RESUMO
BACKGROUND: Pendred syndrome, an autosomal-recessive disorder characterized by deafness and goiter, is caused by a mutation of SLC26A4, which codes for the anion exchanger pendrin. We investigated the relationship between pendrin expression and deafness using mice that have (Slc26a4+/+ or Slc26a4+/-) or lack (Slc26a4-/-) a complete Slc26a4 gene. Previously, we reported that stria vascularis of adult Slc26a4-/- mice is hyperpigmented and that marginal cells appear disorganized. Here we determine the time course of hyperpigmentation and marginal cell disorganization, and test the hypothesis that inflammation contributes to this tissue degeneration. METHODS: Slc26a4-/- and age-matched control (Slc26a4+/+ or Slc26a4+/-) mice were studied at four postnatal (P) developmental stages: before and after the age that marks the onset of hearing (P10 and P15, respectively), after weaning (P28-41) and adult (P74-170). Degeneration and hyperpigmentation stria vascularis was evaluated by confocal microscopy. Gene expression in stria vascularis was analyzed by microarray and quantitative RT-PCR. In addition, the expression of a select group of genes was quantified in spiral ligament, spleen and liver to evaluate whether expression changes seen in stria vascularis are specific for stria vascularis or systemic in nature. RESULTS: Degeneration of stria vascularis defined as hyperpigmentation and marginal cells disorganization was not seen at P10 or P15, but occurred after weaning and was associated with staining for CD68, a marker for macrophages. Marginal cells in Slc26a4-/-, however, had a larger apical surface area at P10 and P15. No difference in the expression of Lyzs, C3 and Cd45 was found in stria vascularis of P15 Slc26a4+/- and Slc26a4-/- mice. However, differences in expression were found after weaning and in adult mice. No difference in the expression of markers for acute inflammation, including Il1a, Il6, Il12a, Nos2 and Nos3 were found at P15, after weaning or in adults. The expression of macrophage markers including Ptprc (= Cd45), Cd68, Cd83, Lyzs, Lgals3 (= Mac2 antigen), Msr2, Cathepsins B, S, and K (Ctsb, Ctss, Ctsk) and complement components C1r, C3 and C4 was significantly increased in stria vascularis of adult Slc26a4-/- mice compared to Slc26a4+/+ mice. Expression of macrophage markers Cd45 and Cd84 and complement components C1r and C3 was increased in stria vascularis but not in spiral ligament, liver or spleen of Slc26a4-/- compared to Slc26a4+/- mice. The expression of Lyzs was increased in stria vascularis and spiral ligament but not in liver or spleen. CONCLUSION: The data demonstrate that hyperpigmentation of stria vascularis and marginal cell reorganization in Slc26a4-/- mice occur after weaning, coinciding with an invasion of macrophages. The data suggest that macrophage invasion contributes to tissue degeneration in stria vascularis, and that macrophage invasion is restricted to stria vascularis and is not systemic in nature. The delayed onset of degeneration of stria vascularis suggests that a window of opportunity exists to restore/preserve hearing in mice and therefore possibly in humans suffering from Pendred syndrome.
Assuntos
Proteínas de Transporte de Ânions/genética , Surdez/genética , Expressão Gênica , Bócio/genética , Macrófagos/fisiologia , Estria Vascular/patologia , Animais , Proteínas de Transporte de Ânions/metabolismo , Biomarcadores/metabolismo , Surdez/metabolismo , Surdez/patologia , Modelos Animais de Doenças , Hiperpigmentação/etiologia , Hiperpigmentação/metabolismo , Canal de Potássio KCNQ1/metabolismo , Camundongos , Camundongos Endogâmicos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Estria Vascular/imunologia , Transportadores de Sulfato , SíndromeRESUMO
For over 100 years after the first description of the disorder, the molecular pathology underlying the deafness and thyroid pathology in Pendred syndrome (PS) remained unknown. In 1997, early progress towards understanding the molecular basis of the disorder was made when we identified the PS gene and found it to belong to the SLC26 family of anion transporters. The realization that an anion transporter was responsible for these clinical features soon highlighted a potential role for pendrin in thyroid hormone biosynthesis. The role of pendrin in deafness, however, remained unclear. Our determination of its expression pattern in the inner ear along with the development of a mouse with a targeted disruption of the Slc26a4 gene has revealed that Slc26a4 is expressed in areas of the endolymphatic compartment known to play a role in endolymph reabsorption and that absence of this protein leads to a profound prenatal endolymphatic hydrops and destruction of many of the epithelial cells surrounding the scala media. The precise mechanisms underlying endolymph reabsorption in the inner ear are not yet known; these studies, however, provide some of the groundwork for allowing the future delineation of these processes.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Orelha Interna/fisiologia , Endolinfa/metabolismo , Homeostase/fisiologia , Animais , Orelha Interna/anormalidades , Camundongos , Filogenia , Potássio/metabolismo , Transportadores de Sulfato , Hormônios Tireóideos/biossínteseRESUMO
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.
