RESUMEN
Calcineurin dephosphorylates nuclear factor of activated T cells transcription factors, thereby facilitating T cell-mediated immune responses. Calcineurin inhibitors are instrumental for immunosuppression after organ transplantation but may cause side effects, including hypertension and electrolyte disorders. Kidneys were recently shown to display activation of the furosemide-sensitive Na-K-2Cl cotransporter (NKCC2) of the thick ascending limb and the thiazide-sensitive Na-Cl cotransporter (NCC) of the distal convoluted tubule upon calcineurin inhibition using cyclosporin A (CsA). An involvement of major hormones like angiotensin II or arginine vasopressin (AVP) has been proposed. To resolve this issue, the effects of CsA treatment in normal Wistar rats, AVP-deficient Brattleboro rats, and cultured renal epithelial cells endogenously expressing either NKCC2 or NCC were studied. Acute administration of CsA to Wistar rats rapidly augmented phosphorylation levels of NKCC2, NCC, and their activating kinases suggesting intraepithelial activating effects. Chronic CsA administration caused salt retention and hypertension, along with stimulation of renin and suppression of renal cyclooxygenase 2, pointing to a contribution of endocrine and paracrine mechanisms at long term. In Brattleboro rats, CsA induced activation of NCC, but not NKCC2, and parallel effects were obtained in cultured cells in the absence of AVP. Stimulation of cultured thick ascending limb cells with AVP agonist restored their responsiveness to CsA. Our results suggest that the direct epithelial action of calcineurin inhibition is sufficient for the activation of NCC, whereas its effect on NKCC2 is more complex and requires concomitant stimulation by AVP.
Asunto(s)
Inhibidores de la Calcineurina/toxicidad , Ciclosporina/toxicidad , Células Epiteliales/efectos de los fármacos , Inmunosupresores/toxicidad , Túbulos Renales Distales/efectos de los fármacos , Asa de la Nefrona/efectos de los fármacos , Miembro 1 de la Familia de Transportadores de Soluto 12/agonistas , Animales , Arginina Vasopresina/farmacología , Células Cultivadas , Ciclooxigenasa 2/metabolismo , Células Epiteliales/metabolismo , Hipertensión/inducido químicamente , Hipertensión/metabolismo , Hipertensión/fisiopatología , Túbulos Renales Distales/metabolismo , Túbulos Renales Distales/fisiopatología , Asa de la Nefrona/metabolismo , Asa de la Nefrona/fisiopatología , Masculino , Ratas Brattleboro , Ratas Wistar , Renina/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/genética , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismo , Miembro 3 de la Familia de Transportadores de Soluto 12/agonistas , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Factores de Tiempo , Equilibrio Hidroelectrolítico/efectos de los fármacosRESUMEN
AIM: This study was designed to assess the potential of the continuous erythropoietin receptor activator (C.E.R.A.) to correct anemia at extended administration intervals in erythropoiesis-stimulating agent-naive patients with chronic kidney disease (CKD) not on dialysis and to determine its optimal starting dose. METHODS: Patients were assigned to subcutaneous C.E.R.A. at 3 doses: 0.15, 0.30 and 0.60 microg/kg/wk. During the first 6 weeks, dose adjustments for efficacy were not permitted in order to assess dose response. Within each of the 3 dose groups, patients were randomized to receive C.E.R.A. QW, Q2W or Q3W; the total dose during the first 6 weeks was the same for a particular dose group across the frequency subgroups. During the next 12 weeks, dose was adjusted according to predefined hemoglobin (Hb) criteria. The primary efficacy parameter was change in Hb over 6 weeks, estimated from regression analysis between baseline and the point at which the patient received a dose change or blood transfusion. It therefore provided an estimate of Hb increase based on starting dose. Other endpoints included Hb response rate (proportion of patients with a Hb increase > 1.0 g/dl on 2 consecutive occasions). A 1-year extension period investigated long term tolerability and efficacy. RESULTS: A dose-dependent relationship was noted in the mean change in Hb from baseline over 6 weeks (p < 0.0001), independent of administration schedule (p = 0.9201). There was also a significant relationship between Hb change and median serum C.E.R.A. concentration (p < 0.0001). Erythropoietic responses were sustained in all groups with mean changes from baseline in Hb > 1.2 g/dl observed at doses > or = 0.30 microg/kg/wk. Hb response rate increased with increasing dose: 67, 72 and 90% with C.E.R.A. 0.15, 0.30 and 0.60 microg/kg/wk, respectively. Generally, the median Hb response time was faster with increasing dose (89, 43 and 31 days, respectively). Response was unrelated to administration frequency. Stable Hb concentrations were maintained throughout the 1-year extension period. C.E.R.A. was generally well tolerated, and the most common adverse events were hypertension, urinary tract infection and renal failure. CONCLUSIONS: C.E.R.A. corrected anemia and maintained sustained and stable control of Hb over 1 year. These results suggest that 0.60 microg/kg subcutaneous C.E.R.A. given twice monthly is a suitable starting dose for further investigation in Phase III studies in patients with CKD not on dialysis.
