (OLF) have been described in some but not all patients with EH. Moreover, ouabain has a hypertrophic influence on isolated cardiac myocytes. Accordingly, we investigated relationships among plasma OLF, left ventricular mass, and cardiac function in patients with EH. Plasma OLF was determined in 110 normotensive subjects and 128 patients with EH. Echocardiographic parameters and humoral determinants were measured in EH. Plasma OLF levels were increased (P<0.0001) in patients with EH (377+/-19 pmol/L) versus normotensive (253+/-53 pmol/L) subjects. The distribution of plasma OLF was unimodal in normotensives, whereas it was bimodal in EH. Twenty-four-hour diastolic ambulatory blood pressure was slighter higher in EH with high OLF compared with EH with normal OLF (93.2+/-1.14 versus 89.4+/-1.33 mm Hg, P=0.03). Left ventricular mass index and stroke volume in EH with high OLF were greater than in EH with normal OLF (101.9+/-3.3 versus 86.1+/-2.5 g/m(2), P=0.0003, and 57.10+/-1.48 versus 52.30+/-1.14 mL/m(2), P=0. 02, respectively), although heart rate was slower (74.2+/-1.3 versus 80.5+/-1.3 bpm, P=0.005). Multiple regression analysis that tested the influence of body mass index, age, gender, 24-hour blood pressure, and OLF on left ventricular mass revealed independent contributions of systolic (13.2%) and diastolic (12.4%) blood pressure and plasma OLF (11.6%) to left ventricular mass. We conclude that approximately 50% of patients with uncomplicated EH have elevated-high circulating OLF levels, higher diastolic blood pressure, greater left ventricular mass and stroke volume, and reduced heart rate. We propose that the OLF affects cardiovascular function and structure and should be considered as a factor that contributes to the risk of morbid events.
Asunto(s)
Hipertensión/fisiopatología , Hipertrofia Ventricular Izquierda , Ouabaína/metabolismo , Volumen Sistólico , Adulto , Femenino , Humanos , Hipertensión/metabolismo , Masculino , Persona de Mediana Edad , Análisis de Regresión , Función VentricularRESUMEN
A genetic alteration in the adducin genes is associated with hypertension and up-regulation of the expression of renal Na, K-ATPase in Milan-hypertensive (MHS) rats, in which increased ouabain-like factor (OLF) levels are also observed. PST 2238, a new antihypertensive compound that antagonizes the pressor effect of ouabain in vivo and normalizes ouabain-dependent up-regulation of the renal Na-K pump, was evaluated for its ability to lower blood pressure and regulate renal Na,K-ATPase activity in MHS genetic hypertension. In this study, we show that PST 2238, given orally at very low doses (1 and 10 microg/kg for 5-6 weeks), reduced the development of hypertension in MHS rats and normalized the increased renal Na,K-ATPase activity and mRNA levels, whereas it did not affect either blood pressure or Na,K-ATPase in Milan-normotensive (MNS) rats. In addition, a similar antihypertensive effect was observed in adult MHS rats after a short-term treatment. In cultured rat renal cells with increased Na-K pump activity at Vmax due to overexpression of the hypertensive variant of adducin, 5 days of incubation with PST 2238 (10(-10-)-10(-9) M) lowered the pump rate to the level of normal wild-type cells, which in turn were not affected by the drug. In conclusion, PST 2238 is a very potent compound that in MHS rats reduces blood pressure and normalizes Na-K pump alterations caused by a genetic alteration of the cytoskeletal adducin. Because adducin gene mutations have been associated with human essential hypertension, it is suggested that PST 2238 may display greater antihypertensive activity in those patients carrying such a genetic alteration.
Asunto(s)
Androstanoles/uso terapéutico , Antihipertensivos/uso terapéutico , Hipertensión/tratamiento farmacológico , Médula Renal/efectos de los fármacos , ATPasa Intercambiadora de Sodio-Potasio/biosíntesis , Animales , Presión Sanguínea/efectos de los fármacos , Proteínas de Unión a Calmodulina/biosíntesis , Proteínas de Unión a Calmodulina/genética , Células Cultivadas , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Hipertensión/enzimología , Hipertensión/genética , Médula Renal/enzimología , Masculino , Mutación , Ouabaína/farmacología , ARN Mensajero/análisis , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/genética , TransfecciónRESUMEN
Endogenous ouabain-like factor (OLF) is present in mammal tissues and after standardized extraction procedure can be similarly quantified by two independent assays: RIA and Na-KATPase inhibition. OLF was quantified both from plasma and tissues obtained from MHS hypertensive and MNS normotensive rats, maintained under the same environmental and dietary conditions, and from plasma of healthy volunteers and essential hypertensive patients. OLF biochemical characterization shows that it behaves like ouabain except for a 1000-fold higher affinity for the ouabain low-affinity Na-KATPase isoforms than ouabain. Tissue and plasma levels of OLF are higher in MHS than in MNS rats and are not influenced by exogenous OLF sources. Plasma OLF is also increased in a subgroup of hypertensive patients. Both in rats and humans a primary cell membrane alteration affecting ion transports seems to be linked to the increased levels of OLF. An antihypertensive compound which selectively antagonizes the pressor effect of OLF and corrects the ion transport defect is under development and can represent a new pharmacological approach to the treatment of hypertension.
