RESUMEN
BACKGROUND: Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (Slc26a4+/+) or lack a complete Slc26a4 gene (Slc26a4-/-). METHODS: Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic K+ concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry. RESULTS: Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in Slc26a4-/- mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4-/- mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K+ channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K+ concentrations were normal and membrane proteins necessary for K+ secretion were present, including the K+ channel KCNQ1 and KCNE1, Na+/2Cl-/K+ cotransporter SLC12A2 and the gap junction GJB2. CONCLUSIONS: These observations demonstrate that pendrin dysfunction leads to a loss of KCNJ10 protein expression and a loss of the endocochlear potential, which may be the direct cause of deafness in Pendred syndrome.
Asunto(s)
Cóclea/química , Sordera/etiología , Proteínas de Transporte de Membrana/análisis , Canales de Potasio de Rectificación Interna/análisis , Vestíbulo del Laberinto/química , Animales , Conexina 26 , Conexinas , Endolinfa/química , Potenciales Evocados Auditivos/fisiología , Bocio , Ratones , Canales de Potasio de Rectificación Interna/metabolismo , ARN Mensajero/análisis , Transportadores de Sulfato , SíndromeRESUMEN
Immunolocalization studies of mouse cochlea and vestibular end-organ were performed to study the expression pattern of pendrin, the protein encoded by the Pendred syndrome gene (PDS), in the inner ear. The protein was restricted to the areas composed of specialized epithelial cells thought to play a key role in regulating the composition and resorption of endolymph. In the cochlea, pendrin was abundant in the apical membrane of cells in the spiral prominence and outer sulcus cells (along with their root processes). In the vestibular end-organ, pendrin was found in the transitional cells of the cristae ampullaris, utriculi, and sacculi as well as in the apical membrane of cells in the endolymphatic sac. Pds-knockout (Pds-/-) mice were found to lack pendrin immunoreactivity in all of these locations. Histological studies revealed that the stria vascularis in Pds-/- mice was only two-thirds the thickness seen in wild-type mice, with the strial marginal cells showing irregular shapes and sizes. Functional studies were also performed to examine the role of pendrin in endolymph homeostasis. Using double-barreled electrodes placed in both the cochlea and the utricle, the endocochlear potential and endolymph potassium concentration were measured in wild-type and Pds-/- mice. Consistent with the altered strial morphology, the endocochlear potential in Pds-/- mice was near zero and did not change during anoxia. On the other hand, the endolymphatic potassium concentration in Pds-/- mice was near normal in the cochlea and utricle. Together, these results suggest that pendrin serves a key role in the functioning of the basal and/or intermediate cells of the stria vascularis to maintain the endocochlear potential, but not in the potassium secretory function of the marginal cells.
Asunto(s)
Proteínas Portadoras/genética , Cóclea/fisiopatología , Sordera/etiología , Sordera/fisiopatología , Bocio/fisiopatología , Proteínas de Transporte de Membrana , Animales , Proteínas Portadoras/metabolismo , Cóclea/metabolismo , Cóclea/patología , Sordera/patología , Endolinfa/fisiología , Saco Endolinfático/metabolismo , Saco Endolinfático/patología , Saco Endolinfático/fisiopatología , Femenino , Técnica del Anticuerpo Fluorescente , Homeostasis/fisiología , Masculino , Ratones , Ratones Noqueados , Potasio/metabolismo , Transportadores de Sulfato , Vestíbulo del Laberinto/metabolismo , Vestíbulo del Laberinto/patología , Vestíbulo del Laberinto/fisiopatologíaRESUMEN
Pendrin is an anion exchanger expressed along the apical plasma membrane and apical cytoplasmic vesicles of type B and of non-A, non-B intercalated cells of the distal convoluted tubule, connecting tubule, and cortical collecting duct. Thus, Pds (Slc26a4) is a candidate gene for the putative apical anion-exchange process of the type B intercalated cell. Because apical anion exchange-mediated transport is upregulated with deoxycorticosterone pivalate (DOCP), we tested whether Pds mRNA and protein expression in mouse kidney were upregulated after administration of this aldosterone analogue by using quantitative real-time polymerase chain reaction as well as light and electron microscopic immunolocalization. In kidneys from DOCP-treated mice, Pds mRNA increased 60%, whereas pendrin protein expression in the apical plasma membrane increased 2-fold in non-A, non-B intercalated cells and increased 6-fold in type B cells. Because pendrin transports HCO3- and Cl-, we tested whether DOCP treatment unmasks abnormalities in acid-base or NaCl balance in Pds (-/-) mice. In the absence of DOCP, arterial pH, systolic blood pressure, and body weight were similar in Pds (+/+) and Pds (-/-) mice. After DOCP treatment, weight gain and hypertension were observed in Pds (+/+) but not in Pds (-/-) mice. Moreover, after DOCP administration, metabolic alkalosis was more severe in Pds (-/-) than Pds (+/+) mice. We conclude that pendrin is upregulated with aldosterone analogues and is critical in the pathogenesis of mineralocorticoid-induced hypertension and metabolic alkalosis.
