ABSTRACT
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.
Subject(s)
Beckwith-Wiedemann Syndrome/genetics , Genomic Imprinting/genetics , Prenatal Diagnosis , Reproductive Techniques, Assisted/adverse effects , Beckwith-Wiedemann Syndrome/epidemiology , Beckwith-Wiedemann Syndrome/physiopathology , DNA Methylation/genetics , Female , Fertilization in Vitro , Fetus/physiopathology , Humans , Male , PregnancyABSTRACT
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.
Subject(s)
Calcium-Binding Proteins/chemistry , Esophagus/abnormalities , Genes, Dominant , Genetic Predisposition to Disease , Hypertelorism/genetics , Hypospadias/genetics , Microfilament Proteins/chemistry , Mutation/genetics , Phosphoproteins/chemistry , Phosphoproteins/genetics , Adult , Base Sequence , DNA Mutational Analysis , Exons/genetics , Family , Female , Genetic Testing , Humans , Infant , Male , Microtubule Proteins/genetics , Molecular Sequence Data , Nuclear Proteins/genetics , Pedigree , Phenotype , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Transcription Factors/genetics , Ubiquitin-Protein Ligases , CalponinsABSTRACT
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.
Subject(s)
Aldosterone/metabolism , Kidney Cortex/metabolism , Kidney Tubules, Collecting/metabolism , MicroRNAs/metabolism , Sodium/metabolism , Aldosterone/genetics , Animals , Ankyrins/metabolism , Biological Transport/physiology , Cell Line , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Epithelial Sodium Channels/metabolism , Kidney Cortex/cytology , Kidney Tubules, Collecting/cytology , Luciferases/genetics , Mice, Inbred C57BL , Nephrons/cytology , Nephrons/metabolism , RNA, Small Interfering/genetics , Ribonuclease III/genetics , Ribonuclease III/metabolism , Signal Transduction/physiologySubject(s)
Angelman Syndrome/diagnosis , Fertilization in Vitro/trends , Genomic Imprinting/genetics , Prenatal Diagnosis , Adult , Angelman Syndrome/genetics , Angelman Syndrome/pathology , Female , Humans , Prader-Willi Syndrome/diagnosis , Prader-Willi Syndrome/genetics , Prader-Willi Syndrome/pathology , PregnancyABSTRACT
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.
Subject(s)
Endosomes/metabolism , Epithelial Sodium Channels/metabolism , Kidney Tubules, Collecting/metabolism , rab GTP-Binding Proteins/metabolism , Animals , Cell Line , Cells, Cultured , Epithelial Cells/cytology , Epithelial Cells/metabolism , Kidney Tubules, Collecting/cytology , Mice , Protein TransportABSTRACT
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.
Subject(s)
Chromosome Deletion , Chromosome Duplication/genetics , Chromosomes, Human, Pair 13/genetics , Chromosomes, Human, Pair 14/genetics , Hypercalcemia/genetics , Humans , Infant, Newborn , Karyotyping , Male , Middle Aged , Pedigree , Phenotype , SyndromeSubject(s)
Chromosome Deletion , Chromosomes, Human, Pair 6 , Cleft Palate/diagnosis , Cleft Palate/genetics , Fingers/abnormalities , Hand Deformities, Congenital/diagnosis , Hand Deformities, Congenital/genetics , Heart Defects, Congenital/diagnosis , Heart Defects, Congenital/genetics , Urogenital Abnormalities/diagnosis , Urogenital Abnormalities/genetics , Chromosome Mapping , Comparative Genomic Hybridization , Facies , Female , Genetic Association Studies , Humans , In Situ Hybridization, Fluorescence , Infant , Phenotype , SyndromeABSTRACT
Acute hormonal regulation of the epithelial sodium channel (ENaC) in tight epithelia increases transcellular Na(+) transport via trafficking of intracellular channels to the apical surface. The fate of the channels removed from the apical surface following agonist washout is less clear. By repetitively stimulating polarized mouse cortical collecting duct (mCCD, (MPK)CCD(14)) epithelia, we evaluated the hypothesis that ENaC recycles through an intracellular pool to be available for reinsertion into the apical membrane. Short circuit current (I(SC)), membrane capacitance (C(T)), and conductance (G(T)) were recorded from mCCD epithelia mounted in modified Ussing chambers. Surface biotinylation of