RESUMEN

Shroom is a family of related proteins linked to the actin cytoskeleton, and one of them, xShroom1, is constitutively expressed in Xenopus laevis oocytes which is required for the expression of the epithelial sodium channel (ENaC). On the other hand, ENaC and the cystic fibrosis transmembrane regulator (CFTR) are co-expressed in many types of cells with a negative or positive interaction depending on the studied tissues. Here, we measured the amiloride-sensitive ENaC currents (INaamil) and CFTR currents (ICFTR) with voltage clamp techniques in oocytes co-injected with ENaC and/or CFTR and xShroom1 antisense oligonucleotides. The objective was to study the mechanism of regulation of ENaC by CFTR when xShroom1 was suppressed and the endocytic traffic of CFTR was blocked. CFTR activation had a measurable negative effect on ENaC and this activation resulted in a greater inhibition of INaamil than with xShroom1 antisense alone. Our results with Dynasore, a drug that acts as an inhibitor of endocytic pathways, suggest that the changes in INaamil by xShroom1 downregulation were probably due to an increment in channel endocytosis. An opposite effect was observed when ICFTR was measured. Thus, when xShroom1 was downregulated, the ICFTR was larger than in the control experiments and this effect is not observed with Dynasore. A speculative explanation could be that xShroom1 exerts a dual effect on the endocytic traffic of ENaC and CFTR and these actions were canceled with Dynasore. In the presence of Dynasore, no difference in either INaamil or ICFTR was observed when xShroom1 was downregulated.

Asunto(s)

Regulador de Conductancia de Transmembrana de Fibrosis Quística , Fibrosis Quística , Animales , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Hidrazonas , Oocitos/metabolismo , Xenopus laevis/metabolismo

RESUMEN

Hypertension (HTN), i.e. abnormally high blood pressure, is a major risk factor for heart attack, stroke, and kidney failure. The Epithelial Sodium Channel (ENaC), one of the main transporters regulates blood pressure by tightly controlling the sodium reabsorption along the nephron. Recently, we have shown an α-ENaC overexpression in platelets from hypertensive patients compared to platelets from normotensive subjects, suggesting it makes a contribution to the activation state of platelets and the physiopathology of hypertension. However, the involvement of the α-ENaC localized in neutrophils to this disease remains unknown. Neutrophils are the first leukocytes to be recruited to an inflammatory site and are equipped with a strong ability to eliminate intra- or extracellular pathogens using reactive oxygen species or antibacterial proteins contained in their granules. Using the Western blotting (Wb), flow cytometry, and qRT-PCR approaches; we determined α-ENaC neutrophil overexpression at the protein and messenger RNA (mRNA) levels. By confocal and cytometry analysis, we determined the α-ENaC distribution and the heterogeneity of HTN neutrophils population, respectively. Immunoprecipitation and Wb assays demonstrated the presence of both α-ENaC and caveolin-1 phosphorylated forms, compared with neutrophils from healthy individuals. Although neutrophils from hypertensive subjects circulating in an activated state were exhibiting important oxidative stress and modifications registered by confocal, atomic force, and scanning electron microscope, they conserved their defense capabilities. The features described above for neutrophils from hypertensive patients could be attributed to α-ENaC overexpression, as its drug inhibition diminished their activation state modulating the actin cytoskeleton reorganization triggered during the activation process.

Asunto(s)

Canales Epiteliales de Sodio/metabolismo , Hipertensión/metabolismo , Hipertensión/patología , Neutrófilos/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Amilorida/farmacología , Antihipertensivos/farmacología , Antihipertensivos/uso terapéutico , Fenómenos Biofísicos/efectos de los fármacos , Estudios de Casos y Controles , Caveolina 1/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Canales Epiteliales de Sodio/genética , Femenino , Humanos , Hipertensión/tratamiento farmacológico , Hipertensión/genética , Masculino , Persona de Mediana Edad , Activación Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/ultraestructura , Estrés Oxidativo/efectos de los fármacos , Fosforilación/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

