RESUMO
OBJECTIVES: In vitro fertilization (IVF) has been linked to an increased risk for imprinting disorders in offspring. The data so far have predominantly been retrospective, comparing the rate of IVF conceptions in affected patients with controls. We describe a series of fetuses with omphalocele that were tested for Beckwith-Wiedemann syndrome (BWS) and subsequently ascertained as to whether pregnancies were conceived by assisted reproductive technologies (ART). METHODS: Fetuses were tested for BWS by Southern blot, PCR based methods, and methylation analysis to identify the imprinting status at primarily the IC2 locus, KCNQ1OT1, as well as IC1, H19/IGF-2. Some fetuses were also tested for uniparental disomy of chromosome 11p. RESULTS: We tested 301 fetuses with omphalocele for BWS. Forty samples were positive. Sixteen were from IVF pregnancies, for an overall rate of 40%. Such as high proportion of IVF pregnancies in a series of BWS-positive fetuses has not been described previously. Possible factors such as twinning and ascertainment bias are discussed. CONCLUSION: We found about a 20-fold overrepresentation of IVF cases in fetuses with BWS/omphalocele when compared with the rate of ART pregnancies in the USA (p < .0001). Our series provides support for an association of IVF and BWS. Patients should be counseled about these risks and made aware of the availability of prenatal diagnosis for detection.
Assuntos
Síndrome de Beckwith-Wiedemann/genética , Impressão Genômica/genética , Diagnóstico Pré-Natal , Técnicas de Reprodução Assistida/efeitos adversos , Síndrome de Beckwith-Wiedemann/epidemiologia , Síndrome de Beckwith-Wiedemann/fisiopatologia , Metilação de DNA/genética , Feminino , Fertilização in vitro , Feto/fisiopatologia , Humanos , Masculino , GravidezAssuntos
Síndrome de Angelman/diagnóstico , Fertilização in vitro/tendências , Impressão Genômica/genética , Diagnóstico Pré-Natal , Adulto , Síndrome de Angelman/genética , Síndrome de Angelman/patologia , Feminino , Humanos , Síndrome de Prader-Willi/diagnóstico , Síndrome de Prader-Willi/genética , Síndrome de Prader-Willi/patologia , GravidezRESUMO
Microalbuminuria provides the earliest clinical marker of diabetic nephropathy among patients with Type 1 diabetes, yet it lacks sensitivity and specificity for early histological manifestations of disease. In recent years microRNAs have emerged as potential mediators in the pathogenesis of diabetes complications, suggesting a possible role in the diagnosis of early stage disease. We used quantiative polymerase chain reaction (qPCR) to evaluate the expression profile of 723 unique microRNAs in the normoalbuminuric urine of patients who did not develop nephropathy (n = 10) relative to patients who subsequently developed microalbuminuria (n = 17). Eighteen microRNAs were strongly associated with the subsequent development of microalbuminuria, while 15 microRNAs exhibited gender-related differences in expression. The predicted targets of these microRNAs map to biological pathways known to be involved in the pathogenesis and progression of diabetic renal disease. A microRNA signature (miR-105-3p, miR-1972, miR-28-3p, miR-30b-3p, miR-363-3p, miR-424-5p, miR-486-5p, miR-495, miR-548o-3p and for women miR-192-5p, miR-720) achieved high internal validity (cross-validated misclassification rate of 11.1%) for the future development of microalbuminuria in this dataset. Weighting microRNA measurements by their number of kidney-relevant targets improved the prognostic performance of the miRNA signature (cross-validated misclassification rate of 7.4%). Future studies are needed to corroborate these early observations in larger cohorts.