ENaC demonstrated an increase in channel number in the apical membrane following cAMP stimulation. This increase was accompanied by a 83 +/- 6% (n = 31) increase in I(SC) and a 15.3 +/- 1.5% (n = 15) increase in C(T). Selective membrane permeabilization demonstrated that the C(T) increase was due to an increase in apical membrane capacitance. I(SC) and C(T) declined to basal levels on stimulus washout. Repetitive cAMP stimulation and washout (approximately 1 h each cycle) resulted in response fatigue; DeltaI(SC) decreased approximately 10% per stimulation-recovery cycle. When channel production was blocked by cycloheximide, DeltaI(SC) decreased approximately 15% per stimulation cycle, indicating that newly synthesized ENaC contributed a relatively small fraction of the channels mobilized to the apical membrane. Selective block of surface ENaC by benzamil demonstrated that channels inserted from a subapical pool made up >90% of the stimulated I(SC), and that on restimulation a large proportion of channels retrieved from the apical surface were reinserted into the apical membrane. Channel recycling was disrupted by brefeldin A, which inhibited ENaC exocytosis, by chloroquine, which inhibited ENaC endocytosis and recycling, and by latrunculin A, which blocked ENaC exocytosis. A compartment model featuring channel populations in the apical membrane and intracellular recycling pool provided an adequate kinetic description of the I(SC) responses to repetitive stimulation. The model supports the concept of ENaC recycling in response to repetitive cAMP stimulation.
Subject(s)
Cyclic AMP/metabolism , Exocytosis/physiology , Ion Channel Gating/physiology , Kidney/physiology , Membrane Potentials/physiology , Models, Biological , Sodium Channels/metabolism , Animals , Cell Line , Colforsin/pharmacology , Computer Simulation , Epithelial Sodium Channels , Exocytosis/drug effects , Kidney/drug effects , MiceABSTRACT
Endogenous serine proteases have been reported to control the reabsorption of Na(+) by kidney- and lung-derived epithelial cells via stimulation of electrogenic Na(+) transport mediated by the epithelial Na(+) channel (ENaC). In this study we investigated the effects of aprotinin on ENaC single channel properties using transepithelial fluctuation analysis in the amphibian kidney epithelium, A6. Aprotinin caused a time- and concentration-dependent inhibition (84 +/- 10.5%) in the amiloride-sensitive sodium transport (I(Na)) with a time constant of 18 min and half maximal inhibition constant of 1 microM. Analysis of amiloride analogue blocker-induced fluctuations in I(Na) showed linear rate-concentration plots with identical blocker on and off rates in control and aprotinin-inhibited conditions. Verification of open-block kinetics allowed for the use of a pulse protocol method (Helman, S.I., X. Liu, K. Baldwin, B.L. Blazer-Yost, and W.J. Els. 1998. Am. J. Physiol. 274:C947-C957) to study the same cells under different conditions as well as the reversibility of the aprotinin effect on single channel properties. Aprotinin caused reversible changes in all three single channel properties but only the change in the number of open channels was consistent with the inhibition of I(Na). A 50% decrease in I(Na) was accompanied by 50% increases in the single channel current and open probability but an 80% decrease in the number of open channels. Washout of aprotinin led to a time-dependent restoration of I(Na) as well as the single channel properties to the control, pre-aprotinin, values. We conclude that protease regulation of I(Na) is mediated by changes in the number of open channels in the apical membrane. The increase in the single channel current caused by protease inhibition can be explained by a hyperpolarization of the apical membrane potential as active Na(+) channels are retrieved. The paradoxical increase in channel open probability caused by protease inhibition will require further investigation but does suggest a potential compensatory regulatory mechanism to maintain I(Na) at some minimal threshold value.