OBJECTIVES: Toll-like receptors and complement are two components of the innate immunity. Complement factor B is essential for the alternative pathway of complement activation. We have recently reported that complement factor B is significantly up-regulated in the kidney and may contribute to acute tubular injury in an animal model of sepsis. This study investigates the mechanisms responsible for the complement factor B up-regulation and its role in sodium transporter expression in tubular cells during sepsis. DESIGN: Animal study. SETTING: Laboratory investigation. SUBJECTS: C57BL/6 J wild-type, complement factor B(-/-), and Nfkb1(tm1Bal) p50(-/-) mice. INTERVENTIONS: Human proximal tubular cells and mouse tubular epithelial cells were stimulated with Toll-like receptor agonists. Bay 11-7082 was used to block nuclear factor-κB pathway. Alternative pathway activation was detected by C3 zymosan deposition. Polymicrobial sepsis was created by cecal ligation and puncture. Sodium transporter gene expression was determined by quantitative reverse transcriptase-polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: The agonists for Toll-like receptor 4 (lipopolysaccharide) or Toll-like receptor 3 (polyinosinic-polycytidylic acid) induced a marked increase in complement factor B expression in human proximal tubular cells and mouse tubular epithelial cells both at gene and protein levels. The Toll-like receptor 1/2 agonist, Pam3cys, induced complement factor B production only in human proximal tubular cells, not in mouse tubular epithelial cells. The Toll-like receptor 9 ligand, CpG oligodeoxynucleotides failed to induce complement factor B production either in human proximal tubular cells or in mouse tubular epithelial cells. Lipopolysaccharide/polyinosinic-polycytidylic acid-induced complement factor B up-regulation was blocked by Bay 11-7082, a potent inhibitor of nuclear factor-κB signaling, and in mouse tubular epithelial cells deficient in p50 subunit of nuclear factor-κB. Media from the lipopolysaccharide-treated mouse tubular epithelial cell cultures contained de novo synthesized complement factor B and led to functional alternative pathway activation. In a cecal ligation and puncture model, wild-type septic mice had down-regulated expression of sodium transporters in the kidney compared with the sham. In comparison, complement factor B mice or mice treated with anti-complement factor B displayed preserved levels of Na⁺/K⁺ ATPase-α1 following sepsis. CONCLUSIONS: 1) Toll-like receptor 3/4 activation is sufficient to induce complement factor B production via nuclear factor-κB pathway and to enhance alternative pathway activation in the kidney tubular epithelial cells. 2) Complement factor B may contribute to the down-regulation of certain sodium transporter expression during sepsis.

Asunto(s)

Factor B del Complemento/biosíntesis , Riñón/fisiopatología , Sepsis/fisiopatología , Animales , Transporte Biológico Activo/fisiología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Canales Epiteliales de Sodio/genética , Femenino , Expresión Génica , Humanos , Túbulos Renales Proximales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/antagonistas & inhibidores , Nitrilos/farmacología , Canales de Potasio/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Sulfonas/farmacología , Receptores Toll-Like/agonistas , Receptores Toll-Like/genética , Regulación hacia Arriba

RESUMEN

OBJECTIVE: To identify the gene mutation in ß and γ subunits of the epithelial sodium channel (ENaC) in an adolescent and family members with Liddle syndrome, an autosomal dominant form of secondary hypertension. STUDY DESIGN: We screened an adolescent with severe hypertension who was clinically diagnosed with Liddle syndrome for mutations in the C-terminus of the SCNN1B and SCNN1G genes. We also screened for these mutations in his family members, in 100 hypertensive patients, and in 100 controls. RESULTS: The index case, a 14-year-old boy, was diagnosed with Liddle syndrome by the identification of a novel missense mutation, P614L, in the PY motif of the ß subunit of the ENaC. Testing of relatives considered at risk revealed 6 subjects heterozygous for the mutation. All genetically affected subjects had a history of severe hypertension as well as hypokalemia. No other variants in the ß or γ subunits of the ENaC were detected. CONCLUSION: Based on direct DNA sequencing, we have detected a novel mutation that causes Liddle syndrome. This confirms the diagnosis and helps guide effective therapy for this adolescent and his affected relatives. These findings provide further evidence that the conserved PY motif is critical to regulation of ENaC activity.