Asunto(s)
Proteínas Portadoras/genética , Desoxicorticosterona/farmacología , Riñón/efectos de los fármacos , Proteínas de Transporte de Membrana , Animales , Análisis de los Gases de la Sangre , Presión Sanguínea/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Proteínas Portadoras/análisis , Cloruros/sangre , Cloruros/orina , Regulación de la Expresión Génica/efectos de los fármacos , Genotipo , Concentración de Iones de Hidrógeno , Hipertensión/inducido químicamente , Hipertensión/genética , Hipertensión/metabolismo , Inmunohistoquímica , Riñón/metabolismo , Riñón/ultraestructura , Masculino , Ratones , Ratones Noqueados , Microscopía Inmunoelectrónica , Mineralocorticoides , Potasio/sangre , Potasio/orina , Sodio/sangre , Sodio/orina , Transportadores de SulfatoRESUMEN
Ammonia is both produced and transported by renal epithelial cells, and it regulates renal ion transport. Recent studies have identified a family of putative ammonium transporters; mRNA for two members of this family, Rh B-glycoprotein (RhBG) and Rh C-glycoprotein (RhCG), is expressed in the kidney. The purpose of this study was to determine the cellular location of RhBG and RhCG protein in the mouse kidney. We generated RhBG- and RhCG-specific anti-peptide antibodies. Immunoblot analysis confirmed that both proteins were expressed in the mouse kidney. RhBG localization with immunohistochemistry revealed discrete basolateral labeling in the connecting segment (CNT) and in the majority of initial collecting tubule (ICT) and cortical collecting duct (CCD) cells. In the outer medullary collecting duct (OMCD) and inner medullary collecting duct (IMCD) only a subpopulation of cells exhibited basolateral immunoreactivity. Colocalization of RhBG with carbonic anhydrase II, the thiazide-sensitive transporter, and the anion exchangers AE1 and pendrin demonstrated RhBG immunoreactivity in all CNT cells and all CCD and ICT principal cells. In the ICT and CCD, basolateral RhBG immunoreactivity is also present in A-type intercalated cells but not in pendrin-positive CCD intercalated cells. In the OMCD and IMCD, only intercalated cells exhibit RhBG immunoreactivity. Immunoreactivity for a second putative ammonium transporter, RhCG, was present in the apical region of cells with almost the same distribution as RhBG. However, RhCG immunoreactivity was present in all CCD cells, and it was present in outer stripe OMCD principal cells, in addition to OMCD and IMCD intercalated cells. Thus the majority of RhBG and RhCG protein expression is present in the same epithelial cell types in the CNT and collecting duct but with opposite polarity. These findings suggest that RhBG and RhCG may play important and cell-specific roles in ammonium transport and signaling in these regions of the kidney.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana , Simportadores , Animales , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Anhidrasa Carbónica II/metabolismo , Femenino , Immunoblotting , Ratones , Ratones Endogámicos BALB C , Receptores de Droga/metabolismo , Simportadores del Cloruro de Sodio , Miembro 3 de la Familia de Transportadores de Soluto 12 , Transportadores de Sulfato , Distribución TisularRESUMEN
Pendrin is an anion exchanger expressed in type B intercalated cells of the cortical collecting duct (CCD). Whether pendrin localizes to other nephron segments with intercalated cells is unknown. Moreover, whether pendrin is expressed in proximal tubule is debated. Thus the distribution of pendrin mRNA and protein expression in mouse kidney was investigated by using light and electron microscopic immunohistochemistry and quantitative real-time PCR. We observed that pendrin mRNA is expressed mainly in cortex. Within cortex, pendrin mRNA is at least fivefold higher in CCD and the connecting tubule (CNT) than in the other segments. Pendrin protein was observed in a subset of cells within the distal convoluted tubule as well as in type B and in non-A-non-B intercalated cells of the CNT and CCD. In type B intercalated cells, pendrin immunoreactivity was highest in apical cytoplasmic vesicles with little immunolabel along the apical plasma membrane. In non-A-non-B intercalated cells, intense pendrin immunoreactivity was detected along the apical plasma membrane. These differences in the subcellular distribution of pendrin immunolabel were confirmed by morphometric analysis. In conclusion, pendrin is expressed in the mouse distal convoluted tubule, CCD, and CNT along the apical plasma membrane of non-A-non-B intercalated cells and in subapical cytoplasmic vesicles of type B intercalated cells.