Asunto(s)
Anemia/tratamiento farmacológico , Anemia/etiología , Eritropoyetina/administración & dosificación , Fallo Renal Crónico/complicaciones , Polietilenglicoles/administración & dosificación , Diálisis Renal , Adulto , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Área Bajo la Curva , Biomarcadores/sangre , Relación Dosis-Respuesta a Droga , Eritropoyesis/efectos de los fármacos , Eritropoyetina/efectos adversos , Eritropoyetina/sangre , Femenino , Ferritinas/sangre , Ferritinas/efectos de los fármacos , Estudios de Seguimiento , Hemoglobinas/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Polietilenglicoles/efectos adversos , Proteínas Recombinantes , Análisis de Regresión , Factores de Tiempo , Resultado del TratamientoRESUMEN
With-No-Lysine [K] (WNKs) are a recently discovered family of serine/threonine protein kinases that contain a uniquely structured catalytic domain. Mutations in the genes encoding two family members, WNK1 and WNK4, cause a chloride-dependent, thiazide-sensitive inherited syndrome of hypertension and hyperkalemia. Over the past 5 years, physiologic studies have demonstrated that these proteins regulate transcellular and paracellular epithelial ion flux. In this mini review, we discuss WNK1 and WNK4 gene products and their regulatory effects on sodium chloride and potassium handling in the aldosterone-sensitive distal nephron. Experimental observations regarding the effects of these proteins on transport processes mediated by the thiazide-sensitive Na-Cl co-transporter, the epithelial sodium channel, the renal outer medullary potassium channel, and the paracellular pathway integrate into a model that suggests an essential role for WNKs in coordinating renal Na-Cl reabsorption and K(+) secretion.
Asunto(s)
Túbulos Renales Distales/metabolismo , Potasio/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Cloruro de Sodio/metabolismo , Aldosterona/fisiología , Transporte Biológico/fisiología , Regulación Enzimológica de la Expresión Génica , Humanos , Hipertensión Renal/genética , Hipertensión Renal/metabolismo , Hipertensión Renal/fisiopatología , Péptidos y Proteínas de Señalización Intracelular , Antígenos de Histocompatibilidad Menor , Mutación , Nefronas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
We sought to assess whether the distal convoluted tubule (DCT) segment of the rabbit nephron expresses a functional epithelial sodium channel. First, the transepithelial voltage (V(te), lumen vs. bath) was measured in isolated perfused DCT segments (assessed separately in the upstream half and the downstream half of the DCT). V(te) was zero and not affected by amiloride or barium in the upstream DCT. V(te) was sometimes negative in the downstream DCT and depolarized by amiloride and hyperpolarized by barium, suggesting inclusion of connecting tubule (CNT) cells. To determine expression of epithelial sodium channel (ENaC) mRNA subunits by the upstream DCT, rabbit alpha-, beta-, and gamma-ENaC cDNA fragments were cloned and primers were selected for single-nephron RT-PCR analysis. Although alpha-ENaC was expressed by the DCT, beta- and gamma-ENaC were not detected in the DCT. In contrast, the CNT, CCD, and outer medullary collecting duct (OMCD) expressed all three subunits. Nedd4 was also not detected in the DCT but was expressed by the CNT, CCD, and OMCD. When upstream DCT fragments were grown to confluent monolayers in primary culture, the epithelia exhibited negative voltages and high transepithelial resistances and expressed mRNA for all three ENaC subunits as well as for Nedd4. The absence of a negative voltage and failure to detect transcript for beta- and gamma-ENaC and Nedd4 in the native rabbit DCT suggest that the sodium channel is not a significant pathway for sodium absorption by this segment. The phenotype conversion observed when DCT cells are grown in culture does not rule out the possibility that there may be conditions in which the DCT in the intact kidney expresses sodium channel activity. The results are consistent with the notion that DCT sodium transport is predominantly, if not exclusively, electroneutral.