Asunto(s)

Canales Epiteliales de Sodio/genética , Síndrome de Liddle/genética , Mutación Missense , Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Linaje , Adulto Joven

RESUMEN

Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in X. oocytes and is required for the expression of amiloride sensitive sodium channels (ENaC). Oocytes were injected with α, ß, and γ mENaC and xShroom1 sense or antisense oligonucleotides. We used voltage clamp techniques to study the amiloride-sensitive Na(+) currents (INa((amil))). We observed a marked reduction in INa((amil)) in oocytes co-injected with xShroom1 antisense. Oocytes expressing a DEG mutant ß-mENaC subunit (ß-S518K) with an open probability of 1 had enhanced INa((amil)) although these currents were also reduced when co-injected with xShroom1 antisense. Addition of low concentration (20 ng/ml) of trypsin which activates the membrane-resident ENaC channels led to a slow increase in INa((amil)) in oocytes with xShroom1 sense but had no effect on the currents in oocytes coinjected with ENaC and xShroom1 antisense. The same results were obtained with higher concentrations of trypsin (2 µg/ml) exposed during 2.5 min. In addition, fluorescence positive staining of plasma membrane in the oocytes expressing α, ß and γ mENaC and xShroom1 sense were observed but not in oocytes coinjected with ENaC and xShroom1 antisense oligonucleotides. On this basis, we suggest that xShroom1-dependent ENaC inhibition may be through the number of channels inserted in the membrane.

Asunto(s)

Canales Epiteliales de Sodio/metabolismo , Canales de Sodio/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , Canales Epiteliales de Sodio/genética , Femenino , Mutación , Oligonucleótidos Antisentido/metabolismo , Oocitos/metabolismo , Oocitos/patología , Oocitos/fisiología , Técnicas de Placa-Clamp , Canales de Sodio/genética , Tripsina/farmacología , Proteínas de Xenopus/genética

RESUMEN

The knowledge of the genetic bases of hypertension has improved over the last decade; this area of research has high priority due to the high incidence of hypertension and its impact on public health. Monogenetic mineralocorticoid hypertension syndromes are associated with suppressed plasma renin activity due to excessive activation of the mineralocorticoid pathway. We review the pathophysiology, phenotype, and method of diagnosis for familial hyperaldosteronism type I and type II, hypertensive forms of congenital adrenal hyperplasia, 11beta-hydroxysteroid dehydrogenase type 2 deficiency, Liddle's syndrome, an activating mutation of the MR, and glucocorticoid resistance. We also review some genes that could contribute to essential hypertension.

Asunto(s)

Hipertensión/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , 11-beta-Hidroxiesteroide Deshidrogenasas/deficiencia , 11-beta-Hidroxiesteroide Deshidrogenasas/genética , Hiperplasia Suprarrenal Congénita/genética , Angiotensinógeno/genética , Resistencia a Medicamentos/fisiología , Canales Epiteliales de Sodio/genética , Glucocorticoides/fisiología , Humanos , Hiperaldosteronismo/genética , Síndrome de Exceso Aparente de Mineralocorticoides/fisiopatología , Peptidil-Dipeptidasa A/genética , Fenotipo , Receptor de Angiotensina Tipo 1/genética , Receptores de Mineralocorticoides/genética , Síndrome

RESUMEN

The present study was performed to assay sodium currents in BeWo cells. These cells comprise a human trophoblast cell line which displays many of the biochemical and morphological properties similar to those reported for the in uterus proliferative cytotrophoblast. For whole-cell patch-clamp experiments, BeWo cells treated for 12 h with 100 nM aldosterone were exposed to 8Br-cAMP, a membrane-permeable cAMP analogue, to induce channel activity. Cells showed an amiloride-sensitive ion current (IC50 of 5.77 microM). Ion substitution experiments showed that the amiloride-sensitive current carried cations with a permeability rank order of Li+ > Na+ > K+ > NMDG (PLi/PNa = 1.3, PK/PNa = 0.6, PNMDG/PNa = 0.2). In cells pretreated with aldosterone, we observed that nearly half of successful patches had sodium channels with a linear conductance of 6.4 +/- 1.8 pS, a low voltage-independent Po and a PK/PNa of 0.19. Using RT-PCR, we determined that control cells express the alpha-, but not beta- and gamma-, epithelial sodium channel (ENaC) mRNA. When cells were treated with aldosterone (100 nM, 12 h), all alpha-, beta- and gamma-ENaC mRNAs were detected. The presence of ENaC subunit proteins in these cells was confirmed by Western blot analysis and immunolocalization with specific ENaC primary antibodies. In summary, our results suggest that BeWo cells express ENaC subunits and that aldosterone was able to modulate a selective response by generating amiloride-sensitive sodium currents similar to those observed in other human tissues.

