RESUMEN
Low Na+ intake activates aldosterone signaling, which increases renal Na+ reabsorption through increased apical activity of the NaCl cotransporter (NCC) and the epithelial Na+ channel (ENaC). Na+ transporter proteins are excreted in urine as an integral part of cell-derived extracellular vesicles (uEVs). It was hypothesized that Na+ transport protein levels in uEVs from healthy humans reflect their physiological regulation by aldosterone. Urine and plasma samples from 10 healthy men (median age: 22.8 yr) were collected after 5 days on a low-Na+ (70 mmol/day) diet and 5 days on a high-Na+ (250 mmol/day) diet. uEVs were isolated by ultracentrifugation and analyzed by Western blot analysis for EV markers (CD9, CD63, and ALIX), transport proteins (Na+-K+-ATPase α1-subunit, NCC, ENaC α- and γ-subunits, and aquaporin 2), and the ENaC-cleaving protease prostasin. Plasma renin and aldosterone concentrations increased during the low-Na+ diet. uEV size and concentration were not different between diets by tunable resistive pulse sensing. EV markers ALIX and CD9 increased with the low-Na+ diet, whereas CD63 and aquaporin 2 excretion were unchanged. Full-length ENaC γ-subunits were generally not detectable in uEVs, whereas ENaC α-subunits, NCC, and phosphorylated NCC were consistently detected but not changed by Na+ intake. Prostasin increased with low Na+ in uEVs. uEV excretion of transporters was not correlated with blood pressure, urinary Na+ and K+ excretion, plasma renin, or aldosterone. In conclusion, apical Na+ transporter proteins and proteases were excreted in uEVs, and while the excretion rate and size of uEVs were not affected, EV markers and prostasin increased in response to the low-Na+ diet.
Asunto(s)
Canales Epiteliales de Sodio/metabolismo , Serina Endopeptidasas/orina , Sodio en la Dieta/farmacología , Adenosina Trifosfatasas/orina , Adulto , Albuminuria/orina , Creatinina/orina , Dieta Hiposódica , Electrólitos/orina , Canales Epiteliales de Sodio/efectos de los fármacos , Exosomas/metabolismo , Vesículas Extracelulares , Humanos , Riñón/patología , Masculino , Sistema Renina-Angiotensina , Miembro 3 de la Familia de Transportadores de Soluto 12/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Adulto JovenRESUMEN
Recently, in addition to epithelial sodium channel alpha-subunit (αENaC), the thiazide-sensitive sodium-chloride cotransporter (NCC) and pendrin, also known as sodium-independent chloride/iodide transporter, were reported to be activated by aldosterone. Here, we investigated whether chloride (Cl-) is responsible for hypertension, inflammation, and renal damage in aldosterone-infused rats. Following left nephrectomy, 8-wk-old male Sprague-Dawley rats were allocated into four groups: 1) drinking 1.0% sodium chloride solution with aldosterone infusion (Aldo/NaCl rats); 2) drinking 1.44% sodium bicarbonate solution with aldosterone infusion (Aldo/NaHCO3 rats); 3) drinking distilled water with aldosterone infusion (Aldo/water rats); and 4) drinking distilled water without aldosterone infusion (sham rats). Additionally, heminephrectomized rats with aldosterone infusion were fed a 0.26% NaCl diet (control); 8.0% NaCl diet (high-Na/high-Cl); or a 4.0% NaCl 6.67% sodium citrate diet (high-Na/half-Cl). Last, Aldo/NaCl rats were treated with or without hydrochlorothiazide. Blood pressure in the Aldo/NaCl rats was significantly higher than in the Aldo/NaHCO3 rats, which was associated with the increased expression of NCC. Expression of markers of inflammation (CD3, CD68, interleukin-17A) and fibrosis (α-smooth muscle actin, collagen 1) were also increased in Aldo/NaCl rats. Similarly, aldosterone-infused rats fed a high-Na/half-Cl diet had lower blood pressure than those fed a high-Na/high-Cl diet, with a reduction of phosphorylated NCC, but not αENaC and pendrin. NCC inhibition with hydrochlorothiazide attenuated interleukin-17A protein expression along with the phosphorylation of NCC in Aldo/NaCl rats. These findings suggest that NCC-mediated Cl- uptake plays important roles in the development of aldosterone-induced hypertension and renal injury.
