Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 21
Filtrar
1.
Pflugers Arch ; 476(6): 911-922, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38538989

RESUMO

Aldosterone is a steroid hormone that is important for maintaining the volume and ionic composition of extracellular fluids and is produced in the zona glomerulosa of the adrenal cortex. The basic mechanisms controlling aldosterone secretion are known. However, more detailed studies on the regulation of aldosterone secretion often fail due to the lack of suitable models: although secretion can be studied in cultured adrenocortical cells under defined conditions, the differentiation status of the cells is difficult to control and the complex anatomy of the adrenal cortex is lost. In living animals, the physiological context is intact, but the influences are manifold and the examination conditions cannot be sufficiently controlled. One method that closes the gap between cell models and studies in living animals is the isolated perfused adrenal gland. In the past, this method has provided important data on the pathophysiology of adrenal glands from larger animals, but the technique was not used in mice. Here, we developed a method for isolation and perfusion of the mouse adrenal gland to study aldosterone secretion. This technique preserves the complex anatomical and functional context of the mouse adrenal cortex, to ensure defined experimental conditions and to minimize extra-adrenal influences. Initial series of experiments with the ex vivo perfused mouse adrenal gland show that this model offers the possibility for unique insights into pathophysiological regulatory principles and is suitable for the use of genetically modified mouse models.


Assuntos
Glândulas Suprarrenais , Aldosterona , Animais , Aldosterona/metabolismo , Camundongos , Glândulas Suprarrenais/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Perfusão/métodos , Zona Glomerulosa/metabolismo
2.
Am J Hum Genet ; 103(5): 808-816, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30388404

RESUMO

Over the last decades, a growing spectrum of monogenic disorders of human magnesium homeostasis has been clinically characterized, and genetic studies in affected individuals have identified important molecular components of cellular and epithelial magnesium transport. Here, we describe three infants who are from non-consanguineous families and who presented with a disease phenotype consisting of generalized seizures in infancy, severe hypomagnesemia, and renal magnesium wasting. Seizures persisted despite magnesium supplementation and were associated with significant intellectual disability. Whole-exome sequencing and conventional Sanger sequencing identified heterozygous de novo mutations in the catalytic Na+, K+-ATPase α1 subunit (ATP1A1). Functional characterization of mutant Na+, K+-ATPase α1 subunits in heterologous expression systems revealed not only a loss of Na+, K+-ATPase function but also abnormal cation permeabilities, which led to membrane depolarization and possibly aggravated the effect of the loss of physiological pump activity. These findings underline the indispensable role of the α1 isoform of the Na+, K+-ATPase for renal-tubular magnesium handling and cellular ion homeostasis, as well as maintenance of physiologic neuronal activity.


Assuntos
Deficiência Intelectual/genética , Mutação/genética , Erros Inatos do Transporte Tubular Renal/genética , Convulsões/genética , ATPase Trocadora de Sódio-Potássio/genética , Criança , Pré-Escolar , Feminino , Células Germinativas , Heterozigoto , Homeostase/genética , Humanos , Lactente , Recém-Nascido , Rim/patologia , Magnésio/metabolismo , Masculino , Fenótipo , Isoformas de Proteínas/genética
3.
Kidney Int ; 97(6): 1208-1218, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32299681

RESUMO

The basolateral potassium channel KCNJ10 (Kir4.1), is expressed in the renal distal convoluted tubule and controls the activity of the thiazide-sensitive sodium chloride cotransporter. Loss-of-function mutations of KCNJ10 cause EAST/SeSAME syndrome with salt wasting and severe hypokalemia. KCNJ10 is also expressed in the principal cells of the collecting system. However, its pathophysiological role in this segment has not been studied in detail. To address this, we generated the mouse model AQP2cre:Kcnj10flox/flox with a deletion of Kcnj10 specifically in the collecting system (collecting system-Kcnj10-knockout). Collecting system-Kcnj10-knockout mice responded normally to standard and high potassium diet. However, this knockout exhibited a higher kaliuresis and lower plasma potassium than control mice when treated with thiazide diuretics. Likewise, collecting systemKcnj10-knockout displayed an inadequately high kaliuresis and renal sodium retention upon dietary potassium restriction. In this condition, these knockout mice became hypokalemic due to insufficient downregulation of the epithelial sodium channel (ENaC) and the renal outer medullary potassium channel (ROMK) in the collecting system. Consistently, the phenotype of collecting system-Kcnj10-knockout was fully abrogated by ENaC inhibition with amiloride and ameliorated by genetic inactivation of ROMK in the collecting system. Thus, KCNJ10 in the collecting system contributes to the renal control of potassium homeostasis by regulating ENaC and ROMK. Hence, impaired KCNJ10 function in the collecting system predisposes for thiazide and low potassium diet-induced hypokalemia and likely contributes to the pathophysiology of renal potassium loss in EAST/SeSAME syndrome.