Asunto(s)
Túbulos Renales Distales/metabolismo , Canales de Sodio/metabolismo , Ubiquitina-Proteína Ligasas , Animales , Northern Blotting , Proteínas de Unión al Calcio/genética , Clonación Molecular , Complejos de Clasificación Endosomal Requeridos para el Transporte , Canales Epiteliales de Sodio , Técnicas In Vitro , Riñón/metabolismo , Ligasas/genética , Datos de Secuencia Molecular , Ubiquitina-Proteína Ligasas Nedd4 , Nefronas , Perfusión , ARN Mensajero/metabolismo , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales de Sodio/genética , Distribución TisularRESUMEN
Treatment of congestive heart failure has changed dramatically during the past 20 years, but diuretic drugs remain an essential component. Diuretics are essential despite the fact that these drugs stimulate the renin-angiotensin-aldosterone (RAA) axis and lead to adaptive responses that may be counterproductive. In this paper, new diuretic drugs and new uses of older drugs are discussed. These approaches emphasize low-dose combination therapy and may prove superior to traditional approaches that rely exclusively on loop diuretics. Such approaches aim to prevent adverse compensatory processes that appear to result from chronic diuretic treatment. These include acute and chronic increases in plasma renin activity and stimulation of the sympathetic nervous system, both of which increase afterload and may tend to increase mortality. They also include adaptive changes in nephron structure and function resulting from diuretic-induced increases in distal sodium load and diuretic-induced neurohormonal stimulation. These adaptations blunt the effectiveness of diuretic therapy. Diuretic strategies that rely on combinations of diuretics are emphasized as a method to prevent resistance. If diuretic resistance does develop, higher-dose combination regimens, continuous diuretic infusions and mechanical ultrafiltration can be used to overcome diuretic adaptations and restore diuretic efficacy. The goal of reducing the extracellular fluid volume with the least stimulation of the RAA axis and minimal changes in nephron architecture can be achieved in many patients.
Asunto(s)
Benzotiadiazinas , Bumetanida/uso terapéutico , Diuréticos/uso terapéutico , Furosemida/uso terapéutico , Insuficiencia Cardíaca/tratamiento farmacológico , Inhibidores de los Simportadores del Cloruro de Sodio/uso terapéutico , Resistencia a Medicamentos , Sinergismo Farmacológico , Quimioterapia Combinada , Espacio Extracelular/efectos de los fármacos , Humanos , Sistema Renina-Angiotensina/efectos de los fármacos , Cloruro de Sodio DietéticoRESUMEN
Elucidation of the gene defects responsible for many disorders of renal fluid and electrolyte homeostasis has provided new insights into normal and abnormal physiology. Identifying the causes of Gitelman's and Bartter's syndromes has greatly enhanced our understanding of ion transport by thick ascending limb and distal convoluted tubule cells. Despite this information, several phenotypic features of these diseases remain confusing, even in the face of molecular insight. Paramount among these are disorders of divalent cation homeostasis. Bartter's syndrome is caused by dysfunction of thick ascending limb cells. It is associated with calcium wasting, but magnesium wasting is usually mild. Loop diuretics, which inhibit ion transport by thick ascending limb cells, markedly increase urinary excretion of both calcium and magnesium. In contrast, Gitelman's syndrome is caused by dysfunction of the distal convoluted tubule. Hypocalciuria and hypomagnesemia are universal parts of this disorder. Yet although thiazide diuretics, which inhibit ion transport by distal convoluted tubule cells, reduce urinary calcium excretion, they have minimal effects on urinary magnesium excretion, when given acutely. This review proposes mechanisms that may account for the differences between the effects of diuretic drugs and the phenotypic features of Gitelman's and Bartter's syndromes. These mechanisms are based on recent insights from another inherited disease of ion transport, inherited magnesium wasting, and from a review of the chronic effects of diuretic drugs in animals and people.