RESUMO
BACKGROUND: Opitz G/BBB syndrome is a heterogeneous disorder characterised by variable expression of midline defects including cleft lip and palate, hypertelorism, laryngealtracheoesophageal anomalies, congenital heart defects, and hypospadias. The X-linked form of the condition has been associated with mutations in the MID1 gene on Xp22. The autosomal dominant form has been linked to chromosome 22q11.2, although the causative gene has yet to be elucidated. METHODS AND RESULTS: In this study, we performed whole exome sequencing on DNA samples from a three-generation family with characteristics of Opitz G/BBB syndrome with negative MID1 sequencing. We identified a heterozygous missense mutation c.1189A>C (p.Thr397Pro) in SPECC1L, located at chromosome 22q11.23. Mutation screening of an additional 19 patients with features of autosomal dominant Opitz G/BBB syndrome identified a c.3247G>A (p.Gly1083Ser) mutation segregating with the phenotype in another three-generation family. CONCLUSIONS: Previously, SPECC1L was shown to be required for proper facial morphogenesis with disruptions identified in two patients with oblique facial clefts. Collectively, these data demonstrate that SPECC1L mutations can cause syndromic forms of facial clefting including some cases of autosomal dominant Opitz G/BBB syndrome and support the original linkage to chromosome 22q11.2.
Assuntos
Proteínas de Ligação ao Cálcio/química , Esôfago/anormalidades , Genes Dominantes , Predisposição Genética para Doença , Hipertelorismo/genética , Hipospadia/genética , Proteínas dos Microfilamentos/química , Mutação/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Adulto , Sequência de Bases , Análise Mutacional de DNA , Éxons/genética , Família , Feminino , Testes Genéticos , Humanos , Lactente , Masculino , Proteínas dos Microtúbulos/genética , Dados de Sequência Molecular , Proteínas Nucleares/genética , Linhagem , Fenótipo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases , CalponinasRESUMO
Non-syndromal X-linked intellectual disability (NS-XLID) represents a broad group of clinical disorders in which ID is the only clinically consistent manifestation. Although in many cases either chromosomal linkage data or knowledge of the >100 existing XLID genes has assisted mutation discovery, the underlying cause of disease remains unresolved in many families. We report the resolution of a large family (K8010) with NS-XLID, with variable macrocephaly and macro-orchidism. Although a previous linkage study had mapped the locus to Xq12-q21, this region contained too many candidate genes to be analyzed using conventional approaches. However, X-chromosome exome sequencing, bioinformatics analysis and segregation analysis revealed a novel missense mutation (c.1012C>T; p.R338W) in ARHGEF9. This gene encodes collybistin (CB), a neuronal GDP-GTP exchange factor previously implicated in several cases of XLID, as well as clustering of gephyrin and GABAA receptors at inhibitory synapses. Molecular modeling of the CB R338W substitution revealed that this change results in the substitution of a long electropositive side-chain with a large non-charged hydrophobic side-chain. The R338W change is predicted to result in clashes with adjacent amino acids (K363 and N335) and disruption of electrostatic potential and local folding of the PH domain, which is known to bind phosphatidylinositol-3-phosphate (PI3P/PtdIns-3-P). Consistent with this finding, functional assays revealed that recombinant CB CB2SH3- (R338W) was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Taken together, these results suggest that the R338W mutation in ARHGEF9 is the underlying cause of NS-XLID in this family.
RESUMO
A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells. Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport. Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein. A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3' untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.