Asunto(s)
Factores Biológicos/metabolismo , Digoxina , Inhibidores Enzimáticos/metabolismo , Hipertensión/genética , Saponinas , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Glándulas Suprarrenales/metabolismo , Animales , Factores Biológicos/aislamiento & purificación , Factores Biológicos/farmacología , Cardenólidos , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Humanos , Hipertensión/tratamiento farmacológico , Hipertensión/metabolismo , Hipotálamo/metabolismo , Hipófisis/metabolismo , Radioinmunoensayo , Ratas , Ratas Endogámicas SHR , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidoresRESUMEN
The inhibition of the long-term pressor effect of ouabain may be useful for the therapy of essential hypertension. Here, for the first time, a selective inhibitor of the ouabain pressor effect is described. In vitro, 17beta-(3-furyl)-5beta-androstane-3beta, 14beta, 17alpha-triol (PST 2238) displaced ouabain from its binding sites on purified sodium, potassium ATPase enzyme (Na-K ATPase) (IC50 1.7 x 10(-6) M) without interacting with other receptors involved in blood pressure regulation or hormonal control. In cultured renal cells, incubation with ouabain (10(-10) to 10(-8) M) for 5 days stimulated the Na-K pump at Vmax, whereas PST 2238 showed the same effect at micromolar concentration. The ouabain-dependent increase in the Na-K pump rate was abolished by PST 2238 at concentrations from 10(-14) to 10(-9) M. In rats made hypertensive by chronic infusion of 50 microg/kg/day of ouabain, PST 2238 given p.o at very low doses (0.1-1 microg/kg/day for 4 weeks) abolished the increase in blood pressure and renal Na-K ATPase activity caused by ouabain. PST 2238 did not affect either blood pressure or renal Na-K ATPase activity in normotensive rats. In conclusion, PST 2238 is a very potent compound that normalizes both blood pressure and alterations in the Na-K pump caused by ouabain. Thus it represents the prototype of a new class of antihypertensive drugs that could be effective in forms of hypertension sustained by the concomitant increase of endogenous ouabain levels and alterations in the Na-K pump.
Asunto(s)
Androstanoles/farmacología , Antihipertensivos/farmacología , Cardiotónicos/antagonistas & inhibidores , Ouabaína/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/efectos de los fármacos , Androstanoles/metabolismo , Animales , Antihipertensivos/metabolismo , Cardiotónicos/metabolismo , Células Cultivadas/efectos de los fármacos , Masculino , Ouabaína/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
The resolution of controversies that concern the detectability of an endogenous ouabain-like factor (OLF) in mammalian tissues and plasma was approached by the application of a standardized method for its extraction and quantification. Two independent assays were used to quantify the OLF: (1) a radioimmunoassay, which used a polyclonal anti-ouabain antiserum, and (2) a radioenzymatic assay based on the inhibition of dog kidney Na+,K+-ATPase. Plasma and tissues were obtained from the Milan hypertensive strain (MHS) and the Milan normotensive strain (MNS) of rats and from healthy human volunteers. Results indicate that (1) a single high-performance liquid chromatography (HPLC) fraction identical to that of ouabain was identified by both assay methods in the rat hypothalamus and hypophysis and in both rat and human plasma; (2) dilution curves of OLF and standard ouabain were parallel and with a similar Kd, both in radioimmunoassay (3 nmol/L) and ATPase assay (14 nmol/L); (3) after HPLC, OLF was similarly quantified by the two methods in the hypothalamus, hypophysis, adrenals, and plasma of rats and in human plasma; (4) OLF was present in larger amounts in the hypothalamus, hypophysis, and plasma of MHS rats than that of MNS rats; (5) the HPLC fraction of human plasma was quantified similarly by both assays (range, 60 to 150 pmol/L); (6) recovery of standard ouabain in pre-HPLC plasma extracts was approximately 90%; and (7) pre-HPLC OLF concentrations in human plasma ranged between 0.05 and 0.75 nmol/L. Rat cerebral tissues and both rat and human plasma contained measurable amounts of OLF, which were quantified similarly by radioimmunoassay and ATPase assay, both before and after HPLC fractionation. The increased MHS tissue and plasma levels of OLF are in keeping with the pathogenetic role of this factor in MHS hypertension.