Asunto(s)
Cationes Bivalentes/metabolismo , Nefronas/metabolismo , Animales , Síndrome de Bartter/metabolismo , Transporte Biológico , Calcio/metabolismo , Humanos , Magnesio/metabolismoRESUMEN
The distal tubule of the mammalian kidney, defined as the region between the macula densa and the collecting duct, is morphologically and functionally heterogeneous. This heterogeneity has stymied attempts to define functional properties of individual cell types and has led to controversy concerning mechanisms and regulation of ion transport. Recently, molecular techniques have been used to identify and localize ion transport pathways along the distal tubule and to identify human diseases that result from abnormal distal tubule function. Results of these studies have clarified the roles of individual distal cell types. They suggest that the basic molecular architecture of the distal nephron is surprisingly similar in mammalian species investigated to date. The results have also reemphasized the role played by the distal tubule in regulating urinary potassium excretion. They have clarified how both peptide and steroid hormones, including aldosterone and estrogen, regulate ion transport by distal convoluted tubule cells. Furthermore, they highlight the central role that the distal tubule plays in systemic calcium homeostasis. Disorders of distal nephron function, such as Gitelman's syndrome, nephrolithiasis, and adaptation to diuretic drug administration, emphasize the importance of this relatively short nephron segment to human physiology. This review integrates molecular and functional results to provide a contemporary picture of distal tubule function in mammals.
Asunto(s)
Enfermedades Renales/fisiopatología , Túbulos Renales Distales/anatomía & histología , Túbulos Renales Distales/fisiología , Animales , Calcio/metabolismo , Cloruros/metabolismo , Desarrollo Embrionario y Fetal , Humanos , Túbulos Renales Distales/fisiopatología , Mamíferos , Potasio/metabolismo , Sodio/metabolismoRESUMEN
Diuretic drugs are usually effective treatment for edema when used judiciously. However, some patients become resistant to their effects. Adaptation to diuretic drugs and diuretic resistance may be caused by similar mechanisms. Diuretic adaptations can be classified as those that occur during diuretic action, those that cause sodium retention in the short term (causing 'post-diuretic NaCl retention'), and those that increase sodium retention chronically (the 'braking phenomenon'). Recent experimental work has indicated ways in which kidneys adapt to chronic diuretic treatment. First, nephron segments downstream from the site of diuretic action increase NaCl reabsorption during diuretic administration because delivered NaCl load is increased. Second, when diuretic concentrations in the tubule decline, the kidney tubules act to retain Na until the next dose of diuretic is administered. Third, the ability of the diuretic to increase renal NaCl excretion declines over time, an effect that results both from depletion of the extracellular fluid volume and from structural and functional changes of kidney tubules themselves. These adaptations all increase the rate of NaCl reabsorption and blunt the effectiveness of diuretic therapy. Many times, a second diuretic drug is effective treatment for diuretic resistance. Recent experimental results suggest that a second drug may act synergistically because it blocks the adaptive processes limiting the effectiveness of the first diuretic. Based on an understanding of the mechanisms of diuretic adaptation and resistance, treatment regimens can be designed to block specific adaptive mechanisms and improve diuretic effectiveness.
Asunto(s)
Diuréticos/administración & dosificación , Adaptación Fisiológica , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Humanos , Riñón/efectos de los fármacos , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
The mammalian distal nephron plays a pivotal role in adjusting urinary sodium excretion. Successive portions of the renal tubule are formed to adapt to this function, and an axial heterogeneity of the distal segments has been defined. The specific transport properties of these epithelia are accomplished by the expression of proteins (cotransporters, exchangers, channels) governing the movement of ions on either cell side. Molecular cloning of these proteins has had a marked impact on the study of their localization and function in the healthy and diseased kidney. Electroneutral cation-chloride cotransporters [Na(K)CC] have been localized to the thick ascending limb and the distal convoluted tubule using specific probes. Proteins implicated in the function of aldosterone target cells, such as the epithelial Na(+) channel (ENaC), the mineralocorticoid receptor (MR) and 11beta-hydroxysteroid dehydrogenase type 2 (11HSD2), an enzyme that confers mineralocorticoid specificity, have been found in the terminal portion of the nephron and the collecting duct. A mineralocorticoid-sensitive component of thiazide-sensitive NaCl transport has been identified in the distal convoluted tubule. Analysis of the ontogeny of these proteins in the maturing kidney has provided a detailed picture of epithelial differentiation and morphological specialization of the renal tubule. The study of mutations of the proteins related with NaCl transport has led to the identification of the molecular causes of inherited human diseases associated with hypo- or hypertension, and the respective sites of an impaired ion transport could be mapped to the renal tubule.