Asunto(s)
Aldosterona , Presión Sanguínea , Cloruros/metabolismo , Hipertensión/metabolismo , Enfermedades Renales/metabolismo , Riñón/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Modelos Animales de Enfermedad , Canales Epiteliales de Sodio/metabolismo , Fibrosis , Hidroclorotiazida/farmacología , Hipertensión/inducido químicamente , Hipertensión/fisiopatología , Hipertensión/prevención & control , Mediadores de Inflamación/metabolismo , Riñón/efectos de los fármacos , Riñón/patología , Riñón/fisiopatología , Enfermedades Renales/inducido químicamente , Enfermedades Renales/patología , Enfermedades Renales/prevención & control , Masculino , Nefrectomía , Fosforilación , Ratas Sprague-Dawley , Bicarbonato de Sodio/administración & dosificación , Bicarbonato de Sodio/metabolismo , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Cloruro de Sodio Dietético , Citrato de Sodio , Miembro 3 de la Familia de Transportadores de Soluto 12/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
The thiazide-sensitive sodium chloride cotransporter NCC is important for maintaining serum sodium (Na+) and, indirectly, serum potassium (K+) levels. Functional studies on NCC have used cell lines with native NCC expression, transiently transfected nonpolarized cell lines, or Xenopus laevis oocytes. Here, we developed the use of polarized Madin-Darby canine kidney type I (MDCKI) mammalian epithelial cell lines with tetracycline-inducible human NCC expression to study NCC activity and membrane abundance in the same system. In radiotracer assays, induced cells grown on filters had robust thiazide-sensitive and chloride dependent sodium-22 (22Na) uptake from the apical side. To minimize cost and maximize throughput, assays were modified to use cells grown on plastic. On plastic, cells had similar thiazide-sensitive 22Na uptakes that increased following preincubation of cells in chloride-free solutions. NCC was detected in the plasma membrane, and both membrane abundance and phosphorylation of NCC were increased by incubation in chloride-free solutions. Furthermore, in cells exposed for 15 min to low or high extracellular K+, the levels of phosphorylated NCC increased and decreased, respectively. To demonstrate that the system allows rapid and systematic assessment of mutated NCC, three phosphorylation sites in NCC were mutated, and NCC activity was examined. 22Na fluxes in phosphorylation-deficient mutants were reduced to baseline levels, whereas phosphorylation-mimicking mutants were constitutively active, even without chloride-free stimulation. In conclusion, this system allows the activity, cellular localization, and abundance of wild-type or mutant NCC to be examined in the same polarized mammalian expression system in a rapid, easy, and low-cost fashion.
Asunto(s)
Polaridad Celular , Cloruros/metabolismo , Células Epiteliales/metabolismo , Mutación , Sodio/metabolismo , Animales , Técnicas de Cultivo de Célula , Perros , Relación Dosis-Respuesta a Droga , Células Epiteliales/efectos de los fármacos , Genotipo , Ensayos Analíticos de Alto Rendimiento , Cinética , Células de Riñón Canino Madin Darby , Fenotipo , Fosforilación , Potasio/metabolismo , Procesamiento Proteico-Postraduccional , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Miembro 3 de la Familia de Transportadores de Soluto 12/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , TransfecciónRESUMEN
Dietary potassium intake is inversely related to blood pressure and mortality. Moreover, the sodium-chloride cotransporter (NCC) plays an important role in blood pressure regulation and urinary potassium excretion in response to potassium intake. Previously, it was shown that NCC is activated by the WNK4-SPAK cascade and dephosphorylated by protein phosphatase. However, the mechanism of NCC regulation with acute potassium intake is still unclear. To identify the molecular mechanism of NCC regulation in response to potassium intake, we used adult C57BL/6 mice fed a 1.7% potassium solution by oral gavage. We confirmed that acute potassium load rapidly dephosphorylated NCC, which was not dependent on the accompanying anions. Mice were treated with tacrolimus (calcineurin inhibitor) and W7 (calmodulin inhibitor) before the oral potassium loads. Dephosphorylation of NCC induced by potassium was significantly inhibited by both tacrolimus and W7 treatment. There was no significant difference in WNK4, OSR1, and SPAK expression after high potassium intake, even after tacrolimus and W7 treatment. Another phosphatase, protein phosphatase 1, and its endogenous inhibitor I-1 did not show a significant change after potassium intake. Hyperkaliuria, induced by high potassium intake, was significantly suppressed by tacrolimus treatment. Thus, calcineurin is activated by an acute potassium load, which rapidly dephosphorylates NCC, leading to increased urinary potassium excretion.