Assuntos
Aldosterona/metabolismo , Córtex Renal/metabolismo , Túbulos Renais Coletores/metabolismo , MicroRNAs/metabolismo , Sódio/metabolismo , Aldosterona/genética , Animais , Anquirinas/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Canais Epiteliais de Sódio/metabolismo , Córtex Renal/citologia , Túbulos Renais Coletores/citologia , Luciferases/genética , Camundongos Endogâmicos C57BL , Néfrons/citologia , Néfrons/metabolismo , RNA Interferente Pequeno/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo , Transdução de Sinais/fisiologiaAssuntos
Deleção Cromossômica , Cromossomos Humanos Par 6 , Fissura Palatina/diagnóstico , Fissura Palatina/genética , Dedos/anormalidades , Deformidades Congênitas da Mão/diagnóstico , Deformidades Congênitas da Mão/genética , Cardiopatias Congênitas/diagnóstico , Cardiopatias Congênitas/genética , Anormalidades Urogenitais/diagnóstico , Anormalidades Urogenitais/genética , Mapeamento Cromossômico , Hibridização Genômica Comparativa , Fácies , Feminino , Estudos de Associação Genética , Humanos , Hibridização in Situ Fluorescente , Lactente , Fenótipo , SíndromeRESUMO
Resveratrol, a naturally occurring phytoalexin, has reported cardioprotective, anti-inflammatory, chemopreventative and antidiabetic properties. Several studies indicate the multiple effects of resveratrol on cellular function are due to its inhibition of class 1A phosphoinositide 3-kinase (PI3K) mediated signaling pathways, but it also activates AMP-activated protein kinase (AMPK). As sodium transport in the kidney via the Epithelial Sodium Channel (ENaC) is highly sensitive to changes in phosphoinositide signaling in the membrane and AMPK, we employed resveratrol to probe the relative effects of phosphatidylinositol species in the plasma membrane and AMPK activity and their impact on ENaC activity in mouse cortical collecting duct (mpkCCDc14) cells. Here we demonstrate that resveratrol acutely reduces amiloride-sensitive current in mpkCCDc14 cells. The time course and dose dependency of this inhibition paralleled depletion of the PI(3,4,5)P3 reporter (AKT-PH) in live-cell microscopy, indicating the early inhibition is likely mediated by resveratrol's known effects on PI3K activity. Additionally, resveratrol induces a late inhibitory effect (4-24 hours) that appears to be mediated via AMPK activation. Resveratrol treatment induces significant AMPK activation compared with vehicle controls after 4 h, which persists through 16 h. Knockdown of AMPK or treatment with the AMPK inhibitor Compound C reduced the late phase of current reduction but had no effect on the early inhibitory activity of resveratrol. Collectively, these data demonstrate that resveratrol inhibits ENaC activity by a dual effect: an early reduction in activity seen within 5 minutes related to depletion of membrane PIP3, and a sustained late (4-24 h) effect secondary to activation of AMPK.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Canais Epiteliais de Sódio/metabolismo , Túbulos Renais Coletores/citologia , Fosfatidilinositóis/metabolismo , Estilbenos/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Amilorida/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Canais Epiteliais de Sódio/genética , Immunoblotting , Camundongos , RNA Interferente Pequeno , ResveratrolRESUMO
The epithelial sodium channel (ENaC) is the rate-limiting step for sodium reabsorption across tight epithelia. Cyclic-AMP (cAMP) stimulation promotes ENaC trafficking to the apical surface to increase channel number and transcellular Na(+) transport. Removal of corticosteroid supplementation in a cultured cortical collecting duct cell line reduced ENaC expression. Concurrently, the number of vesicles trafficked in response to cAMP stimulation, as measured by a change in membrane capacitance, also decreased. Stimulation with aldosterone restored both the basal and cAMP-stimulated ENaC activity and increased the number of exocytosed vesicles. Knocking down ENaC directly decreased both the cAMP-stimulated short-circuit current and capacitance response in the presence of aldosterone. However, constitutive apical recycling of the Immunoglobulin A receptor was unaffected by alterations in ENaC expression or trafficking. Fischer Rat Thyroid cells, transfected with α,ß,γ-mENaC had a significantly greater membrane capacitance response to cAMP stimulation compared to non-ENaC controls. Finally, immunofluorescent labeling and quantitation revealed a smaller number of vesicles in cells where ENaC expression was reduced. These findings indicate that ENaC is not a passive passenger in regulated epithelial vesicle trafficking, but plays a role in establishing and maintaining the pool of vesicles that respond to cAMP stimulation.