Assuntos
Hipopotassemia , Canais de Potássio Corretores do Fluxo de Internalização , Animais , Dieta , Canais Epiteliais de Sódio , Hipopotassemia/induzido quimicamente , Hipopotassemia/genética , Camundongos , Camundongos Knockout , Potássio , Canais de Potássio Corretores do Fluxo de Internalização/genética , Tiazidas
4.
Pflugers Arch ; 467(5): 1027-42, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25339223

RESUMO

The physiological control of steroid hormone secretion from the adrenal cortex depends on the function of potassium channels. The "two-pore domain K(+) channels" (K2P) TWIK-related acid sensitive K(+) channel 1 (TASK1), TASK3, and TWIK-related K(+) channel 1 (TREK1) are strongly expressed in adrenocortical cells. They confer a background K(+) conductance to these cells which is important for the K(+) sensitivity as well as for angiotensin II and adrenocorticotropic hormone-dependent stimulation of aldosterone and cortisol synthesis. Mice with single deletions of the Task1 or Task3 gene as well as Task1/Task3 double knockout mice display partially autonomous aldosterone synthesis. It appears that TASK1 and TASK3 serve different functions: TASK1 affects cell differentiation and prevents expression of aldosterone synthase in the zona fasciculata, while TASK3 controls aldosterone secretion in glomerulosa cells. TREK1 is involved in the regulation of cortisol secretion in fasciculata cells. These data suggest that a disturbed function of K2P channels could contribute to adrenocortical pathologies in humans.


Assuntos
Córtex Suprarrenal/metabolismo , Aldosterona/metabolismo , Angiotensina II/metabolismo , Membrana Celular/metabolismo , Canais de Potássio/metabolismo , Animais , Humanos , Proteínas do Tecido Nervoso/metabolismo
5.
Kidney Int ; 85(6): 1369-81, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24476694

RESUMO

The role of calcium-activated chloride channels for renal function is unknown. By immunohistochemistry we demonstrate dominant expression of the recently identified calcium-activated chloride channels, Anoctamin 1 (Ano1, TMEM16A) in human and mouse proximal tubular epithelial (PTE) cells, with some expression in podocytes and other tubular segments. Ano1-null mice had proteinuria and numerous large reabsorption vesicles in PTE cells. Selective knockout of Ano1 in podocytes (Ano1-/-/Nphs2-Cre) did not impair renal function, whereas tubular knockout in Ano1-/-/Ksp-Cre mice increased urine protein excretion and decreased urine electrolyte concentrations. Purinergic stimulation activated calcium-dependent chloride currents in isolated proximal tubule epithelial cells from wild-type but not from Ano1-/-/Ksp-Cre mice. Ano1 currents were activated by acidic pH, suggesting parallel stimulation of Ano1 chloride secretion with activation of the proton-ATPase. Lack of calcium-dependent chloride secretion in cells from Ano1-/-/Ksp-Cre mice was paralleled by attenuated proton secretion and reduced endosomal acidification, which compromised proximal tubular albumin uptake. Tubular knockout of Ano1 enhanced serum renin and aldosterone concentrations, probably leading to enhanced compensatory distal tubular reabsorption, thus maintaining normal blood pressure levels. Thus, Ano1 has a role in proximal tubular proton secretion and protein reabsorption. The results correspond to regulation of the proton-ATPase by the Ano1-homolog Ist2 in yeast.


Assuntos
Canais de Cloreto/metabolismo , Túbulos Renais Proximais/metabolismo , Podócitos/metabolismo , Reabsorção Renal , Trifosfato de Adenosina/farmacologia , Aldosterona/sangue , Animais , Anoctamina-1 , Células Cultivadas , Canais de Cloreto/deficiência , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/genética , Feminino , Genótipo , Humanos , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/fisiopatologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fenótipo , Podócitos/efeitos dos fármacos , Proteinúria/genética , Proteinúria/metabolismo , Proteinúria/fisiopatologia , Reabsorção Renal/efeitos dos fármacos , Renina/sangue , Fatores de Tempo , ATPases Vacuolares Próton-Translocadoras/metabolismo
6.
Hum Mol Genet ; 21(22): 4922-9, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22918120

RESUMO

Primary aldosteronism (PA, autonomous aldosterone production from the adrenal cortex) causes the most common form of secondary arterial hypertension (HT), which is also the most common curable form of HT. Recent studies have highlighted an important role of mutations in genes encoding potassium channels in the pathogenesis of PA, both in human disease and in animal models. Here, we have exploited the unique features of the hyperaldosteronemic phenotype of Kcnk3 null mice, which is dependent on sexual hormones, to identify genes whose expression is modulated in the adrenal gland according to the dynamic hyperaldosteronemic phenotype of those animals. Genetic inactivation of one of the genes identified by our strategy, dickkopf-3 (Dkk3), whose expression is increased by calcium influx into adrenocortical cells, in the Kcnk3 null background results in the extension of the low-renin, potassium-rich diet insensitive hyperaldosteronemic phenotype to the male sex. Compound Kcnk3/Dkk3 animals display an increased expression of Cyp11b2, the rate-limiting enzyme for aldosterone biosynthesis in the adrenal zona glomerulosa (ZG). Our data show that Dkk3 can act as a modifier gene in a mouse model for altered potassium channel function and suggest its potential involvement in human PA syndromes.


