RESUMEN
Podocytes are highly specialized epithelial cells essentially required to establish and maintain the kidney filtration barrier. Due to their complex cellular architecture these cells rely on an elaborated cytoskeletal apparatus providing plasticity as well as adaptive adhesion properties to withstand significant physical filtration forces. However, our knowledge about podocyte specific components of the cytoskeletal machinery is still incomplete. Employing cross-analysis of various quantitative omics-data sets we identify the WD40-domain containing protein CORO2B as a podocyte enriched protein. Furthermore, we demonstrate the distinct localization pattern of CORO2B to the ventral actin cytoskeleton serving as a physical linkage module to cell-matrix adhesion sites. Analysis of a novel Coro2b knockout mouse revealed that CORO2B modulates stress response of podocytes in an experimental nephropathy model. Using quantitative focal adhesome proteomics we identify the recruitment of CFL1 via CORO2B to focal adhesions as an underlying mechanism. Thus, we describe CORO2B as a novel podocyte enriched protein influencing cytoskeletal plasticity and stress adaptation.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Podocitos/metabolismo , Repeticiones WD40 , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Animales , Cofilina 1/metabolismo , Adhesiones Focales/metabolismo , Adhesiones Focales/ultraestructura , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Modelos Biológicos , Podocitos/ultraestructura , Estrés Fisiológico , Análisis de SupervivenciaRESUMEN
The serum- and glucocorticoid-inducible kinase SGK1 is an ubiquitously expressed kinase with the ability to regulate a variety of transport systems. Recent observations point to a role of SGK1 in the regulation of diverse physiological functions such as epithelial transport and cardiac and neuronal excitability. At least partially through its effect on transport, SGK1 contributes to a number of pathophysiological conditions including metabolic syndrome and fibrosing disease.
Asunto(s)
Transporte Biológico/fisiología , Proteínas Nucleares/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Activación Enzimática , Proteínas Inmediatas-Precoces , Proteínas Nucleares/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Epithelial Na+ channel (ENaC) activity in kidney and colon is stimulated by aldosterone acting on the mineralocorticoid receptor (MR). MR and the glucocorticoid receptor (GR) show high homology in their DNA-binding domain and have similar affinities to mineralo- and glucocorticoids. We therefore asked whether the glucocorticoid-mediated activation of ENaC is restricted to the presence of MR and used the MR knockout mouse model to address this question. Due to their MR deficiency and the consecutive reduction of ENaC activity these mice die as neonates, and even after appropriate substitution therapy adult MR knockout mice suffer from high Na+ loss and hyperkalemia. In the present study, glucocorticoid treatment restored plasma K+ and almost normalized the fractional excretions of Na+ (FENa+) and K+ (FEK+) in adult salt-substituted MR knockout mice, while the effect of amiloride on FENa+ and FEK+ was augmented in these animals. In order to estimate ENaC activity, measurements of transepithelial equivalent short-circuit current (Isc) were performed. Glucocorticoids induced an amiloride-sensitive Na+ absorption in renal cortical collecting duct and distal colon of MR-/- of about 25% and 50% of the currents observed in glucocorticoid-treated wild-type mice, respectively. In the colon glucocorticoid treatment increased the mRNA abundance of all three ENaC subunits, in the kidney only alpha-ENaC was increased. The regulation of ENaC expression was the same in both genotypes and thus irrespective of the presence of MR. These data show that MR is no prerequisite for the activation of ENaC transcription and activity, and that the respective mechanisms can be stimulated via GR.
Asunto(s)
Glucocorticoides/farmacología , Receptores de Mineralocorticoides/fisiología , Canales de Sodio/metabolismo , Triamcinolona/farmacología , Amilorida/farmacología , Animales , Animales Recién Nacidos/orina , Presión Sanguínea/efectos de los fármacos , Agua Corporal/metabolismo , Colon/efectos de los fármacos , Colon/metabolismo , Corticosterona/orina , Diuréticos/farmacología , Electrólitos/sangre , Canales Epiteliales de Sodio , Homeostasis/efectos de los fármacos , Técnicas In Vitro , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/metabolismo , Ratones , Ratones Noqueados/genética , ARN Mensajero/metabolismo , Receptores de Mineralocorticoides/deficiencia , Receptores de Mineralocorticoides/genética , Canales de Sodio/genéticaRESUMEN
The gene KCNQ1 encodes a K(+) channel alpha-subunit important for cardiac repolarization, formerly known as K(v)LQT1. In large and small intestine a channel complex consisting of KCNQ1 and the beta-subunit KCNE3 (MiRP2) is known to mediate the cAMP-activated basolateral K(+) current, which is essential for luminal Cl(-) secretion. Northern blot experiments revealed an expression of both subunits in lung tissue. However, previous reports suggested a role of KCNE1 (minK, Isk) but not KCNE3 in airway epithelial cells. Here we give evidence that KCNE1 is not detected in murine tracheal epithelial cells and that Cl(-) secretion by these cells is not reduced by the knock-out of the KCNE1 gene. In contrast we show that a complex consisting of KCNQ1 and KCNE3 probably forms a basolateral K(+) channel in murine tracheal epithelial cells. As described for colonic epithelium, the current through KCNQ1 complexes in murine trachea is specifically inhibited by the chromanol 293B. A 293B-sensitive current was present after stimulation with forskolin and agonists that increase Ca(2+) as well as after administration of the pharmacological K(+) channel activator, 1-EBIO. A 293B-inhibitable current was already present under control conditions and reduced after administration of amiloride indicating a role of this K(+) channel not only for Cl(-) secretion but also for Na(+) reabsorption. We conclude that at least in mice a KCNQ1 channel complex seems to be the dominant basolateral K(+) conductance in tracheal epithelial cells.