Assuntos
Vesículas Citoplasmáticas/metabolismo , Canais Epiteliais de Sódio/metabolismo , Aldosterona/fisiologia , Animais , Polaridade Celular , Células Cultivadas , Colforsina/farmacologia , Meios de Cultura , AMP Cíclico/fisiologia , Capacitância Elétrica , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Canais Epiteliais de Sódio/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Camundongos , Transporte Proteico , Interferência de RNA , Ratos , Ratos Endogâmicos F344RESUMO
Expression of the epithelial sodium channel (ENaC) at the apical membrane of cortical collecting duct (CCD) principal cells is modulated by regulated trafficking mediated by vesicle insertion and retrieval. Small GTPases are known to facilitate vesicle trafficking, recycling, and membrane fusion events; however, little is known about the specific Rab family members that modify ENaC surface density. Using a mouse CCD cell line that endogenously expresses ENaC (mpkCCD), the channel was localized to both Rab11a- and Rab11b-positive endosomes by immunoisolation and confocal fluorescent microscopy. Expression of a dominant negative (DN) form of Rab11a or Rab11b significantly reduced the basal and cAMP-stimulated ENaC-dependent sodium (Na(+)) transport. The greatest reduction in Na(+) transport was observed with the expression of DN-Rab11b. Furthermore, small interfering RNA-mediated knockdown of each Rab11 isoform demonstrated the requirement for Rab11b in ENaC surface expression. These data indicate that Rab11b, and to a lesser extent Rab11a, is involved in establishing the constitutive and cAMP-stimulated Na(+) transport in mpkCCD cells.
Assuntos
Endossomos/metabolismo , Canais Epiteliais de Sódio/metabolismo , Túbulos Renais Coletores/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Túbulos Renais Coletores/citologia , Camundongos , Transporte ProteicoRESUMO
During infancy, this 50-year-old man with a previously undiagnosed multiple congenital anomalies/intellectual disability (MCA/MR) syndrome had grossly symptomatic hypercalcemia and was (briefly) thought to have Williams syndrome. Results of studies with the cytogenetic methods of the 1960s and 1970s were apparently normal. He matured late, but is high-functioning and healthy. Over 50 years he remained a diagnostic enigma. Thus, it came as a surprise when recent high-resolution banding methods showed an abnormality of the terminal portion of 13q, determined on array-comparative genomic hybridization to constitute an unbalanced chromosome rearrangement with a 0.35 Mb loss of 13q34-ter and 7.67 Mb gain of 14q32.2q32.33 translocated to 13q34. This apparently de novo genomic abnormality must be presumed as the cause of this previously undescribed MCA/MR syndrome which, however, may remain a private syndrome in this family. Williams syndrome was ruled out, and presently it is not possible to ascribe this patient's severely symptomatic infantile hypercalcemia to any gene on the deleted or duplicated chromosome segments. This "case" does underscore the importance of re-studying previously obscure but evidently genetic conditions, of long-term follow-up and documentation of natural history, and of providing, at last, a causal explanation to the family.