Assuntos
Córtex Suprarrenal/metabolismo , Aldosterona/biossíntese , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Córtex Suprarrenal/patologia , Animais , Cálcio/metabolismo , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Inativação Gênica , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Fenótipo
7.
Nephron Physiol ; 124(3-4): 7-13, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24296675

RESUMO

Genetically modified mice represent important models for elucidating renal pathophysiology, but gene deletions frequently cause severe failure to thrive. In such cases, the analysis of the phenotype is often limited to the first weeks of life when renal excretory function undergoes dramatic physiological changes. Here, we investigated the postnatal dynamics of urinary ion excretion in mice. The profiles of urinary electrolyte excretion of mice were examined from birth until after weaning using an automated ion chromatography system. Postnatally, mice grew about 0.4 g/day, except during two phases with slower weight gain: (i) directly after birth during adaptation to extrauterine conditions (P0-P2) and (ii) during the weaning period (P15-P21), when nutrition changed from mother's milk to solid chow and water. During the first 3 days after birth, remarkable changes in urinary Na(+), Ca(2+), Mg(2+), and phosphate concentrations occurred, whereas K(+) and Cl(-) concentrations hardly changed. From days 4-14 after birth, Na(+), Ca(2+), Mg(2+), K(+), and Cl(-) concentrations remained relatively stable at low levels. Urinary concentrations of creatinine, NH4(+), phosphate, and sulfate constantly increased from birth until after weaning. Profiles of salt excretion in KCNJ10(-/-) mice exemplified the relevance of age-dependent analysis of urinary excretion. In conclusion, the most critical phases for analysis of renal ion excretion during the first weeks of life are directly after birth and during the weaning period. The age dependence of urinary excretion varies for the different ions. This should be taken into consideration when the renal phenotype of mice is investigated during the first weeks of life.


Assuntos
Animais Recém-Nascidos/crescimento & desenvolvimento , Creatinina/urina , Íons/urina , Rim/fisiologia , Fatores Etários , Animais , Cromatografia por Troca Iônica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Desmame
8.
Proc Natl Acad Sci U S A ; 107(32): 14490-5, 2010 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-20651251

RESUMO

Mutations of the KCNJ10 (Kir4.1) K(+) channel underlie autosomal recessive epilepsy, ataxia, sensorineural deafness, and (a salt-wasting) renal tubulopathy (EAST) syndrome. We investigated the localization of KCNJ10 and the homologous KCNJ16 in kidney and the functional consequences of KCNJ10 mutations found in our patients with EAST syndrome. Kcnj10 and Kcnj16 were found in the basolateral membrane of mouse distal convoluted tubules, connecting tubules, and cortical collecting ducts. In the human kidney, KCNJ10 staining was additionally observed in the basolateral membrane of the cortical thick ascending limb of Henle's loop. EM of distal tubular cells of a patient with EAST syndrome showed reduced basal infoldings in this nephron segment, which likely reflects the morphological consequences of the impaired salt reabsorption capacity. When expressed in CHO and HEK293 cells, the KCNJ10 mutations R65P, G77R, and R175Q caused a marked impairment of channel function. R199X showed complete loss of function. Single-channel analysis revealed a strongly reduced mean open time. Qualitatively similar results were obtained with coexpression of KCNJ10/KCNJ16, suggesting a dominance of KCNJ10 function in native renal KCNJ10/KCNJ16 heteromers. The decrease in the current of R65P and R175Q was mainly caused by a remarkable shift of pH sensitivity to the alkaline range. In summary, EAST mutations of KCNJ10 lead to impaired channel function and structural changes in distal convoluted tubules. Intriguingly, the metabolic alkalosis present in patients carrying the R65P mutation possibly improves residual function of KCNJ10, which shows higher activity at alkaline pH.