Asunto(s)

Canales Epiteliales de Sodio/metabolismo , Trofoblastos/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Aldosterona/fisiología , Amilorida/farmacología , Línea Celular Tumoral , Bloqueadores del Canal de Sodio Epitelial , Canales Epiteliales de Sodio/genética , Femenino , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Técnicas de Placa-Clamp , ARN Mensajero/análisis , ARN Mensajero/antagonistas & inhibidores , Bloqueadores de los Canales de Sodio/farmacología , Trofoblastos/efectos de los fármacos

RESUMEN

Pseudohypoaldosteronism type 1 (PHA1) is a rare genetic disease characterized by neonatal renal salt wasting, vomiting, dehydration and failure to thrive. Affected patients present hyponatremia, hyperkalemia, associated with high levels of plasma renin and aldosterone resulting from a renal or systemic resistance to aldosterone. The systemic form of PHA1 results in a severe phenotype, and high doses of salt supplementation are necessary. The symptoms are life-long recurrent. This form is associated with autosomal recessive transmission. Homozygous or compound heterozygous loss of function mutations in the genes coding for the epithelial sodium channel (ENaC) subunities are responsible for this disease. The renal form of PHA1 results in a mild phenotype. Low doses of salt supplementation are required and usually the symptoms remit at the end of the first year of life. Heterozygous loss-of-function mutations in the mineralocorticoid receptor (MR) gene are associated with the renal form of PHA1 in the majority of the affected families but sporadic cases have been reported. In this review the mechanisms of aldosterone action and its effects are discussed. Additionally, clinical and molecular findings of a Brazilian family with the renal form of PHA1 caused by a nonsense mutation (R947X) in the MR gene are presented.

Asunto(s)

Aldosterona/sangre , Seudohipoaldosteronismo/genética , Receptores de Mineralocorticoides/genética , Aldosterona/fisiología , Canales Epiteliales de Sodio/genética , Humanos , Recién Nacido , Masculino , Mutación , Linaje , Seudohipoaldosteronismo/metabolismo , Seudohipoaldosteronismo/fisiopatología , Receptores de Mineralocorticoides/metabolismo , Transcripción Genética

RESUMEN

BACKGROUND: The epithelial sodium channel (ENaC) is a candidate gene associated with the development of essential hypertension. A potentially polymorphic repetitive region (GT dinucleotide short tandem repeat [STR]) was identified in intron 8 of beta-ENaC gene (SCNN1B). The aim of this study was to identify the prevalence and distribution of a polymorphic GT-STR in SCNN1B in Chilean essential hypertensive (EH) patients and to analyze the correlation between the different genotypes with plasma renin activity (PRA) and serum aldosterone (SA), and furthermore, to evaluate the beta-ENaC gene expression in vitro. METHODS: We studied 133 patients with EH and 69 normotensive (NT). In both EH and NT subjects we measured PRA, SA, urine sodium, and genotyped them according to the GT-STR length using sequencing analysis. We detected 11, 13 and 14 GT alleles in EH and NT subjects. Both groups were classified according to genotype: 14/14, 14/13, 13/13, 13/11, and 11/11. Influence of the GT-STR on beta-ENaC minigene expression was evaluated by real-time polymerase chain reaction. RESULTS: In EH, PRA decreased with the length of the STR region 11/13, 1.40 +/- 0.69; 13/13, 1.16 +/- 0.61; 13/14, 0.90 +/- 0.56; 14/14, 0.32 +/- 0.09 ng/mL/h; P < .01. Likewise, PRA in patients with EH with 14/14 or 14/13 genotypes were lower than EH with 13/13 or 13/11 genotypes (0.77 +/- 0.5 v 1.24 +/- 0.6 ng/mL/h; P < .01). Real-time polymerase chain reaction demonstrated an increased beta-ENaC expression in minigenes containing 14 GT-STR. CONCLUSIONS: We have identified a polymorphic GT-STR in the beta-ENaC gene, which is present in the EH and NT Chilean population. Biochemical analysis showed a possible linkage between this polymorphic region and low renin hypertension. The in vitro assay suggests that GT-STR could regulate the beta-ENaC expression.