Asunto(s)
Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Túbulos Renales Colectores/química , Túbulos Renales Distales/química , Proteínas de Transporte de Membrana , Animales , Aniones/metabolismo , Proteínas Portadoras/metabolismo , Expresión Génica , Túbulos Renales Colectores/fisiología , Túbulos Renales Colectores/ultraestructura , Túbulos Renales Distales/fisiología , Túbulos Renales Distales/ultraestructura , Ratones , Microscopía Inmunoelectrónica , ARN Mensajero/análisis , Transportadores de SulfatoRESUMEN
Pendred syndrome is a major cause of congenital deafness, goiter and defective iodide organification. Mutations in the transmembrane protein, pendrin, cause diminished export of iodide from thyroid follicular cells to the colloid and are associated with the syndrome. We used green fluorescent protein (GFP) chimeras of wild-type (WT) pendrin and three common natural mutants (L236P, T416P and G384) to study their intracellular trafficking in living cells. Time-lapse imaging, dual color labeling and fluorescent recovery after photobleaching (FRAP) studies demonstrated that GFP-WT pendrin targets to the plasma membrane. In contrast, all three mutant pendrins were retained in the endoplasmic reticulum (ER) in co-localization studies with ER and Golgi markers. The ER retention of L236P appeared to be selective as this mutant did not prevent a viral membrane protein, VSVGtsO45 or wild-type pendrin from targeting the plasma membrane. These findings suggest that ER retention and defective plasma membrane targeting of pendrin mutants play a key role in the pathogenesis of Pendred syndrome.
Asunto(s)
Anomalías Múltiples/genética , Proteínas Portadoras/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Transporte de Membrana , Transporte de Proteínas/fisiología , Anomalías Múltiples/metabolismo , Sustitución de Aminoácidos , Genes Reporteros/fisiología , Humanos , Transportadores de SulfatoRESUMEN
Pendrin is an anion exchanger in the cortical collecting duct of the mammalian nephron that appears to mediate apical Cl(-)/HCO3(-) exchange in bicarbonate-secreting intercalated cells. The goals of this study were to determine 1) if pendrin immunoreactivity was present in the gills of a euryhaline elasmobranch (Atlantic stingray, Dasyatis sabina), and 2) if branchial pendrin immunoreactivity was influenced by environmental salinity. Immunoblots detected pendrin immunoreactivity in Atlantic stingray gills; pendrin immunoreactivity was greatest in freshwater stingrays compared with freshwater stingrays acclimated to seawater (seawater acclimated) and marine stingrays. Using immunohistochemistry, pendrin-positive cells were detected on both gill lamellae and interlamellar regions of freshwater stingrays but were more restricted to interlamellar regions in seawater-acclimated and marine stingray gills. Pendrin immunolabeling in freshwater stingray gills was more apical, discrete, and intense compared with seawater-acclimated and marine stingrays. Regardless of salinity, pendrin immunoreactivity occurred on the apical region of cells rich with basolateral vacuolar-proton-ATPase, and not in Na(+)-K(+)-ATPase-rich cells. We suggest that a pendrin-like transporter may contribute to apical Cl(-)/HCO3(-) exchange in gills of Atlantic stingrays from both freshwater and marine environments.
Asunto(s)
Proteínas Portadoras/metabolismo , Elasmobranquios/metabolismo , Branquias/metabolismo , Proteínas de Transporte de Membrana , Animales , Región Branquial/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Epitelio/metabolismo , Branquias/citología , Immunoblotting , Inmunohistoquímica , Agua de Mar , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Transportadores de Sulfato , Distribución Tisular , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
The Pendred syndrome gene encodes a 780-amino acid putative transmembrane protein (pendrin) that is expressed in the apical membrane of thyroid follicular cells. Although pendrin was shown to transport iodide and chloride using Xenopus laevis oocytes and Sf9 insect cells, there is no report using mammalian cells to study its role in thyroid function. We show here, using COS-7 cells and Chinese hamster ovary cells transfected with expression vectors encoding sodium iodide symporter or human Pendred syndrome gene cDNA and by comparison with studies using rat thyroid FRTL-5 cells, that pendrin is an iodide-specific transporter in mammalian cells and is responsible for iodide efflux in the thyroid.
Asunto(s)
Proteínas Portadoras/fisiología , Yoduros/farmacocinética , Proteínas de Transporte de Membrana , Glándula Tiroides/metabolismo , Animales , Transporte Biológico/fisiología , Células CHO , Células COS , Línea Celular , Membrana Celular/metabolismo , Cloruros/farmacología , Cricetinae , Humanos , Yodo/farmacocinética , Radioisótopos de Yodo , Ratas , Transportadores de Sulfato , Simportadores/fisiología , Glándula Tiroides/citologíaRESUMEN
Expression of the Pendred syndrome gene (PDS/Pds) is thought to be responsible for the iodide transport in the thyroid as well as the formation and function of the inner ear. Its mRNA is also expressed in the kidney and placenta. We report here that PDS and its encoded protein (pendrin) are also expressed in the endometrium. The RNA levels of rat PDS in the endometrium and kidney were much higher than those of the thyroid, opposite of the pattern of RNA expression in humans. In human endometrium, pendrin localization changed from the basal to apical surfaces of the epithelium during progression of the menstrual cycle. This suggests a possible role for pendrin in cationic ion transport required to maintain the physiological function of the endometrium. Since there is no evidence of endometrial abnormalities in patients with Pendred syndrome, it suggests the existence of a compensatory mechanisms for pendrin's function in the uterus.