Assuntos
Anormalidades Múltiplas/genética , Mutação de Sentido Incorreto , Canais de Potássio Corretores do Fluxo de Internalização/genética , Animais , Ataxia , Linhagem Celular , Epilepsia , Perda Auditiva Neurossensorial , Humanos , Nefropatias , Túbulos Renais Distais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Canais de Potássio Corretores do Fluxo de Internalização/análise , Síndrome , Transfecção
9.
Proc Natl Acad Sci U S A ; 107(5): 2325-30, 2010 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-20133877

RESUMO

Task2 K(+) channel expression in the central nervous system is surprisingly restricted to a few brainstem nuclei, including the retrotrapezoid (RTN) region. All Task2-positive RTN neurons were lost in mice bearing a Phox2b mutation that causes the human congenital central hypoventilation syndrome. In plethysmography, Task2(-/-) mice showed disturbed chemosensory function with hypersensitivity to low CO(2) concentrations, leading to hyperventilation. Task2 probably is needed to stabilize the membrane potential of chemoreceptive cells. In addition, Task2(-/-) mice lost the long-term hypoxia-induced respiratory decrease whereas the acute carotid-body-mediated increase was maintained. The lack of anoxia-induced respiratory depression in the isolated brainstem-spinal cord preparation suggested a central origin of the phenotype. Task2 activation by reactive oxygen species generated during hypoxia could silence RTN neurons, thus contributing to respiratory depression. These data identify Task2 as a determinant of central O(2) chemoreception and demonstrate that this phenomenon is due to the activity of a small number of neurons located at the ventral medullary surface.


Assuntos
Dióxido de Carbono/fisiologia , Oxigênio/fisiologia , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Centro Respiratório/fisiologia , Animais , Animais Recém-Nascidos , Tronco Encefálico/patologia , Tronco Encefálico/fisiologia , Tronco Encefálico/fisiopatologia , Células Quimiorreceptoras/patologia , Células Quimiorreceptoras/fisiologia , Modelos Animais de Doenças , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Humanos , Hipercapnia/fisiopatologia , Hipóxia/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Pletismografia Total , Canais de Potássio de Domínios Poros em Tandem/deficiência , Canais de Potássio de Domínios Poros em Tandem/genética , Gravidez , Fenômenos Fisiológicos Respiratórios , Apneia do Sono Tipo Central/etiologia , Apneia do Sono Tipo Central/genética , Apneia do Sono Tipo Central/fisiopatologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
10.
Nat Genet ; 55(10): 1623-1631, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37709865

RESUMO

Primary aldosteronism (PA) is the most common form of endocrine hypertension and is characterized by inappropriately elevated aldosterone production via a renin-independent mechanism. Driver somatic mutations for aldosterone excess have been found in approximately 90% of aldosterone-producing adenomas (APAs). Other causes of lateralized adrenal PA include aldosterone-producing nodules (APNs). Using next-generation sequencing, we identified recurrent in-frame deletions in SLC30A1 in four APAs and one APN (p.L51_A57del, n = 3; p.L49_L55del, n = 2). SLC30A1 encodes the ubiquitous zinc efflux transporter ZnT1 (zinc transporter 1). The identified SLC30A1 variants are situated close to the zinc-binding site (His43 and Asp47) in transmembrane domain II and probably cause abnormal ion transport. Cases of PA with SLC30A1 mutations showed male dominance and demonstrated increased aldosterone and 18-oxocortisol concentrations. Functional studies of the SLC30A151_57del variant in a doxycycline-inducible adrenal cell system revealed pathological Na+ influx. An aberrant Na+ current led to depolarization of the resting membrane potential and, thus, to the opening of voltage-gated calcium (Ca2+) channels. This resulted in an increase in cytosolic Ca2+ activity, which stimulated CYP11B2 mRNA expression and aldosterone production. Collectively, these data implicate zinc transporter alterations as a dominant driver of aldosterone excess in PA.


Assuntos
Adenoma , Neoplasias do Córtex Suprarrenal , Adenoma Adrenocortical , Proteínas de Transporte de Cátions , Hiperaldosteronismo , Masculino , Humanos , Aldosterona/genética , Adenoma Adrenocortical/genética , Hiperaldosteronismo/genética , Adenoma/genética , Adenoma/complicações , Mutação , Zinco/metabolismo , Neoplasias do Córtex Suprarrenal/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Proteínas de Transporte de Cátions/genética
11.
EMBO J ; 27(1): 179-87, 2008 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-18034154

RESUMO

TASK1 (KCNK3) and TASK3 (KCNK9) are two-pore domain potassium channels highly expressed in adrenal glands. TASK1/TASK3 heterodimers are believed to contribute to the background conductance whose inhibition by angiotensin II stimulates aldosterone secretion. We used task1-/- mice to analyze the role of this channel in adrenal gland function. Task1-/- exhibited severe hyperaldosteronism independent of salt intake, hypokalemia, and arterial 'low-renin' hypertension. The hyperaldosteronism was fully remediable by glucocorticoids. The aldosterone phenotype was caused by an adrenocortical zonation defect. Aldosterone synthase was absent in the outer cortex normally corresponding to the zona glomerulosa, but abundant in the reticulo-fasciculata zone. The impaired mineralocorticoid homeostasis and zonation were independent of the sex in young mice, but were restricted to females in adults. Patch-clamp experiments on adrenal cells suggest that task3 and other K+ channels compensate for the task1 absence. Adrenal zonation appears as a dynamic process that even can take place in adulthood. The striking changes in the adrenocortical architecture in task1-/- mice are the first demonstration of the causative role of a potassium channel in development/differentiation.