Asunto(s)

Aldosterona/sangre , Canales Epiteliales de Sodio/genética , Hipertensión/genética , Renina/sangre , Adulto , Presión Sanguínea/genética , Estudios de Casos y Controles , Chile , Repeticiones de Dinucleótido , Canales Epiteliales de Sodio/metabolismo , Femenino , Expresión Génica , Frecuencia de los Genes , Genotipo , Humanos , Hipertensión/epidemiología , Intrones , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Prevalencia , ARN Mensajero/metabolismo , Renina/deficiencia , Sodio/orina

RESUMEN

Pseudo-hipoaldosteronismo tipo 1 (PHA1) é uma doença genética rara, caracterizada por vômitos, desidratação, baixo ganho pôndero-estatural e perda urinária de sal no período neonatal. Indivíduos afetados apresentam hiponatremia, hipercalemia, aumento da atividade de renina plasmática e concentrações muito elevadas de aldosterona plasmática, secundárias a uma resistência renal ou sistêmica à aldosterona. A forma sistêmica do PHA1 é a mais grave, havendo necessidade de reposição de doses altas de NaCl. Os sintomas persistem por toda a vida. Mutações inativadoras nos genes codificadores das sub-unidades do canal de sódio sensível à amilorida (ENaC) em homozigose ou heterozigose composta são responsáveis pelo quadro clínico de PHA1 sistêmico. A forma renal do PHA1 tem apresentação clínica mais leve, com necessidade de suplementação de doses baixas de NaCl. Os sintomas regridem no final do primeiro ano de vida. Mutações inativadoras do gene do receptor do mineralocorticóide (MR) estão associadas à forma renal do PHA1 em várias famílias afetadas. O padrão de herança é autossômico dominante, entretanto casos esporádicos têm sido relatados. No presente trabalho, discutimos as ações e os mecanismos de ação da aldosterona, e os aspectos clínicos e fisiopatológicos envolvidos nas síndromes de resistência aos mineralocorticóides. Adicionalmente, os aspectos clínicos e moleculares de uma família brasileira com PHA1 secundário à mutação R947X no gene do MR são discutidos.

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Pseudohypoaldosteronism type 1 (PHA1) is a rare genetic disease characterized by neonatal renal salt wasting, vomiting, dehydration and failure to thrive. Affected patients present hyponatremia, hyperkalemia, associated with high levels of plasma renin and aldosterone resulting from a renal or systemic resistance to aldosterone. The systemic form of PHA1 results in a severe phenotype, and high doses of salt supplementation are necessary. The symptoms are life-long recurrent. This form is associated with autosomal recessive transmission. Homozygous or compound heterozygous loss of function mutations in the genes coding for the epithelial sodium channel (ENaC) subunities are responsible for this disease. The renal form of PHA1 results in a mild phenotype. Low doses of salt supplementation are required and usually the symptoms remit at the end of the first year of life. Heterozygous loss-of-function mutations in the mineralocorticoid receptor (MR) gene are associated with the renal form of PHA1 in the majority of the affected families but sporadic cases have been reported. In this review the mechanisms of aldosterone action and its effects are discussed. Additionally, clinical and molecular findings of a Brazilian family with the renal form of PHA1 caused by a nonsense mutation (R947X) in the MR gene are presented.

Asunto(s)

Humanos , Recién Nacido , Masculino , Aldosterona/sangre , Seudohipoaldosteronismo/genética , Receptores de Mineralocorticoides/genética , Aldosterona/fisiología , Canales Epiteliales de Sodio/genética , Mutación , Linaje , Seudohipoaldosteronismo/metabolismo , Seudohipoaldosteronismo/fisiopatología , Receptores de Mineralocorticoides/metabolismo , Transcripción Genética