Asunto(s)
Canales de Potasio con Entrada de Voltaje , Canales de Potasio/fisiología , Tráquea/química , Adenosina Trifosfato/farmacología , Animales , Calcio/metabolismo , Cloruros/metabolismo , AMP Cíclico/fisiología , Indoles/farmacología , Canales de Potasio KCNQ , Canal de Potasio KCNQ1 , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Canales de Potasio/química , Canales de Potasio/genética , Subunidades de ProteínaRESUMEN
The voltage-dependent K(+) channel responsible for the slowly activating delayed K(+) current I(Ks) is composed of pore-forming KCNQ1 and regulatory KCNE1 subunits, which are mutated in familial forms of cardiac long QT syndrome. Because KCNQ1 and KCNE1 genes also are expressed in epithelial tissues, such as the kidneys and the intestine, we have investigated the adaptation of KCNE1-deficient mice to different K(+) and Na(+) intakes. On a normal K(+) diet, homozygous kcne1(-/-) mice exhibit signs of chronic volume depletion associated with fecal Na(+) and K(+) wasting and have lower plasma K(+) concentration and higher levels of aldosterone than wild-type mice. Although plasma aldosterone can be suppressed by low K(+) diets or stimulated by low Na(+) diets, a high K(+) diet provokes a tremendous increase of plasma aldosterone levels in kcne1(-/-) mice as compared with wild-type mice (7.1-fold vs. 1.8-fold) despite lower plasma K(+) in kcne1(-/-) mice. This exacerbated aldosterone production in kcne1(-/-) mice is accompanied by an abnormally high plasma renin concentration, which could partly explain the hyperaldosteronism. In addition, we found that KCNE1 and KCNQ1 mRNAs are expressed in the zona glomerulosa of adrenal glands where I(Ks) may directly participate in the control of aldosterone production by plasma K(+). These results, which show that KCNE1 and I(Ks) are involved in K(+) homeostasis, might have important implications for patients with I(Ks)-related long QT syndrome, because hypokalemia is a well known risk factor for the occurrence of torsades de pointes ventricular arrhythmia.
Asunto(s)
Aldosterona/metabolismo , Síndrome de QT Prolongado/congénito , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/fisiología , Potasio/metabolismo , Aldosterona/sangre , Animales , Presión Sanguínea , Colon/metabolismo , Modelos Animales de Enfermedad , Electrocardiografía , Heces , Expresión Génica , Humanos , Iones/metabolismo , Canales de Potasio KCNQ , Canal de Potasio KCNQ1 , Síndrome de QT Prolongado/metabolismo , Ratones , Ratones Noqueados , Potasio/sangre , Canales de Potasio/genética , Renina/sangre , Sodio/metabolismo , Sodio/orina , Distribución TisularRESUMEN
BACKGROUND & AIMS: Gastric H+ secretion via the H+/K+-adenosine triphosphatase is coupled to the uptake of K+. However, the molecular identity of luminal K+ channels enabling K+ recycling in parietal cells is unknown. This study was aimed to investigate these luminal K+ channels. METHODS: Acid secretion was measured in vivo and in vitro; KCNQ1 protein localization was assessed by immunofluorescence, and acid-sensitivity of KCNQ1 by patch-clamp. RESULTS: We identified KCNQ1, which is mutated in cardiac long QT syndrome, as a K+ channel located in tubulovesicles and apical membrane of parietal cells, where it colocalized with H+/K+-adenosine triphosphatase. Blockade of KCNQ1 current by 293B led to complete inhibition of acid secretion. The putative KCNQ1 subunits, KCNE2 and KCNE3, were abundant in human stomach; KCNE1, however, was absent. Coexpression of KCNE3/KCNQ1 in COS cells led to an acid-insensitive current; KCNE2/KCNQ1 was activated by low extracellular pH. CONCLUSIONS: We identified KCNQ1 as the missing luminal K+ channel in parietal cells and characterized its crucial role in acid secretion. Because KCNE3 and KCNE2 are expressed in human stomach, one or both are candidates to coassemble with KCNQ1 in parietal cells. Thus, stomach- and subunit-specific inhibitors of KCNQ1 might offer new therapeutical perspectives for peptic ulcer disease.