Asunto(s)
Proteínas Portadoras/fisiología , Nefronas/química , Nefronas/fisiología , Sodio/fisiología , Animales , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , HumanosRESUMEN
Gitelman's syndrome is an autosomal recessive disorder of salt wasting and hypokalemia caused by mutations in the thiazide-sensitive Na-Cl cotransporter. To investigate the pathogenesis of Gitelman's syndrome, eight disease mutations were introduced into the mouse thiazide-sensitive Na-Cl cotransporter and studied by functional expression in Xenopus oocytes. Sodium uptake into oocytes that expressed the wild-type clone was more than sevenfold greater than uptake into control oocytes. Uptake into oocytes that expressed the mutated transporters was not different from control. Hydrochlorothiazide reduced Na uptake by oocytes expressing the wild-type gene to control values but had no effect on oocytes expressing the mutant clones. Western blots of oocytes injected with the wild-type clone showed bands representing glycosylated (125 kDa) and unglycosylated (110 kDa) forms of the transport protein. Immunoblot of oocytes expressing the mutated clones showed only the unglycosylated protein, indicating that protein processing was disrupted. Immunocytochemistry with an antibody against the transport protein showed intense membrane staining of oocytes expressing the wild-type protein. Membrane staining was completely absent from oocytes expressing mNCC(R948X); instead, diffuse cytoplasmic staining was evident. In summary, the results show that several mutations that cause Gitelman's syndrome are nonfunctional because the mutant thiazide-sensitive Na-Cl cotransporter is not processed normally, probably activating the "quality control" mechanism of the endoplasmic reticulum.
Asunto(s)
Alcalosis/metabolismo , Proteínas Portadoras/metabolismo , Hipopotasemia/metabolismo , Hipotensión/metabolismo , Procesamiento Proteico-Postraduccional , Receptores de Droga/metabolismo , Simportadores , Alcalosis/genética , Animales , Proteínas Portadoras/genética , Clonación Molecular , Femenino , Hipopotasemia/genética , Hipotensión/genética , Ratones , Ratones Endogámicos BALB C , Mutación/fisiología , Oocitos/metabolismo , Receptores de Droga/genética , Simportadores del Cloruro de Sodio , Miembro 3 de la Familia de Transportadores de Soluto 12 , Síndrome , Xenopus laevisRESUMEN
During the past several years, sites of expression of ion transport proteins in tubules from adult kidneys have been described and correlated with functional properties. Less information is available concerning sites of expression during tubule morphogenesis, although such expression patterns may be crucial to renal development. In the current studies, patterns of renal axial differentiation were defined by mapping the expression of sodium transport pathways during nephrogenesis in the rat. Combined in situ hybridization and immunohistochemistry were used to localize the Na-Pi cotransporter type 2 (NaPi2), the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2), the thiazide-sensitive Na-Cl cotransporter (NCC), the Na/Ca exchanger (NaCa), the epithelial sodium channel (rENaC), and 11beta-hydroxysteroid dehydrogenase (11HSD). The onset of expression of these proteins began in post-S-shape stages. NKCC2 was initially expressed at the macula densa region and later extended into the nascent ascending limb of the loop of Henle (TAL), whereas differentiation of the proximal tubular part of the loop of Henle showed a comparatively retarded onset when probed for NaPi2. The NCC was initially found at the distal end of the nascent distal convoluted tubule (DCT) and later extended toward the junction with the TAL. After a period of changing proportions, subsegmentation of the DCT into a proximal part expressing NCC alone and a distal part expressing NCC together with NaCa was evident. Strong coexpression of rENaC and 11HSD was observed in early nascent connecting tubule (CNT) and collecting ducts and later also in the distal portion of the DCT. Ontogeny of the expression of NCC, NaCa, 11HSD, and rENaC in the late distal convolutions indicates a heterogenous origin of the CNT. These data present a detailed analysis of the relations between the anatomic differentiation of the developing renal tubule and the expression of tubular transport proteins.