Assuntos
Glândulas Suprarrenais/metabolismo , Homeostase/genética , Mineralocorticoides/antagonistas & inibidores , Mineralocorticoides/metabolismo , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Canais de Potássio de Domínios Poros em Tandem/deficiência , Canais de Potássio de Domínios Poros em Tandem/genética , Glândulas Suprarrenais/patologia , Aldosterona/sangue , Aldosterona/metabolismo , Animais , Feminino , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/antagonistas & inibidores , Potássio/sangue , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Renina/sangue
12.
N Engl J Med ; 360(19): 1960-70, 2009 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-19420365

RESUMO

BACKGROUND: Five children from two consanguineous families presented with epilepsy beginning in infancy and severe ataxia, moderate sensorineural deafness, and a renal salt-losing tubulopathy with normotensive hypokalemic metabolic alkalosis. We investigated the genetic basis of this autosomal recessive disease, which we call the EAST syndrome (the presence of epilepsy, ataxia, sensorineural deafness, and tubulopathy). METHODS: Whole-genome linkage analysis was performed in the four affected children in one of the families. Newly identified mutations in a potassium-channel gene were evaluated with the use of a heterologous expression system. Protein expression and function were further investigated in genetically modified mice. RESULTS: Linkage analysis identified a single significant locus on chromosome 1q23.2 with a lod score of 4.98. This region contained the KCNJ10 gene, which encodes a potassium channel expressed in the brain, inner ear, and kidney. Sequencing of this candidate gene revealed homozygous missense mutations in affected persons in both families. These mutations, when expressed heterologously in xenopus oocytes, caused significant and specific decreases in potassium currents. Mice with Kcnj10 deletions became dehydrated, with definitive evidence of renal salt wasting. CONCLUSIONS: Mutations in KCNJ10 cause a specific disorder, consisting of epilepsy, ataxia, sensorineural deafness, and tubulopathy. Our findings indicate that KCNJ10 plays a major role in renal salt handling and, hence, possibly also in blood-pressure maintenance and its regulation.


Assuntos
Ataxia/genética , Epilepsia/genética , Perda Auditiva Neurossensorial/genética , Mutação de Sentido Incorreto , Canais de Potássio Corretores do Fluxo de Internalização/genética , Erros Inatos do Transporte Tubular Renal/genética , Sequência de Aminoácidos , Animais , Pré-Escolar , Cromossomos Humanos Par 1 , Feminino , Genes Recessivos , Humanos , Escore Lod , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Linhagem , Fenótipo , Potássio/metabolismo , Análise de Sequência de DNA , Sódio/metabolismo , Síndrome
13.
Pflugers Arch ; 461(4): 423-35, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21221631

RESUMO

Mutations in the K+ channel gene KCNJ10 (Kir4.1) cause the autosomal recessive EAST syndrome which is characterized by epilepsy, ataxia, sensorineural deafness, and a salt-wasting tubulopathy. The renal salt-wasting pathology of EAST syndrome is caused by transport defects in the distal convoluted tubule where KCNJ10 plays a pivotal role as a basolateral K+ channel. This review on EAST syndrome outlines the molecular aspects of the physiology and pathophysiology of KCNJ10 in the distal convoluted tubule.


Assuntos
Nefropatias/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Sais/metabolismo , Ataxia/genética , Epilepsia/genética , Humanos , Nefropatias/fisiopatologia , Túbulos Renais Distais/fisiopatologia , Mutação/genética , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Síndrome
14.
Endocrinology ; 161(10)2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32785697