Assuntos
Deleção Cromossômica , Duplicação Cromossômica/genética , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 14/genética , Hipercalcemia/genética , Humanos , Recém-Nascido , Cariotipagem , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , SíndromeRESUMO
We report the identification of a recurrent, 520-kb 16p12.1 microdeletion associated with childhood developmental delay. The microdeletion was detected in 20 of 11,873 cases compared with 2 of 8,540 controls (P = 0.0009, OR = 7.2) and replicated in a second series of 22 of 9,254 cases compared with 6 of 6,299 controls (P = 0.028, OR = 2.5). Most deletions were inherited, with carrier parents likely to manifest neuropsychiatric phenotypes compared to non-carrier parents (P = 0.037, OR = 6). Probands were more likely to carry an additional large copy-number variant when compared to matched controls (10 of 42 cases, P = 5.7 x 10(-5), OR = 6.6). The clinical features of individuals with two mutations were distinct from and/or more severe than those of individuals carrying only the co-occurring mutation. Our data support a two-hit model in which the 16p12.1 microdeletion both predisposes to neuropsychiatric phenotypes as a single event and exacerbates neurodevelopmental phenotypes in association with other large deletions or duplications. Analysis of other microdeletions with variable expressivity indicates that this two-hit model might be more generally applicable to neuropsychiatric disease.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 16 , Deficiências do Desenvolvimento/genética , Modelos Genéticos , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Cromossomos Humanos Par 16/genética , Hibridização Genômica Comparativa/métodos , Família , Frequência do Gene , Humanos , Lactente , Análise de Sequência com Séries de Oligonucleotídeos , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Recidiva , Índice de Gravidade de DoençaRESUMO
Alkalosis impairs the natriuretic response to diuretics, but the underlying mechanisms are unclear. The soluble adenylyl cyclase (sAC) is a chemosensor that mediates bicarbonate-dependent elevation of cAMP in intracellular microdomains. We hypothesized that sAC may be an important regulator of Na(+) transport in the kidney. Confocal images of rat kidney revealed specific immunolocalization of sAC in collecting duct cells, and immunoblots confirmed sAC expression in mouse cortical collecting duct (mpkCCD(c14)) cells. These cells exhibit aldosterone-stimulated transepithelial Na(+) currents that depend on both the apical epithelial Na(+) channel (ENaC) and basolateral Na(+),K(+)-ATPase. RNA interference-mediated 60-70% knockdown of sAC expression comparably inhibited basal transepithelial short circuit currents (I(sc)) in mpkCCD(c14) cells. Moreover, the sAC inhibitors KH7 and 2-hydroxyestradiol reduced I(sc) in these cells by 50-60% within 30 min. 8-Bromoadenosine-3',5'-cyclic-monophosphate substantially rescued the KH7 inhibition of transepithelial Na(+) current. Aldosterone doubled ENaC-dependent I(sc) over 4 h, an effect that was abolished in the presence of KH7. The sAC contribution to I(sc) was unaffected with apical membrane nystatin-mediated permeabilization, whereas the sAC-dependent Na(+) current was fully inhibited by basolateral ouabain treatment, suggesting that the Na(+),K(+)-ATPase, rather than ENaC, is the relevant transporter target of sAC. Indeed, neither overexpression of sAC nor treatment with KH7 modulated ENaC currents in Xenopus oocytes. ATPase and biotinylation assays in mpkCCD(c14) cells demonstrated that sAC inhibition decreases catalytic activity rather than surface expression of the Na(+),K(+)-ATPase. In summary, these results suggest that sAC regulates both basal and agonist-stimulated Na(+) reabsorption in the kidney collecting duct, acting to enhance Na(+),K(+)-ATPase activity.
Assuntos
Adenilil Ciclases/metabolismo , Epitélio/metabolismo , Túbulos Renais Coletores/metabolismo , Sódio/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Inibidores de Adenilil Ciclases , Adenilil Ciclases/genética , Aldosterona/farmacologia , Animais , Biotinilação , Células Cultivadas , Canais Epiteliais de Sódio/metabolismo , Epitélio/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/farmacologia , Imunofluorescência , Immunoblotting , Transporte de Íons , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Masculino , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Bloqueadores dos Canais de Sódio/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Xenopus laevisRESUMO
The epithelial Na(+) channel (ENaC) is a major regulator of salt and water reabsorption in a number of epithelial tissues. Abnormalities in ENaC function have been directly linked to several human disease states including Liddle's syndrome, psuedohypoaldosteronism, and cystic fibrosis and may be implicated in states as diverse as salt-sensitive hypertension, nephrosis, and pulmonary edema. ENaC activity in epithelial cells is highly regulated both by open probability and number of channels. Open probability is regulated by a number of factors, including proteolytic processing, while ENaC number is regulated by cellular trafficking. This review discusses current understanding of apical membrane delivery, cell surface stability, endocytosis, retrieval, and recycling of ENaC and the molecular partners that have so far been shown to participate in these processes. We review known sites and mechanisms of hormonal regulation of trafficking by aldosterone, vasopressin, and insulin. While many details of the regulation of ENaC trafficking remain to be elucidated, knowledge of these mechanisms may provide further insights into ENaC activity in normal and disease states.