Asunto(s)
Inhibidores de la Calcineurina/farmacología , Calcineurina/metabolismo , Riñón/efectos de los fármacos , Potasio en la Dieta/metabolismo , Eliminación Renal/efectos de los fármacos , Tacrolimus/farmacología , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Concentración de Iones de Hidrógeno , Riñón/metabolismo , Masculino , Ratones Endogámicos C57BL , Fosforilación , Potasio en la Dieta/sangre , Potasio en la Dieta/orina , Inhibidores de Proteínas Quinasas/farmacología , Proteína Fosfatasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Sulfonamidas/farmacología , Factores de Tiempo , Factores de Transcripción/metabolismoRESUMEN
The thiazide-sensitive NaCl cotransporter (NCC), located apically in distal convoluted tubule epithelia, regulates the fine-tuning of renal sodium excretion. Three isoforms of NCC are generated through alternative splicing of the transcript, of which the third isoform has been the most extensively investigated in pathophysiological conditions. The aim of this study was to investigate the effect of different anti-hypertensive treatments on the abundance and phosphorylation of all three NCC isoforms in urinary extracellular vesicles (uEVs) of essential hypertensive patients. In uEVs isolated from patients (n = 23) before and after hydrochlorothiazide or valsartan treatment, the abundance and phosphorylation of the NCC isoforms was determined. Additionally, clinical biochemistry and blood pressure of the patients was assessed. Our results show that NCC detected in human uEVs has a glycosylated and oligomeric structure, comparable to NCC present in human kidney membrane fractions. Despite the inhibitory action of hydrochlorothiazide on NCC activity, immunoblot analysis of uEVs showed significantly increased abundance of NCC isoforms 1 and 2 (NCC1/2), total NCC (NCC1-3), and the phosphorylated form of total NCC (pNCC1-3-T55/T60) in essential hypertensive patients treated with hydrochlorothiazide but not with valsartan. This study highlights that NCC1/2, NCC1-3, and pNCC1-3-T55/T60 are upregulated by hydrochlorothiazide, and the increase in NCC abundance in uEVs of essential hypertensive patients correlates with the blood pressure response to hydrochlorothiazide.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Antihipertensivos/uso terapéutico , Vesículas Extracelulares/efectos de los fármacos , Hidroclorotiazida/uso terapéutico , Hipertensión/tratamiento farmacológico , Riñón/efectos de los fármacos , Inhibidores de los Simportadores del Cloruro de Sodio/uso terapéutico , Valsartán/uso terapéutico , Adolescente , Adulto , Anciano , Biomarcadores/orina , Presión Sanguínea/efectos de los fármacos , Estudios Cruzados , Vesículas Extracelulares/metabolismo , Femenino , Glicosilación , Humanos , Hipertensión/fisiopatología , Hipertensión/orina , Riñón/metabolismo , Riñón/fisiopatología , Masculino , Persona de Mediana Edad , Países Bajos , Fosforilación , Estudios Prospectivos , Isoformas de Proteínas , Miembro 3 de la Familia de Transportadores de Soluto 12/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/orina , Resultado del Tratamiento , Regulación hacia Arriba , Adulto JovenRESUMEN
We assessed effects of acute volume reductions induced by administration of diuretics in rats. Direct block of Na+ transport produced changes in urinary electrolyte excretion. Adaptations to these effects appeared as alterations in the expression of protein for the distal nephron Na+ transporters NCC and ENaC. Two hours after a single injection of furosemide (6 mg/kg) or hydrochlorothiazide (HCTZ; 30 mg/kg) Na+ and K+ excretion increased but no changes in the content of activated forms of NCC (phosphorylated on residue T53) or ENaC (cleaved γ-subunit) were detected. In contrast, amiloride (0.6 mg/kg) evoked a similar natriuresis that coincided with decreased pT53NCC and increased cleaved γENaC. Alterations in posttranslational membrane protein processing correlated with an increase in plasma K+ of 0.6-0.8 mM. Decreased pT53NCC occurred within 1 h after amiloride injection, whereas changes in γENaC were slower and were blocked by the mineralocorticoid receptor antagonist spironolactone. Increased γENaC cleavage correlated with elevation of the surface expression of the subunit as assessed by in situ biotinylation. Na depletion induced by 2 h of furosemide or HCTZ treatment increases total NCC expression without affecting ENaC protein. However, restriction of Na intake for 10 h (during the day) or 18 h (overnight) increased the abundance of both total NCC and of cleaved α- and γENaC. We conclude that the kidneys respond acutely to hyperkalemic challenges by decreasing the activity of NCC while increasing that of ENaC. They respond to hypovolemia more slowly, increasing Na+ reabsorptive capacities of both of these transporters.