Subject(s)
Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/pharmacology , Sodium Channels/metabolism , Amiloride/analogs & derivatives , Amiloride/pharmacology , Amphibians , Animals , Aprotinin/pharmacology , Cell Line , Dose-Response Relationship, Drug , Epithelial Sodium Channels , Kidney , Membrane Potentials , Sodium/metabolism , Sodium Channel Blockers/pharmacology , Sodium Channels/chemistry , Sodium Channels/drug effects , Time FactorsABSTRACT
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.
ABSTRACT
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.
ABSTRACT
We review the clinical and histologic features of 17 patients with cryoglobulinemia and renal disease. Most cases were associated with evidence of hepatitis C virus (HCV), although a significant minority had no evidence of HCV. The most common histologic pattern for renal involvement was membranoproliferative glomerulonephritis, which was seen in both HCV-positive and HVC-negative patients. Clinical presentation was variable, including nephrotic syndrome, unexplained elevations of serum creatinine, acute renal failure, or extrarenal manifestations. All patients had type II or type III cryoglobulins and all had low serum complements at presentation. Liver function abnormalities in HCV-positive patients were mild. No clinical or laboratory features beyond hepatitis serologies were helpful in distinguishing between HCV-positive and HCV-negative patients. All but 1 HCV-positive patient were treated with interferon (IFN) in either standard or high dosage, and this treatment was largely ineffective. Five of 11 HCV-positive patients progressed to renal failure. HCV patients treated with cyclophosphamide did not develop active liver disease. In all HCV-negative patients, renal function stabilized or improved, and 5 of 6 were treated with cyclophosphamide. In our series, there is limited experience with IFN-ribavirin therapy, which was not well tolerated. Renal cryoglobulinemia is an uncommon illness of diverse etiologies and clinical presentations. Morphologic presentation is also varied. IFN alone is often inadequate therapy for HCV-associated cryoglobulinemia. Experience with IFN-ribavirin in this entity is limited, but has shown promise in hepatic disease and has shown efficacy in HCV-associated cryoglobulinemia. Cyclophosphamide is the treatment of choice for HCV-negative patients and can be used safely in most HCV-positive patients if they fail IFN or IFN-ribavirin therapy, or if they require more aggressive therapy during periods of rapid clinical progression.
Subject(s)
Cryoglobulinemia/complications , Cryoglobulinemia/pathology , Kidney Diseases/etiology , Kidney Diseases/pathology , Adult , Aged , Antiviral Agents/therapeutic use , Cryoglobulinemia/drug therapy , Cyclophosphamide/therapeutic use , Female , Hepatitis C/complications , Humans , Immunosuppressive Agents/therapeutic use , Interferons/therapeutic use , Kidney Diseases/drug therapy , Male , Middle Aged , Pennsylvania/epidemiology , Retrospective StudiesABSTRACT
The authors report the case of a 47-year-old woman in whom a systemic illness developed characterized by fever, malaise, abnormal liver function results, and acute renal failure after treatment for presumed urinary tract infection with levofloxacin. Because of suspicion of an allergic drug reaction, all medications were discontinued, but the patient remained febrile with renal failure for 18 days. Complete workup for presumed vasculitis, autoimmune illness, or infectious etiologies was negative, and the patient underwent both renal and liver biopsy. Liver biopsy results showed nonspecific changes. Renal biopsy disclosed extensive granulomatous interstitial nephritis with associated granulomatous vasculitis. The patient was begun on oral steroids with rapid defervescence of fever and progressive normalization of renal function. The authors discuss the association of granulomatous nephritis with drugs and review the known nephrotoxicity of fluoroquinolones.