Asunto(s)
Envejecimiento/metabolismo , Nefronas/metabolismo , Sodio/metabolismo , Simportadores , 11-beta-Hidroxiesteroide Deshidrogenasas , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Proteínas Portadoras/metabolismo , Canales Epiteliales de Sodio , Hidroxiesteroide Deshidrogenasas/metabolismo , Nefronas/crecimiento & desarrollo , Ratas , Ratas Sprague-Dawley , Canales de Sodio/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo II , Simportadores de Cloruro de Sodio-PotasioRESUMEN
Mineralocorticoid hormones regulate salt transport along the distal nephron by binding to intracellular receptors and activating gene transcription. Previous experiments showed that systemic aldosterone infusions stimulate thiazide-sensitive Na and Cl transport by distal convoluted tubule (DCT) cells; this effect could have been direct or secondary to systemic hormonal effects. Aldosterone target tissues express both mineralocorticoid receptors and the metabolic enzyme 11beta-hydroxysteroid dehydrogenase type 2. Mineralocorticoid receptors have been localized to the DCT in some experiments, but not in others. Expression of 11beta-hydroxysteroid dehydrogenase type 2 by DCT cells has not been investigated. The present experiments were designed to test the hypothesis that rat DCT cells are targets of aldosterone action. Patterns of mineralocorticoid receptor, 11beta-hydroxysteroid dehydrogenase, thiazide-sensitive Na-Cl cotransporter, and Na/Ca exchanger expression along the distal tubule were examined. A polyclonal antibody was generated to localize the thiazide-sensitive Na-Cl cotransporter. Thiazide-sensitive Na-Cl cotransporter and 11beta-hydroxysteroid dehydrogenase expression were examined using both in situ hybridization and immunocytochemistry; Na/Ca exchanger and mineralocorticoid receptor expression were examined by immunocytochemistry. The results indicate that 11beta-hydroxysteroid dehydrogenase is expressed by DCT cells, as well as connecting tubule cells and principal cells of the collecting duct; expression levels are low near the junction with the thick ascending limb and rise near the transition to the connecting tubule. Mineralocorticoid receptors are expressed by DCT cells, as well as along the thick ascending limb, connecting tubule, and collecting duct. The results indicate that components of the mineralocorticoid receptor system are expressed by DCT cells, suggesting that these cells are targets of aldosterone action.
Asunto(s)
Proteínas Portadoras/metabolismo , Hidroxiesteroide Deshidrogenasas/metabolismo , Túbulos Renales Distales/metabolismo , Receptores de Mineralocorticoides/metabolismo , Simportadores , 11-beta-Hidroxiesteroide Deshidrogenasas , Aldosterona/metabolismo , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Cartilla de ADN/genética , Expresión Génica , Hidroxiesteroide Deshidrogenasas/genética , Inmunohistoquímica , Hibridación in Situ , Túbulos Renales Distales/citología , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Receptores de Droga/genética , Receptores de Droga/metabolismo , Receptores de Mineralocorticoides/genética , Simportadores del Cloruro de Sodio , Miembro 3 de la Familia de Transportadores de Soluto 12RESUMEN
BACKGROUND: Although the renal cortical collecting duct (CCD) is a principal target for aldosterone, recent evidence suggests that salt transport by other nephron segments may also be regulated by aldosterone. Electroneutral and thiazide-sensitive NaCl cotransport by the distal convoluted tubule (DCT) of the rat is increased in animals deprived of dietary NaCl. We tested the hypothesis that the DCT of the rabbit is an aldosterone target tissue. METHODS: The single-nephron reverse-transcriptase/polymerase chain reaction (RT-PCR) technique was used to determine mRNA expression of NaCl cotransporter and 11 beta-HSD 2 in dissected nephron segments. The rabbit NaCl cotransporter was first cloned and rabbit-specific primers selected. A micro-assay was developed to assess 11 beta-HSD 2 enzyme activity in 0.5 mm samples of the same nephron segments. RESULTS: NaCl cotransporter was expressed in 0 of 6 proximal tubule (PT), 6 of 6 DCT and 3 of 6 CCD samples, while 11 beta-HSD was found in 0 of 7 PT, 7 of 7 DCT and 9 of 9 CCD samples. Corticosterone was converted to 11-dehydrocorticosterone at a high rate and to a similar extent by both the DCT and CCD, but not the PT. CONCLUSIONS: We conclude that the DCT is a target tissue for the action of aldosterone. Axial heterogeneity of electroneutral (in DCT) and electrogenic (in CCD) Na transporters along the distal nephron may improve sodium recovery in low salt and volume states.