Asunto(s)
Diuréticos/farmacología , Canales Epiteliales de Sodio/efectos de los fármacos , Hiperpotasemia/metabolismo , Hipovolemia/metabolismo , Nefronas/efectos de los fármacos , Potasio/metabolismo , Sodio/metabolismo , Amilorida/farmacología , Animales , Diuréticos/toxicidad , Canales Epiteliales de Sodio/metabolismo , Femenino , Furosemida/farmacología , Hidroclorotiazida/farmacología , Hiperpotasemia/sangre , Hiperpotasemia/inducido químicamente , Hiperpotasemia/orina , Hipovolemia/sangre , Hipovolemia/inducido químicamente , Hipovolemia/orina , Masculino , Modelos Biológicos , Nefronas/metabolismo , Fosforilación , Potasio/sangre , Potasio/orina , Ratas Sprague-Dawley , Eliminación Renal/efectos de los fármacos , Sodio/sangre , Sodio/orina , Miembro 3 de la Familia de Transportadores de Soluto 12/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Espironolactona/farmacologíaRESUMEN
Gitelman's syndrome is characterized by hypocalciuria, severe hypomagnesemia, and prominent muscular involvements such as fatigue, weakness, cramps, and tetany. It is due to mutations in the thiazide sensitive NaCl co-transporter in the distal convoluted tubule. The administration of thiazide diuretics may induce a subnormal increase of urinary Cl(-) excretion in patients with Gitelman's syndrome, consistent with the hypothesis that less than normal Cl(-) is reabsorbed by the thiazide-inhibitable transporter in Gitelman's syndrome. Thus, we report a case of Gitelman's syndrome presenting with quadriparesis diagnosed by using thiazide clearance test.
Asunto(s)
Síndrome de Gitelman/diagnóstico , Cuadriplejía/etiología , Inhibidores de los Simportadores del Cloruro de Sodio/administración & dosificación , Predisposición Genética a la Enfermedad , Síndrome de Gitelman/complicaciones , Síndrome de Gitelman/tratamiento farmacológico , Síndrome de Gitelman/genética , Humanos , Masculino , Persona de Mediana Edad , Mutación , Fenotipo , Valor Predictivo de las Pruebas , Miembro 3 de la Familia de Transportadores de Soluto 12/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Resultado del TratamientoRESUMEN
Recent studies have implicated a role of norepinephrine (NE) in the activation of the sodium chloride cotransporter (NCC) to drive the development of salt-sensitive hypertension. However, the interaction between NE and increased salt intake on blood pressure remains to be fully elucidated. This study examined the impact of a continuous NE infusion on sodium homeostasis and blood pressure in conscious Sprague-Dawley rats challenged with a normal (NS; 0.6% NaCl) or high-salt (HS; 8% NaCl) diet for 14 days. Naïve and saline-infused Sprague-Dawley rats remained normotensive when placed on HS and exhibited dietary sodium-evoked suppression of peak natriuresis to hydrochlorothiazide. NE infusion resulted in the development of hypertension, which was exacerbated by HS, demonstrating the development of the salt sensitivity of blood pressure [MAP (mmHg) NE+NS: 151 ± 3 vs. NE+HS: 172 ± 4; P < 0.05]. In these salt-sensitive animals, increased NE prevented dietary sodium-evoked suppression of peak natriuresis to hydrochlorothiazide, suggesting impaired NCC activity contributes to the development of salt sensitivity [peak natriuresis to hydrochlorothiazide (µeq/min) Naïve+NS: 9.4 ± 0.2 vs. Naïve+HS: 7 ± 0.1; P < 0.05; NE+NS: 11.1 ± 1.1; NE+HS: 10.8 ± 0.4). NE infusion did not alter NCC expression in animals maintained on NS; however, dietary sodium-evoked suppression of NCC expression was prevented in animals challenged with NE. Chronic NCC antagonism abolished the salt-sensitive component of NE-mediated hypertension, while chronic ANG II type 1 receptor antagonism significantly attenuated NE-evoked hypertension without restoring NCC function. These data demonstrate that increased levels of NE prevent dietary sodium-evoked suppression of the NCC, via an ANG II-independent mechanism, to stimulate the development of salt-sensitive hypertension.