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Factores Biológicos/análisis , Factores Biológicos/sangre , Digoxina , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/sangre , Saponinas , Glándulas Suprarrenales/química , Animales , Cardenólidos , Cromatografía Líquida de Alta Presión , Perros , Humanos , Hipotálamo/química , Sueros Inmunes/inmunología , Masculino , Métodos , Concentración Osmolar , Ouabaína/análisis , Ouabaína/inmunología , Hipófisis/química , Radioinmunoensayo , Ratas , Ratas Endogámicas , ATPasa Intercambiadora de Sodio-Potasio/análisis , Extractos de Tejidos/químicaRESUMEN
Milan hypertensive rats (MHS) develop hypertension because of a primary renal alteration. Both apical and basolateral sodium transport are faster in membrane vesicles derived from renal tubules of MHS than in those of Milan normotensive control rats (MNS). These findings suggest that the increased renal sodium retention and concomitant development of hypertension in MHS may be linked to an altered transepithelial sodium transport. Since this transport is mainly under the control of the Na-K pump, we investigated whether an alteration of the enzymatic activity and/or protein expression of the renal Na,K-ATPase is detectable in prehypertensive MHS. We measured the Na,K-ATPase activity, Rb+ occlusion, turnover number, alpha 1- and beta 1-subunit protein abundance, and alpha 1 and beta 1 mRNA levels in microsomes from renal outer medulla of young (prehypertensive) and adult (hypertensive) MHS and in age-matched MNS. In both young and adult MHS, the Na,K-ATPase activity was significantly higher because of an enhanced number of active pump sites, as determined by Rb+ occlusion maximal binding. The higher number of pump sites was associated with a significant pretranslational increase of alpha 1 and beta 1 mRNA levels that preceded the development of hypertension in MHS. Since a molecular alteration of the cytoskeletal protein adducin is genetically associated with hypertension in MHS and is able to affect the actin-cytoskeleton and Na-K pump activity in transfected renal cells, we propose that the in vivo upregulation of Na-K pump in MHS is primary and linked to a genetic alteration of adducin.
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Hipertensión/enzimología , Hipertensión/genética , Médula Renal/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Envejecimiento/metabolismo , Animales , Técnicas de Cultivo , Microsomas/metabolismo , ARN Mensajero/metabolismo , Ratas , Sodio/metabolismoRESUMEN
AIM: Previous studies have demonstrated that the hypothalamus of the adult Milan hypertensive rat strain (MHS) contains a higher proportion of ouabain-like factor than Milan normotensive (MNS) controls. The present study was designed to demonstrate that the rat standard diet contains a ouabain-like factor similar to that extracted from rat tissue and to investigate the influence of low or high dietary ouabain-like factor content on tissue ouabain-like factor levels at different ages in MHS and MNS rats. MATERIALS AND METHODS: MHS and MNS rats were reared on two controlled batches of standard rat diet containing low (batch A 0.09 mu g/kg) and high (batch B 0.7 mu g/kg) concentrations of ouabain-like factor. The mothers of these rats had also been fed with the diet throughout pregnancy and lactation. The hypothalamic content of ouabain-like factor was measured in both strains at 21, 30 and 90 days of age by high performance liquid chromatography fractionation. RESULTS: (1) The dietary ouabain-like factor content did not influence either the hypothalamic ouabain-like factor yield or systolic blood pressure, either in MHS or MNS rats. (2) As a function of age, the hypothalamic ouabain-like factor content was constant between 21 and 30 days of age in MHS rats, and then decreased by 60% at 90 days. In MNS rats, ouabain-like factor was decreased by 80 and 90%, respectively, at 30 and 90 days, compared to the age of 21 days. (3) At the age of 21 days, MHS rats had 30% lower levels of ouabain-like factor than MNS rats, but 60% higher levels at 30 and 90 days. CONCLUSIONS: Hypothalamic ouabain-like factor and systolic blood pressure are not influenced by dietary ouabain-like factor, thus excluding a process of passive tissue accumulation. Different mechanisms regulate the age-dependent endogenous ouabain-like factor production and accumulation in MHS and MNS rats, suggesting that the maintenance of higher ouabain-like factor levels in MHS than in MNS at the age of 30 and 90 days contributes to the development and maintenance of hypertension in this strain.
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Factores Biológicos/metabolismo , Dieta , Digoxina , Hipertensión/metabolismo , Hipotálamo/metabolismo , Saponinas , Factores de Edad , Animales , Presión Sanguínea , Cardenólidos , Hipertensión/fisiopatología , RatasRESUMEN
The Milan hypertensive strain of rats (MHS) develops a genetic form of renal hypertension that, when compared to its normotensive control (MNS), shows renal dysfunction similar to that of a subset of human patients with primary hypertension. MHS and MNS were shown to be homozygous by multilocus minisatellite analysis and monolocus microsatellite markers. We show here that one point mutation in each of two genes coding for the membrane skeleton protein adducin is associated with blood pressure in the Milan strain of rats. Adducin is a heterodimer formed by alpha and beta subunits that promotes the assembly of actin with spectrin. MHS and MNS differ, respectively, by the amino acids Y and F at position 316 of the alpha subunit. In the beta-adducin locus, MHS is always homozygous for R at position 529 while in MNS either R or Q occurs in that position. The R/Q heterozygotes showed lower blood pressure than any of the homozygotes. In vitro phosphorylation studies suggest that both of these amino acid substitutions occur within protein kinase recognition sites. Analysis of an F2 generation demonstrated that Y alleles segregated with a significant increment in blood pressure. This effect is modulated by the presence of the R allele of the beta subunit. Taken together, these findings strongly support a role for adducin polymorphisms in causing variation of blood pressure in the Milan strain of rats.