Assuntos
Membrana Celular/fisiologia , Células Epiteliais/fisiologia , Canais Epiteliais de Sódio/fisiologia , Transporte Proteico/fisiologia , Células Epiteliais/citologia , Exocitose/fisiologia , Humanos , Vesículas Transportadoras/fisiologiaRESUMO
We have previously shown that IkappaB kinase-beta (IKKbeta) interacts with the epithelial Na+ channel (ENaC) beta-subunit and enhances ENaC activity by increasing its surface expression in Xenopus oocytes. Here, we show that the IKKbeta-ENaC interaction is physiologically relevant in mouse polarized kidney cortical collecting duct (mpkCCDc14) cells, as RNA interference-mediated knockdown of endogenous IKKbeta in these cells by approximately 50% resulted in a similar reduction in transepithelial ENaC-dependent equivalent short circuit current. Although IKKbeta binds to ENaC, there was no detectable phosphorylation of ENaC subunits by IKKbeta in vitro. Because IKKbeta stimulation of ENaC activity occurs through enhanced channel surface expression and the ubiquitin-protein ligase Nedd4-2 has emerged as a central locus for ENaC regulation at the plasma membrane, we tested the role of Nedd4-2 in this regulation. IKKbeta-dependent phosphorylation of Xenopus Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for regulation of Nedd4-2 and thus ENaC activity. 32P labeling studies utilizing wild-type or mutant forms of Xenopus Nedd4-2 demonstrated that Ser-444, a key SGK1 and protein kinase A-phosphorylated residue, is also an important IKKbeta phosphorylation target. ENaC stimulation by IKKbeta was preserved in oocytes expressing wild-type Nedd4-2 but blocked in oocytes expressing either a dominant-negative (C938S) or phospho-deficient (S444A) Nedd4-2 mutant, suggesting that Nedd4-2 function and phosphorylation by IKKbeta are required for IKKbeta regulation of ENaC. In summary, these results suggest a novel mode of ENaC regulation that occurs through IKKbeta-dependent Nedd4-2 phosphorylation at a recognized SGK1 and protein kinase A target site.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Regulação da Expressão Gênica/fisiologia , Quinase I-kappa B/metabolismo , Túbulos Renais Coletores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Linhagem Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte , Canais Epiteliais de Sódio/genética , Humanos , Quinase I-kappa B/genética , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Túbulos Renais Coletores/citologia , Camundongos , Mutação , Ubiquitina-Proteína Ligases Nedd4 , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Ubiquitina-Proteína Ligases/genética , Proteínas de Xenopus/genética , Xenopus laevisAssuntos
Canais Epiteliais de Sódio/metabolismo , Hipertensão/metabolismo , Subunidades Proteicas/imunologia , Pseudo-Hipoaldosteronismo/metabolismo , Sódio/metabolismo , Animais , Canais Epiteliais de Sódio/genética , Humanos , Hipertensão/genética , Transporte de Íons , Camundongos , Mutação , Especificidade de Órgãos/genética , Subunidades Proteicas/genética , Pseudo-Hipoaldosteronismo/genéticaRESUMO
The epithelial sodium channel (ENaC) is ubiquitinated by the E3 ligase Nedd4-2 at the apical membranes of polarized cortical collecting duct (CCD) epithelial cells. This leads to ENaC endocytosis and possible degradation. Because ENaC is known to recycle at the apical membranes of CCD cells, deubiquitinating enzymes (DUBs) are likely involved in regulating ENaC surface density by facilitating ENaC recycling as opposed to degradation. Using a chemical probe approach to tag active DUBs, we identified ubiquitin C-terminal hydrolase (UCH) isoform L3 as the predominant DUB in endosomal compartments of CCD cells. Blocking UCH-L3 activity or reducing its expression by selective knockdown increased ENaC ubiquitination and resulted in its removal from the apical membranes of CCD cells. Functionally this caused a rapid reduction in transepithelial Na(+) currents across the CCD epithelia. Surface biotinylation demonstrated the loss of ENaC from the apical surface when UCH-L3 was inhibited. Whole cell or apical surface immunoprecipitation demonstrated increased ENaC ubiquitination with UCH-L3 inhibition. This constitutes a novel function for UCH in epithelia and in the regulation of ion channels and demonstrates the dynamic regulation of apically located ENaC by recycling, which is facilitated by this DUB.