Asunto(s)
Hipertensión/metabolismo , Riñón/metabolismo , Norepinefrina , Cloruro de Sodio Dietético , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Antihipertensivos/farmacología , Presión Sanguínea , Modelos Animales de Enfermedad , Hidroclorotiazida/farmacología , Hipertensión/tratamiento farmacológico , Hipertensión/etiología , Hipertensión/fisiopatología , Riñón/efectos de los fármacos , Riñón/fisiopatología , Losartán/farmacología , Masculino , Natriuresis , Ratas Sprague-Dawley , Sistema Renina-Angiotensina , Inhibidores de los Simportadores del Cloruro de Sodio/farmacología , Cloruro de Sodio Dietético/sangre , Miembro 3 de la Familia de Transportadores de Soluto 12/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Sistema Nervioso Simpático/metabolismo , Sistema Nervioso Simpático/fisiopatología , Factores de TiempoRESUMEN
The renal thiazide-sensitive NaCl cotransporter, NCC, is the major pathway for salt reabsorption in the distal convoluted tubule. The activity of this cotransporter is critical for regulation of several physiological variables such as blood pressure, serum potassium, acid base metabolism, and urinary calcium excretion. Therefore, it is not surprising that numerous hormone-signaling pathways regulate NCC activity to maintain homeostasis. In this review, we will provide an overview of the most recent evidence on NCC modulation by aldosterone, angiotensin II, vasopressin, glucocorticoids, insulin, norepinephrine, estradiol, progesterone, prolactin, and parathyroid hormone.
Asunto(s)
Hormonas/metabolismo , Túbulos Renales Distales/metabolismo , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Animales , Hormonas Esteroides Gonadales/metabolismo , Hormonas/farmacología , Humanos , Túbulos Renales Distales/efectos de los fármacos , Receptores de Péptidos/efectos de los fármacos , Receptores de Péptidos/metabolismo , Sistema Renina-Angiotensina , Miembro 3 de la Familia de Transportadores de Soluto 12/efectos de los fármacosRESUMEN
Unique situations in female physiology require volume retention. Accordingly, a dimorphic regulation of the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) has been reported, with a higher activity in females than in males. However, little is known about the hormones and mechanisms involved. Here, we present evidence that estrogens, progesterone, and prolactin stimulate NCC expression and phosphorylation. The sex difference in NCC abundance, however, is species dependent. In rats, NCC phosphorylation is higher in females than in males, while in mice both NCC expression and phosphorylation is higher in females, and this is associated with increased expression and phosphorylation of full-length STE-20 proline-alanine-rich kinase (SPAK). Higher expression/phosphorylation of NCC was corroborated in humans by urinary exosome analysis. Ovariectomy in rats resulted in decreased expression and phosphorylation of the cotransporter and promoted the shift of SPAK isoforms toward the short inhibitory variant SPAK2. Conversely, estradiol or progesterone administration to ovariectomized rats restored NCC phosphorylation levels and shifted SPAK expression and phosphorylation towards the full-length isoform. Estradiol administration to male rats induced a significant increase in NCC phosphorylation. NCC is also modulated by prolactin. Administration of this peptide hormone to male rats induced increased phosphorylation of NCC, an effect that was observed even using the ex vivo kidney perfusion strategy. Our results indicate that estradiol, progesterone, and prolactin, the hormones that are involved in sexual cycle, pregnancy and lactation, upregulate the activity of NCC.