Asunto(s)
Proteínas Portadoras/genética , Hidroxiesteroide Deshidrogenasas/genética , Túbulos Renales Distales/metabolismo , ARN Mensajero/análisis , Simportadores , 11-beta-Hidroxiesteroide Deshidrogenasas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Conejos , Simportadores del Cloruro de SodioRESUMEN
Sodium and chloride transport by the macula densa and thick ascending limb of Henle's loop participates importantly in extracellular fluid volume homeostasis, urinary concentration and dilution, control of glomerular filtration, and control of renal hemodynamics. Transepithelial Na and Cl transport across the apical membrane of thick ascending limb (TALH) cells is mediated predominantly by a loop diuretic sensitive Na-K-2Cl cotransport pathway. The corresponding transport protein has recently been cloned. Functional studies suggest that the cotransporter is expressed by macula densa cells as well as by TALH cells. The current studies were designed to identify sites of Na-K-2Cl cotransporter expression along distal nephron in rabbit and rat. Non-isotopic high-resolution in situ hybridization, using an antisense probe for the apical form of the Na-K-2Cl cotransporter identified expression throughout the TALH, from the junction between inner and outer medulla to the transition to distal convoluted tubule. Expression by macula densa cells was confirmed by colocalization using markers specific for macula densa cells. First, Na-K-2Cl cotransporter mRNA was detected in macula densa cells that did not stain with anti-Tamm-Horsfall protein antibodies. Second, Na-K-2Cl cotransporter mRNA was detected in macula densa cells that show positive NADPH-diaphorase reaction, indicating high levels of constitutive nitric oxide synthase activity. In rat, levels of Na-K-2Cl cotransporter mRNA expression were similar in TALH and macula densa cells. In rabbit, expression levels were higher in macula densa cells than in surrounding TALH cells. The present data provide morphological support for a previously established functional concept that Na-K-2Cl cotransport at the TALH is accomplished by the expression of a well-defined cotransporter. At the macula densa, this transporter may establish a crucial link between tubular salt load and glomerular vascular regulation.
Asunto(s)
Proteínas Portadoras/genética , Cloruros/metabolismo , Túbulos Renales Distales/química , Asa de la Nefrona/química , Potasio/metabolismo , ARN Mensajero/análisis , Sodio/metabolismo , Animales , Masculino , Conejos , Ratas , Ratas Sprague-Dawley , Simportadores de Cloruro de Sodio-PotasioRESUMEN
The current experiments were designed to test the hypothesis that adrenal steroids increase thiazide-sensitive Na and Cl transport by the mammalian renal distal convoluted tubule (DCT). Male Sprague-Dawley rats were adrenalectomized and received steroid hormones by osmotic pumps. Six groups of animals were studied as follows: group I, no hormones; group II, replacement levels of dexamethasone only; group III, replacement levels of aldosterone only; group IV, replacement levels of both hormones; group V; replacement levels of aldosterone and high levels of dexamethasone; and group VI, replacement levels of dexamethasone and high levels of aldosterone. Circulating levels of both hormones were found to be in the high physiological range when infused at the high rate. In vivo microperfusion of distal tubules was performed to determine rates of Na and Cl transport. Chlorothiazide was used to assess the magnitude of electroneutral Na-Cl cotransport. Both aldosterone and dexamethasone stimulated thiazide-sensitive Na and Cl transport by the distal tubule by more than fivefold. [3H]metolazone binding was measured to assess the number of thiazide-sensitive Na-Cl cotransporters in renal cortex. Each steroid also increased the number of [3H]metolazone binding sites in kidney cortex more than threefold. The results are consistent with the presence of both mineralocorticoid and glucocorticoid receptors in the mammalian DCT. Physiological changes in circulating levels of adrenal steroids may affect renal NaCl excretion in part by regulating the rate of electroneutral Na-Cl absorption by the DCT.