RESUMO

The physiological stimulation of aldosterone production in adrenocortical glomerulosa cells by angiotensin II and high plasma K+ depends on the depolarization of the cell membrane potential and the subsequent Ca2+ influx via voltage-activated Ca2+ channels. Germline mutations of the low-voltage activated T-type Ca2+ channel CACNA1H (Cav3.2) have been found in patients with primary aldosteronism. Here, we investigated the electrophysiology and Ca2+ signaling of adrenal NCI-H295R cells overexpressing CACNA1H wildtype and mutant M1549V in order to understand how mutant CACNA1H alters adrenal cell function. Whole-cell patch-clamp measurements revealed a strong activation of mutant CACNA1H at the resting membrane potential of adrenal cells. Both the expression of wildtype and mutant CACNA1H led to a depolarized membrane potential. In addition, cells expressing mutant CACNA1H developed pronounced action potential-like membrane voltage oscillations. Ca2+ measurements showed an increased basal Ca2+ activity, an altered K+ sensitivity, and abnormal oscillating Ca2+ changes in cells with mutant CACNA1H. In addition, removal of extracellular Na+ reduced CACNA1H current, voltage oscillations, and Ca2+ levels in mutant cells, suggesting a role of the partial Na+ conductance of CACNA1H in cellular pathology. In conclusion, the pathogenesis of stimulus-independent aldosterone production in patients with CACNA1H mutations involves several factors: i) a loss of normal control of the membrane potential, ii) an increased Ca2+ influx at basal conditions, and iii) alterations in sensitivity to extracellular K+ and Na+. Finally, our findings underline the importance of CACNA1H in the control of aldosterone production and support the concept of the glomerulosa cell as an electrical oscillator.


Assuntos
Glândulas Suprarrenais/fisiopatologia , Canais de Cálcio Tipo T/genética , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologia , Aldosterona/metabolismo , Animais , Células CHO , Cálcio/metabolismo , Canais de Cálcio Tipo T/metabolismo , Cricetinae , Cricetulus , Humanos , Hiperaldosteronismo/patologia , Hiperaldosteronismo/fisiopatologia , Potenciais da Membrana , Mutação , Técnicas de Patch-Clamp , Sódio/metabolismo , Células Tumorais Cultivadas , Zona Glomerulosa/metabolismo , Zona Glomerulosa/patologia , Zona Glomerulosa/fisiopatologia
15.
Biochim Biophys Acta ; 1771(7): 853-63, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17531529

RESUMO

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used for a detailed analysis of cellular phospholipid and cholesterol efflux in free cholesterol (FC) loaded human primary fibroblasts and human monocyte-derived macrophages (HMDM) loaded with enzymatically modified LDL (E-LDL). Although both cell models differed significantly in their cellular lipid composition, a higher apoA-I specific efflux was found for monounsaturated phosphatidylcholine (PC) species together with a decreased contribution of polyunsaturated PC species in both cell types. Moreover, medium chain sphingomyelin (SPM) species SPM 14:0 and SPM 16:1 were translocated preferentially to apoA-I in both cell types. In contrast to fibroblasts, HMDM displayed a considerable proportion of cholesteryl esters (CE) in basal and apoA-I specific efflux media, most likely due to secretion of CE associated to apoE. Analysis of HDL(3) mediated lipid efflux from HMDM using D(9)-choline and (13)C(3)-FC stable isotope labeling revealed significantly different D(9)-PC and D(9)-SPM species pattern for apoA-I and HDL(3) specific efflux media, which indicates a contribution of distinct cellular phospholipid pools to apoA-I and HDL(3) mediated efflux. Together with a partial loading of fibroblasts and HMDM with HDL(3)-derived CE species, these data add further evidence for retroendocytosis of HDL. In summary, analysis of apoA-I/ABCA1 and HDL(3) mediated lipid efflux by ESI-MS/MS demonstrated a preferential efflux of monounsaturated PC and medium chain SPM to apoA-I. Moreover, this is the first study, which provides evidence for distinct cellular phospholipid pools used for lipid transfer to apoA-I and HDL(3) from the analysis of phospholipid species pattern in HMDM.


Assuntos
Apolipoproteína A-I/farmacologia , Fibroblastos/efeitos dos fármacos , Lipoproteínas HDL3/farmacologia , Monócitos/efeitos dos fármacos , Fosfatidilcolinas/metabolismo , Esfingomielinas/metabolismo , Transporte Biológico/efeitos dos fármacos , Isótopos de Carbono , Células Cultivadas , Colesterol/metabolismo , Meios de Cultura , Deutério , Fibroblastos/química , Fibroblastos/metabolismo , Humanos , Marcação por Isótopo , Monócitos/química , Monócitos/metabolismo , Fosfatidilcolinas/química , Espectrometria de Massas por Ionização por Electrospray , Esfingomielinas/química
16.
J Clin Endocrinol Metab ; 100(1): E114-8, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25322277