Assuntos
Proteínas de Transporte/genética , Endométrio/fisiologia , Expressão Gênica , Proteínas de Membrana Transportadoras , Animais , Epitélio/metabolismo , Feminino , Humanos , Ciclo Menstrual/metabolismo , Camundongos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transportadores de Sulfato , Distribuição TecidualRESUMO
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.
Assuntos
Cóclea/química , Surdez/etiologia , Proteínas de Membrana Transportadoras/análise , Canais de Potássio Corretores do Fluxo de Internalização/análise , Vestíbulo do Labirinto/química , Animais , Conexina 26 , Conexinas , Endolinfa/química , Potenciais Evocados Auditivos/fisiologia , Bócio , Camundongos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , RNA Mensageiro/análise , Transportadores de Sulfato , SíndromeRESUMO
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.
Assuntos
Proteínas de Transporte/genética , Cóclea/fisiopatologia , Surdez/etiologia , Surdez/fisiopatologia , Bócio/fisiopatologia , Proteínas de Membrana Transportadoras , Animais , Proteínas de Transporte/metabolismo , Cóclea/metabolismo , Cóclea/patologia , Surdez/patologia , Endolinfa/fisiologia , Saco Endolinfático/metabolismo , Saco Endolinfático/patologia , Saco Endolinfático/fisiopatologia , Feminino , Imunofluorescência , Homeostase/fisiologia , Masculino , Camundongos , Camundongos Knockout , Potássio/metabolismo , Transportadores de Sulfato , Vestíbulo do Labirinto/metabolismo , Vestíbulo do Labirinto/patologia , Vestíbulo do Labirinto/fisiopatologiaRESUMO
Pendrin (Slc26a4) localizes to type B and non-A, non-B intercalated cells in the distal convoluted tubule, the connecting tubule, and the cortical collecting duct (CCD), where it mediates apical Cl(-)/HCO(3)(-) exchange. The purpose of this study was to determine whether angiotensin II increases transepithelial net chloride transport, J(Cl), in mouse CCD through a pendrin-dependent mechanism. J(Cl) and transepithelial voltage, V(T), were measured in CCDs perfused in vitro from wild-type and Slc26a4 null mice ingesting a NaCl-replete diet or a NaCl-replete diet and furosemide. In CCDs from wild-type mice ingesting a NaCl-replete diet, V(T) and J(Cl) were not different from zero either in the presence or absence of angiotensin II (10(-8) M) in the bath. Thus further experiments employed mice given the high-NaCl diet and furosemide to upregulate renal pendrin expression. CCDs from furosemide-treated wild-type mice had a lumen-negative V(T) and absorbed Cl(-). With angiotensin II in the bath, Cl(-) absorption doubled although V(T) did not become more lumen negative. In contrast, in CCDs from furosemide-treated Slc26a4 null mice, Cl(-) secretion and a V(T) of approximately 0 were observed, neither of which changed with angiotensin II application. Inhibiting ENaC with benzamil abolished V(T) although J(Cl) fell only approximately 50%. Thus substantial Cl(-) absorption is observed in the absence of an electromotive force. Attenuating apical anion exchange with the peritubular application of the H(+)-ATPase inhibitor bafilomycin abolished benzamil-insensitive Cl(-) absorption. In conclusion, angiotensin II increases transcellular Cl(-) absorption in the CCD through a pendrin- and H(+)-ATPase-dependent process.
Assuntos
Angiotensina II/farmacologia , Proteínas de Transporte de Ânions/fisiologia , Cloretos/metabolismo , Túbulos Renais Coletores/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Nitrogênio da Ureia Sanguínea , Inibidores Enzimáticos/farmacologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Feminino , Furosemida/farmacologia , Transporte de Íons/efeitos dos fármacos , Rim/anatomia & histologia , Rim/efeitos dos fármacos , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Túbulos Renais Coletores/efeitos dos fármacos , Macrolídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Tamanho do Órgão/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Cloreto de Sódio/farmacologia , Transportadores de Sulfato , Vasoconstritores/farmacologiaRESUMO
Pendrin (encoded by Pds, Slc26a4) is a Cl(-)/HCO(3)(-) exchanger expressed in the apical regions of type B and non-A, non-B intercalated cells of kidney and mediates renal Cl(-) absorption, particularly when upregulated. Aldosterone increases blood pressure by increasing absorption of both Na(+) and Cl(-) through increased protein abundance and function of Na(+) transporters, such as the epithelial Na(+) channel (ENaC) and the Na(+)-Cl(-) cotransporter (NCC), as well as Cl(-) transporters, such as pendrin. Because aldosterone analogs do not increase blood pressure in Slc26a4(-/-) mice, we asked whether Na(+) excretion and Na(+) transporter protein abundance are altered in kidneys from these mutant mice. Thus wild-type and Slc26a4-null mice were given a NaCl-replete, a NaCl-restricted, or NaCl-replete diet and aldosterone or aldosterone analogs. Abundance of the major renal Na(+) transporters was examined with immunoblots and immunohistochemistry. Slc26a4-null mice showed an impaired ability to conserve Na(+) during dietary NaCl restriction. Under treatment conditions in which circulating aldosterone is increased, alpha-, beta-, and 85-kDa gamma-ENaC subunit protein abundances were reduced 15-35%, whereas abundance of the 70-kDa fragment of gamma-ENaC was reduced approximately 70% in Slc26a4-null relative to wild-type mice. Moreover, ENaC-dependent changes in transepithelial voltage were much lower in cortical collecting ducts from Slc26a4-null than from wild-type mice. Thus, in kidney, ENaC protein abundance and function are modulated by pendrin or through a pendrin-dependent downstream event. The reduced ENaC protein abundance and function observed in Slc26a4-null mice contribute to their lower blood pressure and reduced ability to conserve Na(+) during NaCl restriction.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Pressão Sanguínea/fisiologia , Canais Epiteliais de Sódio/metabolismo , Rim/metabolismo , Glândulas Suprarrenais/fisiologia , Aldosterona/sangue , Aldosterona/farmacologia , Amilorida/farmacologia , Animais , Proteínas de Transporte de Ânions/genética , Colo/metabolismo , Canais Epiteliais de Sódio/efeitos dos fármacos , Canais Epiteliais de Sódio/genética , Feminino , Hipotálamo/fisiologia , Masculino , Camundongos , Camundongos Knockout , Sódio/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Simportadores de Cloreto de Sódio/metabolismo , Transportadores de Sulfato , Glândula Tireoide/metabolismoRESUMO
Slc26a4 (Pds) encodes pendrin, a Cl(-)/HCO(3)(-) exchanger expressed in the apical region of type B and non-A, non-B cells, which mediates secretion of OH(-) equivalents. Thus genetic disruption of Slc26a4 leads to systemic alkalosis in some treatment models. However, humans and mice with genetic disruption of Slc26a4 have normal acid-base balance under basal conditions. Thus we asked: 1) Is net acid excretion altered in Slc26a4 (-/-) mice under basal conditions? 2) In the absence of pendrin-mediated OH(-) secretion, are increases in intracellular and systemic pH minimized through changes in intercalated cell subtype abundance or intercalated cell H(+)/OH(-) transporter expression? To answer these questions, net acid excretion and H(+)/OH(-) transporter expression were examined in Slc26a4 (-/-) and Slc26a4 (+/+) mice using balance studies, immunolocalization, and immunoblotting. Excretion of ammonium, titratable acid, and citrate were the same in Slc26a4 null and wild-type mice. However, urinary pH and Pco(2) were much lower in Slc26a4 null relative to wild-type mice due to reduced urinary buffering of secreted H(+) by HCO(3)(-). Abundance of non-A, but not type A intercalated cells, was reduced within the cortical collecting ducts of Slc26a4 null mice. Moreover, kidneys from Slc26a4 null mice had reduced H(+)-ATPase, NBC3 and RhBG total protein expression, particularly within type B and non-A, non-B intercalated cells, although RhCG protein expression was unchanged. Reduced intercalated cell H(+)/OH(-) transporter expression is observed in Slc26a4 null mice, which likely attenuates the rise in intracellular and systemic pH expected with genetic disruption of Slc26a4.
Assuntos
Proteínas de Transporte de Ânions/deficiência , Proteínas de Transporte de Ânions/fisiologia , ATPases Translocadoras de Prótons/biossíntese , Simportadores de Sódio-Bicarbonato/biossíntese , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Camundongos , Camundongos Knockout , ATPases Translocadoras de Prótons/metabolismo , Transportadores de SulfatoRESUMO
A significant fraction of active chloride reabsorption across the apical membrane of the proximal tubule is mediated by a chloride/formate exchange process, whereby intracellular formate drives the transport of chloride into the cell. When chloride/formate exchange operates in parallel with Na(+)/H(+) exchange and H(+)-coupled recycling of formate, the net result is electroneutral NaCl reabsorption. Pendrin is the protein product of the PDS gene (SLC26A4) and functions in several different anion exchange modes, including chloride/formate exchange. Pendrin is expressed in the kidney and may serve as the transporter responsible for formate-dependent NaCl reabsorption. In the present study, Pds-knockout mice were used to determine the role of pendrin in proximal tubule chloride reabsorption. We show that formate-dependent NaCl absorption in microperfused proximal tubules is similar between wild-type and pendrin-deficient mice. In addition, there is no difference in the rate of formate-mediated chloride transport in brush-border membrane vesicles isolated from wild-type and pendrin-deficient mice. These studies demonstrate that pendrin is not responsible for formate-dependent NaCl reabsorption in the proximal tubule.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas de Membrana Transportadoras , Cloreto de Sódio/metabolismo , Animais , Transporte Biológico/fisiologia , Feminino , Formiatos/farmacocinética , Hemostáticos/farmacocinética , Masculino , Camundongos , Camundongos Knockout , Microvilosidades/metabolismo , Transportadores de SulfatoRESUMO
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.
Assuntos
Proteínas de Transporte/genética , Desoxicorticosterona/farmacologia , Rim/efeitos dos fármacos , Proteínas de Membrana Transportadoras , Animais , Gasometria , Pressão Sanguínea/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Proteínas de Transporte/análise , Cloretos/sangue , Cloretos/urina , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Concentração de Íons de Hidrogênio , Hipertensão/induzido quimicamente , Hipertensão/genética , Hipertensão/metabolismo , Imuno-Histoquímica , Rim/metabolismo , Rim/ultraestrutura , Masculino , Camundongos , Camundongos Knockout , Microscopia Imunoeletrônica , Mineralocorticoides , Potássio/sangue , Potássio/urina , Sódio/sangue , Sódio/urina , Transportadores de SulfatoRESUMO
Slc26a4 (Pds, pendrin) is an anion transporter expressed in the apical region of type B and non-A, non-B intercalated cells of the distal nephron. It is upregulated by aldosterone analogues and is critical in the development of mineralocorticoid-induced hypertension. Thus, Slc26a4 expression and its role in blood pressure and fluid and electrolyte homeostasis was explored during NaCl restriction, a treatment model in which aldosterone is appropriately increased. Ultrastructural immunolocalization, balance studies, and cortical collecting ducts (CCDs) perfused in vitro were used. With moderate physiological NaCl restriction, Slc26a4 expression in the apical plasma membrane increased 2- to 3-fold in type B intercalated cells. Because Slc26a4 transports Cl-, we tested whether NaCl balance differs in Slc26a4(+/+) and Slc26a4(-/-) mice during NaCl restriction. Cl- absorption was observed in CCDs from Slc26a4(+/+) but not from Slc26a4(-/-) mice. After moderate NaCl restriction, urinary volume and Cl- excretion were increased in Slc26a4(-/-) relative to Slc26a4(+/+) mice. Moreover, Slc26a4(-/-) mice had evidence of relative vascular volume depletion because they had a higher arterial pH, hematocrit, and blood urea nitrogen than wild-type mice. With moderate NaCl restriction, blood pressure was similar in Slc26a4(+/+) and Slc26a4(-/-) mice. However, on a severely restricted intake of NaCl, Slc26a4(-/-) mice were hypotensive relative to wild-type mice. We conclude that Slc26a4 is upregulated with NaCl restriction and is critical in the maintenance of acid-base balance and in the renal conservation of Cl- and water during NaCl restriction.