Assuntos
Cisteína Endopeptidases/fisiologia , Canais Epiteliais de Sódio/metabolismo , Ubiquitina Tiolesterase/fisiologia , Ubiquitina/química , Animais , Biotinilação , Membrana Celular/metabolismo , Clatrina/química , Cisteína Endopeptidases/metabolismo , Endossomos/metabolismo , Células Epiteliais , Túbulos Renais Coletores/metabolismo , Camundongos , Modelos Biológicos , Isoformas de Proteínas , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Ubiquitina Tiolesterase/metabolismoRESUMO
Ubiquitination of ENaC subunits has been shown to negatively regulate the cell surface expression of ENaC channels. We have previously demonstrated that epsin links ubiquitinated ENaC to clathrin adaptors for clathrin-mediated endocytosis. Epsin is thought to directly modify the curvature of membranes upon binding to phosphatidylinositol 4,5-bisphosphate (PIP2) where it recruits clathrin and stimulates lattice assembly. Murine phosphatidylinositol 4-phosphate 5-kinase alpha (PI5KIalpha) has been shown to enhance endocytosis in a PIP2-dependent manner. We tested the hypothesis that PI5KIalpha-mediated PIP2 production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KIalpha decreased ENaC currents in Xenopus oocytes by 80%, entirely because of a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5Kalpha had no effect on ENaC activity. Expression of the PIP2 binding region of epsin increased ENaC current in oocytes, an effect completely reversed by co-expression of PI5KIalpha. Overexpression of epsin reduced amiloride-sensitive current in CCD cells. Overexpression of PI5KIalpha enhanced membrane PIP2 levels and reduced apical surface expression of ENaC in CCD cells, down-regulating amiloride-sensitive current. Knockdown of PI5KIalpha with isoform-specific siRNA resulted in a 4-fold enhancement of ENaC activity. PI5KIalpha localized exclusively to the apical plasma membrane domain when overexpressed in mouse CCD cells, consistent for a role in regulating PIP2 production at the apical plasma membrane. We conclude that membrane turnover events regulating ENaC surface expression and activity in oocytes and CCD cells can be regulated by PI5KIalpha.
Assuntos
Endocitose/fisiologia , Canais Epiteliais de Sódio/biossíntese , Túbulos Renais Coletores/fisiologia , Potenciais da Membrana/fisiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Amilorida/farmacologia , Animais , Linhagem Celular Transformada , Membrana Celular/enzimologia , Clatrina/genética , Clatrina/metabolismo , Endocitose/efeitos dos fármacos , Canais Epiteliais de Sódio/genética , Expressão Gênica , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Túbulos Renais Coletores/citologia , Potenciais da Membrana/genética , Camundongos , Antígenos de Histocompatibilidade Menor , Oócitos/citologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Interferente Pequeno , Bloqueadores dos Canais de Sódio/farmacologia , Xenopus laevisRESUMO
We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct ((MPK)CCD(14)) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl-beta-cyclodextrin added apically to (MPK)CCD(14) cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 +/- 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of (MPK)CCD(14) cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.