Asunto(s)
Estradiol/metabolismo , Riñón/metabolismo , Ovario/metabolismo , Progesterona/metabolismo , Prolactina/metabolismo , Animales , Estradiol/administración & dosificación , Terapia de Reemplazo de Estrógeno , Femenino , Humanos , Isoenzimas , Riñón/efectos de los fármacos , Masculino , Ratones Noqueados , Ovariectomía , Fosforilación , Progesterona/administración & dosificación , Prolactina/administración & dosificación , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas Wistar , Receptores de Prolactina/genética , Receptores de Prolactina/metabolismo , Factores Sexuales , Transducción de Señal , Miembro 3 de la Familia de Transportadores de Soluto 12/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Regulación hacia ArribaRESUMEN
Guanylin (GN) inhibited water absorption and short-circuit current (Isc) in seawater eel intestine. Similar inhibition was observed after bumetanide, and the effect of bumetanide was abolished by GN or vice versa, suggesting that both act on the same target, Na(+)-K(+)-2Cl(-) cotransporter (NKCC), which is a key player for the Na(+)-K(+)-Cl(-) transport system responsible for water absorption in marine teleost intestine. However, effect of GN was always greater than that of bumetanide: 10% greater in middle intestine (MI) and 40% in posterior intestine (PI) for Isc, and 25% greater in MI and 34% in PI for water absorption. After treatment with GN, Isc decreased to zero, but 20-30% water absorption still remained. The remainder may be due to the Cl(-)/HCO3 (-) exchanger and Na(+)-Cl(-) cotransporter (NCC), since inhibitors for these transporters almost nullified the remaining water absorption. Quantitative PCR analysis revealed the presence of major proteins involved in water absorption; the NKCC2ß and AQP1 genes whose expression was markedly upregulated after seawater acclimation. The SLC26A6 (anion exchanger) and NCCß genes were also expressed in small amounts. Consistent with the inhibitors' effect, expression of NKCC2ß was MI > PI, and that of NCCß was MI << PI. The present study showed that GN not only inhibits the bumetanide-sensitive Na(+)-K(+)-Cl(-) transport system governed by NKCC2ß, but also regulates unknown ion transporters different from GN-insensitive SLC26A6 and NCC. A candidate is cystic fibrosis transmembrane conductance regulator Cl(-) channel, as demonstrated in mammals, but its expression is low in eel intestine, and its role may be minor, as indicated by the small effect of its inhibitors.
Asunto(s)
Anguilas/metabolismo , Proteínas de Peces/antagonistas & inhibidores , Hormonas Gastrointestinales/farmacología , Intestinos/efectos de los fármacos , Péptidos Natriuréticos/farmacología , Agua de Mar , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Miembro 1 de la Familia de Transportadores de Soluto 12/antagonistas & inhibidores , Agua/metabolismo , Adaptación Fisiológica , Animales , Acuaporina 1/genética , Acuaporina 1/metabolismo , Bumetanida/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Anguilas/genética , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Mucosa Intestinal/metabolismo , Transporte Iónico , Cinética , Potenciales de la Membrana , 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/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/genética , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
BACKGROUND/AIMS: Cyclosporine (CsA) is a calcineurin inhibitor widely used as an immunosuppressant in organ transplantation. Previous studies demonstrated the relationship between CsA and renal sodium transporters such as the Na-K-2Cl cotransporter in the loop of Henle (NKCC2). Experimental models of CsA-induced hypertension have shown an increase in renal NKCC2. METHODS: Using immunoblotting of urinary exosomes, we investigated in CsA-treated kidney transplant patients (n = 39) the excretion of NKCC2 and Na-Cl cotransporter (NCC) and its association with blood pressure (BP) level. We included 8 non-CsA-treated kidney transplant patients as a control group. Clinical data, immunosuppression and hypertension treatments, blood and 24-hour urine tests, and 24-hour ambulatory BP monitoring were recorded. RESULTS: CsA-treated patients tended to excrete a higher amount of NKCC2 than non-CsA-treated patients (mean ± SD, 175 ± 98 DU and 90 ± 70.3 DU, respectively; p = 0.05) and showed higher BP values (24-hour systolic BP 138 ± 17 mm Hg and 112 ± 12 mm Hg, p = 0.003; 24-hour diastolic BP, 83.8 ± 9.8 mm Hg and 72.4 ± 5.2 mm Hg, p = 0.015, respectively). Within the CsA-treated group, there was no correlation between either NKCC2 or NCC excretion and BP levels. This was confirmed by a further analysis including potential confounding factors. On the other hand, a significant positive correlation was observed between CsA blood levels and the excretion of NKCC2 and NCC. CONCLUSION: Overall, these results support the hypothesis that CsA induces an increase in NKCC2 and NCC in urinary exosomes of renal transplant patients. The fact that the increase in sodium transporters in urine did not correlate with the BP level suggests that in kidney transplant patients, other mechanisms could be implicated in CsA-induced hypertension.