Asunto(s)
Aldosterona/farmacología , Proteínas Portadoras/metabolismo , Clorotiazida/farmacología , Dexametasona/farmacología , Túbulos Renales Distales/metabolismo , Simportadores , Adrenalectomía , Animales , Sitios de Unión , Combinación de Medicamentos , Corteza Renal/metabolismo , Masculino , Metolazona/metabolismo , Ratas , Ratas Sprague-Dawley , Simportadores del Cloruro de SodioRESUMEN
An electroneutral thiazide-sensitive Na-Cl cotransport pathway (TSC) has been localized functionally to the distal convoluted tubule (DCT), although the TSC has also been detected in the connecting tubule (CNT), the cortical collecting duct, and the medullary collecting tubule as well. The present experiments were designed to localize expression of message for the TSC in rat and human kidney. A riboprobe, generated from the mouse TSC, was used for in situ hybridization. Simultaneous immunocytochemistry, using antibodies to Tamm-Horsfall protein, band 3, and the Na+/Ca2+ exchanger, permitted delineation of specific nephron segments. In rat, message for the TSC was highly expressed in DCT cells but not elsewhere. The transition from thick ascending limb to DCT was abrupt, whereas the transition to CNT was gradual. In the more distal region of rat DCT (DCT-2), which contained few intercalated cells, both TSC message and Na+/Ca2+ exchanger immunoreactivity were present. Treatment of rats with furosemide for 5 days increased expression of TSC message within the DCT but did not induce its expression elsewhere. In humans, expression of TSC message was also highest in cells of the DCT. In humans, however, expression extended well into the CNT. These experiments indicate that the TSC is expressed predominantly by DCT cells in both rat and humans, although expression extends into the CNT cells in humans. They also show that the TSC and Na+/Ca2+ exchanger are coexpressed by a subpopulation of DCT cells near the junction with the CNT.
Asunto(s)
Proteínas Portadoras/metabolismo , Hidroclorotiazida/farmacología , Riñón/metabolismo , Ratas/metabolismo , Simportadores , Animales , Proteínas Portadoras/genética , Furosemida/farmacología , Humanos , Inmunohistoquímica , Hibridación in Situ , Riñón/efectos de los fármacos , Masculino , Ratones , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Simportadores del Cloruro de Sodio , Distribución TisularRESUMEN
A thiazide-sensitive Na-Cl cotransporter contributes importantly to mammalian salt homeostasis by mediating Na-Cl transport along the renal distal tubule. Although it has been accepted that thiazide-sensitive Na-Cl cotransport occurs predominantly along the distal convoluted tubule in rats and mice, sites of expression in the rabbit have been controversial. A commonly accepted model of rabbit distal nephron transport pathways identifies the connecting tubule, not the distal convoluted tubule, as the predominant site of thiazide-sensitive Na-Cl cotransport. The thiazide-sensitive Na-Cl cotransporter has been cloned recently. The present experiments were designed to localize sites of thiazide-sensitive Na-Cl cotransporter mRNA expression along the rabbit distal nephron. Nonradioactive in situ hybridization with a thiazide-sensitive Na-Cl cotransporter probe was combined with immunocytochemistry with an antibody that recognizes distal convoluted tubule cells and with a Na+/Ca2+ exchanger antibody that recognizes only connecting tubule cells. The results indicate that thiazide-sensitive Na-Cl cotransporter mRNA is highly expressed by cells of the distal convoluted tubule and not by connecting tubule cells. Segments that stain with the Na+/Ca2+ exchanger antibody (connecting tubules) do not demonstrate thiazide-sensitive Na-Cl cotransporter mRNA expression. We conclude that the predominant site of thiazide-sensitive Na-Cl cotransporter mRNA expression in rabbit distal nephron is the distal convoluted tubule and that sites of mRNA expression of electroneutral Na and Cl transport are similar in rabbits, rats, and mice.