Subject(s)
Granuloma/chemically induced , Levofloxacin , Nephritis, Interstitial/chemically induced , Ofloxacin/adverse effects , Female , Humans , Middle Aged , Ofloxacin/therapeutic use , Urinary Tract Infections/drug therapyABSTRACT
OBJECTIVE: Patients with critical illness commonly develop acute renal failure requiring mechanical support in the form of either continuous renal replacement therapy (CRRT) or intermittent hemodialysis (IRRT). As controversy exists regarding which modality should be used for most patients with critically illness, we sought to determine whether CRRT or IRRT is associated with better survival. DESIGN: We performed a meta-analysis of all prior randomized and observational studies that compared CRRT with IRRT. Studies were identified through a MEDLINE search, the authors' files, bibliographies of review articles, abstracts and proceedings of scientific meetings. Studies were assessed for baseline characteristics, intervention, outcome and overall quality through blinded review. The primary end-point was hospital mortality, assessed by cumulative relative risk (RR). MEASUREMENTS AND RESULTS: We identified 13 studies ( n=1400), only three of which were randomized. Overall there was no difference in mortality (RR 0.93 (0.79-1.09), p=0.29). However, study quality was poor and only six studies compared groups of equal severity of illness at baseline (time of enrollment). Adjusting for study quality and severity of illness, mortality was lower in patients treated with CRRT (RR 0.72 (0.60-0.87), p<0.01). In the six studies with similar baseline severity, unadjusted mortality was also lower with CRRT (RR 0.48 (0.34 -0.69), p<0.0005). CONCLUSIONS: Current evidence is insufficient to draw strong conclusions regarding the mode of replacement therapy for acute renal failure in the critically ill. However, the life-saving potential with CRRT suggested in our secondary analyses warrants further investigation by a large, randomized trial.
Subject(s)
Renal Dialysis/methods , Respiratory Distress Syndrome , Critical Care , Hospital Mortality , Humans , Randomized Controlled Trials as Topic , Respiratory Distress Syndrome/classification , Respiratory Distress Syndrome/mortality , Respiratory Distress Syndrome/therapy , Severity of Illness Index , Survival AnalysisABSTRACT
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.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Epithelial Sodium Channels/metabolism , Kidney Tubules, Collecting/cytology , Phosphatidylinositols/metabolism , Stilbenes/pharmacology , AMP-Activated Protein Kinases/genetics , Amiloride/pharmacology , Animals , Cell Line , Cells, Cultured , Epithelial Sodium Channels/genetics , Immunoblotting , Mice , RNA, Small Interfering , ResveratrolABSTRACT
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.
Subject(s)
Cytoplasmic Vesicles/metabolism , Epithelial Sodium Channels/metabolism , Aldosterone/physiology , Animals , Cell Polarity , Cells, Cultured , Colforsin/pharmacology , Culture Media , Cyclic AMP/physiology , Electric Capacitance , Epithelial Cells/metabolism , Epithelial Cells/physiology , Epithelial Sodium Channels/genetics , Gene Expression , Gene Knockdown Techniques , Mice , Protein Transport , RNA Interference , Rats , Rats, Inbred F344ABSTRACT
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.
Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 16 , Developmental Disabilities/genetics , Models, Genetic , Adult , Case-Control Studies , Child , Child, Preschool , Chromosomes, Human, Pair 16/genetics , Comparative Genomic Hybridization/methods , Family , Gene Frequency , Humans , Infant , Oligonucleotide Array Sequence Analysis , Pedigree , Phenotype , Polymorphism, Single Nucleotide , Recurrence , Severity of Illness IndexABSTRACT
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.
Subject(s)
Cell Membrane/physiology , Epithelial Cells/physiology , Epithelial Sodium Channels/physiology , Protein Transport/physiology , Epithelial Cells/cytology , Exocytosis/physiology , Humans , Transport Vesicles/physiologyABSTRACT
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.
Subject(s)
Epithelial Sodium Channels/metabolism , Gene Expression Regulation/physiology , I-kappa B Kinase/metabolism , Kidney Tubules, Collecting/metabolism , Ubiquitin-Protein Ligases/metabolism , Xenopus Proteins/metabolism , Animals , Cell Line , Cell Membrane/genetics , Cell Membrane/metabolism , Cell Polarity/physiology , Endosomal Sorting Complexes Required for Transport , Epithelial Sodium Channels/genetics , Humans , I-kappa B Kinase/genetics , Immediate-Early Proteins/genetics , Immediate-Early Proteins/metabolism , Kidney Tubules, Collecting/cytology , Mice , Mutation , Nedd4 Ubiquitin Protein Ligases , Phosphorylation/physiology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA Interference , Ubiquitin-Protein Ligases/genetics , Xenopus Proteins/genetics , Xenopus laevisABSTRACT
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.