RESUMO

CONTEXT: Familial hyperaldosteronism type III (FH-III) is a rare and clinically heterogeneous condition, that can display mild as well as severe phenotypes. Point mutations in the KCNJ5 gene, affecting the ion selectivity of the inward rectifier K(+) channel 4 (Kir3.4), underlie the molecular basis of FH-III. OBJECTIVE: The objective of the study was to investigate the effects of a de novo germline KCNJ5 mutation. PATIENTS AND METHODS: We describe the case of a girl who came to medical attention at the age of 2 years because of polydipsia, polyuria, and failure to thrive. The patient, affected by hypertension and hypokalemia, was diagnosed with primary aldosteronism on the basis of extremely high aldosterone levels and suppressed plasma renin activity. Genomic DNA was isolated and KCNJ5 sequenced. Human adrenocortical cells were used as an in vitro model for the functional characterization of the mutant channel. RESULTS: KCNJ5 sequencing in the index case and her parents revealed a de novo p.Glu145Gln germline mutation. The substitution resulted in Na(+)-dependent depolarization of adrenal cells and increased intracellular calcium concentration, which activated the transcription of NR4A2 and, in turn, CYP11B2. Pharmacological studies revealed that the mutant channel was insensitive to tertiapin-Q and calcium-channel blocker verapamil. CONCLUSIONS: Herein we report the identification of a novel KCNJ5 germline mutation responsible for severe hyperaldosteronism that presented in infancy with symptoms of diabetes insipidus. The findings of this study further elucidate the etiology of FH-III and expand our knowledge of this rare condition.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Hiperaldosteronismo/genética , Mutação , Pré-Escolar , Insuficiência de Crescimento/genética , Feminino , Humanos , Polidipsia/genética , Poliúria/genética
17.
J Clin Endocrinol Metab ; 99(9): E1765-73, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25057880

RESUMO

CONTEXT: Understanding the function of the KCNJ5 potassium channel through characterization of naturally occurring novel mutations is key for dissecting the mechanism(s) of autonomous aldosterone secretion in primary aldosteronism. OBJECTIVE: We sought for such novel KCNJ5 channel mutations in a large database of patients with aldosterone-producing adenomas (APAs). METHODS: We discovered a novel somatic c.446insAAC insertion, resulting in the mutant protein KCNJ5-insT149, in a patient with severe drug-resistant hypertension among 195 consecutive patients with a conclusive diagnosis of APA, 24.6% of whom showed somatic KCNJ5 mutations. By site-directed mutagenesis, we created the mutated cDNA that was transfected, along with KCNJ3 cDNA, in mammalian cells. We also localized CYP11B2 in the excised adrenal gland with immunohistochemistry and immunofluorescence using an antibody specific to human CYP11B2. Whole-cell patch clamp recordings, CYP11B2 mRNA, aldosterone measurement, and molecular modeling were performed to characterize the novel KCNJ5-insT149 mutation. RESULTS: Compared with wild-type and mock-transfected adrenocortical cells, HAC15 cells expressing the mutant KCNJ5 showed increased CYP11B2 expression and aldosterone secretion. Mammalian cells expressing the mutated KCNJ5-insT149 channel exhibited a strong Na(+) inward current and, in parallel, a substantial rise in intracellular Ca(2+), caused by activation of voltage-gated Ca(2+) channels and reduced Ca(2+) elimination by Na(+)/Ca(2+) exchangers, as well as an increased production of aldosterone. CONCLUSIONS: This novel mutation shows pathological Na(+) permeability, membrane depolarization, raised cytosolic Ca(2+), and increased aldosterone synthesis. Hence, a novel KCNJ5 channelopathy located after the pore α-helix preceding the selectivity filter causes constitutive secretion of aldosterone with ensuing resistant hypertension in a patient with a small APA.


Assuntos
Adenoma/genética , Adenoma Adrenocortical/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Hiperaldosteronismo/genética , Hipertensão/genética , Potássio/metabolismo , Adenoma/metabolismo , Córtex Suprarrenal/citologia , Córtex Suprarrenal/metabolismo , Adenoma Adrenocortical/metabolismo , Idoso , Aldosterona/metabolismo , Anti-Hipertensivos/uso terapêutico , Linhagem Celular , Resistência a Medicamentos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Humanos , Hiperaldosteronismo/metabolismo , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Masculino , Mutação Puntual
18.
Endocrinology ; 154(8): 2712-22, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23698720

RESUMO

Task3 K(+) channels are highly expressed in the adrenal cortex and contribute to the angiotensin II and K(+) sensitivity of aldosterone-producing glomerulosa cells. Adult Task3(-/-) mice display a partially autonomous aldosterone secretion, subclinical hyperaldosteronism, and salt-sensitive hypertension. Here, we investigated the age dependence of the adrenal phenotype of Task3(-/-) mice. Compared with adults, newborn Task3(-/-) mice displayed a severe adrenal phenotype with strongly increased plasma levels of aldosterone, corticosterone, and progesterone. This adrenocortical dysfunction was accompanied by a modified gene expression profile. The most strongly up-regulated gene was the protease renin. Real-time PCR corroborated the strong increase in adrenal renin expression, and immunofluorescence revealed renin-expressing cells in the zona fasciculata. Together with additional factors, activation of the local adrenal renin system is probably causative for the severely disturbed steroid hormone secretion of neonatal Task3(-/-) mice. The changes in gene expression patterns of neonatal Task3(-/-) mice could also be relevant for other forms of hyperaldosteronism.