Asunto(s)
Ciclosporina/uso terapéutico , Exosomas/metabolismo , Rechazo de Injerto/prevención & control , Inmunosupresores/uso terapéutico , Trasplante de Riñón , Riñón/metabolismo , Sodio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismo , Adulto , Anciano , Presión Sanguínea , Estudios de Casos y Controles , Ciclosporina/farmacología , Exosomas/efectos de los fármacos , Femenino , Humanos , Inmunosupresores/farmacología , Riñón/efectos de los fármacos , Masculino , Persona de Mediana Edad , Miembro 1 de la Familia de Transportadores de Soluto 12/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Orina , Adulto JovenRESUMEN
Modulation of Na(+)-Cl(-) cotransporter (NCC) activity is essential to adjust K(+) excretion in the face of changes in dietary K(+) intake. We used previously characterized genetic mouse models to assess the role of Ste20-related proline-alanine-rich kinase (SPAK) and with-no-lysine kinase (WNK)4 in the modulation of NCC by K(+) diets. SPAK knockin and WNK4 knockout mice were placed on normal-, low-, or high-K(+)-citrate diets for 4 days. The low-K(+) diet decreased and high-K(+) diet increased plasma aldosterone levels, but both diets were associated with increased phosphorylation of NCC (phospho-NCC, Thr(44)/Thr(48)/Thr(53)) and phosphorylation of SPAK/oxidative stress responsive kinase 1 (phospho-SPAK/OSR1, Ser(383)/Ser(325)). The effect of the low-K(+) diet on SPAK phosphorylation persisted in WNK4 knockout and SPAK knockin mice, whereas the effects of ANG II on NCC and SPAK were lost in both mouse colonies. This suggests that for NCC activation by ANG II, integrity of the WNK4/SPAK pathway is required, whereas for the low-K(+) diet, SPAK phosphorylation occurred despite the absence of WNK4, suggesting the involvement of another WNK (WNK1 or WNK3). Additionally, because NCC activation also occurred in SPAK knockin mice, it is possible that loss of SPAK was compensated by OSR1. The positive effect of the high-K(+) diet was observed when the accompanying anion was citrate, whereas the high-KCl diet reduced NCC phosphorylation. However, the effect of the high-K(+)-citrate diet was aldosterone dependent, and neither metabolic alkalosis induced by bicarbonate, nor citrate administration in the absence of K(+) increased NCC phosphorylation, suggesting that it was not due to citrate-induced metabolic alkalosis. Thus, the accompanying anion might modulate the NCC response to the high-K(+) diet.
Asunto(s)
Potasio en la Dieta/farmacología , Transducción de Señal/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/metabolismo , Aldosterona/metabolismo , Angiotensina II/farmacología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Fosforilación , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Hypertension is a leading cause of morbidity and mortality worldwide, and disordered sodium balance has long been implicated in its pathogenesis. Aldosterone is perhaps the key regulator of sodium balance and thus blood pressure. The sodium chloride cotransporter (NCC) in the distal convoluted tubule of the kidney is a major site of sodium reabsorption and plays a key role in blood pressure regulation. Chronic exposure to aldosterone increases NCC protein expression and function. However, more acute effects of aldosterone on NCC are unknown. In our salt-abundant modern society where chronic salt deprivation is rare, understanding the acute effects of aldosterone is critical. Here, we examined the acute effects (12-36 h) of aldosterone on NCC in the rodent kidney and in a mouse distal convoluted tubule cell line. Studies demonstrated that aldosterone acutely stimulated NCC activity and phosphorylation without affecting total NCC abundance or surface expression. This effect was dependent upon the presence of the mineralocorticoid receptor and serum- and glucocorticoid-regulated kinase 1 (SGK1). Furthermore, STE20/SPS-1-related proline/alanine-rich kinase (SPAK) phosphorylation also increased, and gene silencing of SPAK eliminated the effect of aldosterone on NCC activity. Aldosterone administration via a minipump in adrenalectomized rodents confirmed an increase in NCC phosphorylation without a change in NCC total protein. These data indicate that acute aldosterone-induced SPAK-dependent phosphorylation of NCC increases individual transporter activity.