Assuntos
Glândulas Suprarrenais/metabolismo , Hiperaldosteronismo/genética , Canais de Potássio/genética , Sistema Renina-Angiotensina/genética , Aldosterona/sangue , Aldosterona/metabolismo , Animais , Animais Recém-Nascidos , Corticosterona/sangue , Corticosterona/metabolismo , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Feminino , Imunofluorescência , Perfilação da Expressão Gênica , Hiperaldosteronismo/sangue , Hiperaldosteronismo/metabolismo , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Canais de Potássio/deficiência , Progesterona/sangue , Progesterona/metabolismo , Renina/sangue , Renina/genética , Renina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Zona Fasciculada/metabolismo
19.
Endocrinology ; 153(10): 4740-8, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22878402

RESUMO

Task1 and Task3 potassium channels (Task: tandem of P domains in a weak inward rectifying K(+) channel-related acid-sensitive K(+) channel) are believed to control the membrane voltage of aldosterone-producing adrenal glomerulosa cells. This study aimed at understanding the role of Task3 for the control of aldosterone secretion. The adrenal phenotype of Task3(-/-) mice was investigated using electrophysiology, adrenal slices, and blood pressure measurements. Primary adrenocortical cells of Task3(-/-) mice were strongly depolarized compared with wild-type (-52 vs. -79 mV), and in fresh adrenal slices Ca(2+) signaling of Task3(-/-) glomerulosa cells was abnormal. In living Task3(-/-) mice, the regulation of aldosterone secretion showed specific deficits: Under low Na(+) and high K(+) diets, protocols known to increase aldosterone, and under standard diet, Task3 inactivation was compensated and aldosterone was normal. However, high Na(+) and low K(+) diets, two protocols known to lower aldosterone, failed to lower aldosterone in Task3(-/-) mice. The physiological regulation of aldosterone was disturbed: aldosterone-renin ratio, an indicator of autonomous aldosterone secretion, was 3-fold elevated at standard and high Na(+) diets. Isolated adrenal glands of Task3(-/-) produced 2-fold more aldosterone. As a consequence, Task3(-/-) mice showed salt-sensitive arterial hypertension (plus 10 mm Hg). In conclusion, Task3 plays an important role in the adaptation of aldosterone secretion to dietary salt intake.


Assuntos
Glândulas Suprarrenais/metabolismo , Pressão Sanguínea/genética , Hipertensão/genética , Canais de Potássio/genética , Renina/sangue , Cloreto de Sódio na Dieta , Adaptação Fisiológica/genética , Glândulas Suprarrenais/citologia , Aldosterona/sangue , Animais , Cálcio/metabolismo , Células Cultivadas , Hipertensão/metabolismo , Camundongos , Camundongos Knockout , Fenótipo , Canais de Potássio/metabolismo , Zona Glomerulosa/citologia , Zona Glomerulosa/metabolismo
20.
Hypertension ; 59(2): 235-40, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22203740

RESUMO

Primary aldosteronism is the most frequent cause of endocrine hypertension. Three forms of familial hyperaldosteronism (FH) have been described, named FH-I to -III. Recently, a mutation of KCNJ5 has been shown to be associated with FH-III, whereas the cause of FH-II is still unknown. In this study we searched for mutations in KCNJ5 in 46 patients from 21 families with FH, in which FH-I was excluded. We identified a new germline G151E mutation in 2 primary aldosteronism-affected subjects from an Italian family and 3 somatic mutations in aldosterone-producing adenomas, T158A described previously as a germline mutation associated with FH-III, and G151R and L168R both described as somatic mutations in aldosterone-producing adenoma. The phenotype of the family with the G151E mutation was remarkably milder compared with the previously described American family, in terms of both clinical and biochemical parameters. Furthermore, patients with somatic KCNJ5 mutations displayed a phenotype indistinguishable from that of sporadic primary aldosteronism. The functional characterization of the effects of the G151E mutation in vitro showed a profound alteration of the channel function, with loss of K(+) selectivity, Na(+) influx, and membrane depolarization. These alterations have been postulated to be responsible for voltage gate Ca(2+) channel activation, increase in cytosolic calcium, and stimulation of aldosterone production and adrenal cell proliferation. In conclusion, we describe herein a new mutation in the KCNJ5 potassium channel associated with FH-III, responsible for marked alterations of channel function but associated with a mild clinical and hormonal phenotype.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Glucocorticoides/uso terapêutico , Hiperaldosteronismo/tratamento farmacológico , Hiperaldosteronismo/genética , Mutação/genética , Adulto , Europa (Continente) , Feminino , Humanos , Hiperaldosteronismo/classificação , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Falha de Tratamento , População Branca/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA