Involvement of αENaC and Nedd4-2 in the conversion from lung fluid secretion to fluid absorption at birth in the rat as assayed by RNA interference analysis

To explore interactions between the epithelial Na channel (ENaC) and neural precursor expressed, developmentally downregulated protein 4-2 (Nedd4-2) at the conversion of the rat lung from fluid secretion to absorption at birth, we used small-interfering RNA (siRNA) against αENaC and Nedd4-2. siRNA-generating plasmid DNA (pDNA) was administered via trans-thoracic intrapulmonary (ttip) injection 24 h before ENaC and Nedd4-2 expression, extravascular lung water, and mortality were measured. αENaC mRNA and protein were specifically reduced by ∼65% after pSi-4 injection. Nedd4-2 mRNA and protein were reduced by ∼60% after pSi-N1 injection. Interestingly, αENaC and βENaC mRNA and protein expression were increased after Nedd4-2 silencing. Extravascular lung water was significantly increased after αENaC silencing and reduced after Nedd4-2 silencing. αENaC silencing resulted in a fourfold increase in newborn mortality, whereas silencing Nedd4-2 did not affect mortality. We also isolated distal lung epithelial (DLE) cells after in vivo αENaC or Nedd4-2 silencing and measured αENaC or Nedd4-2 expression in freshly isolated DLE cells. In these DLE cells, there were attenuated αENaC or Nedd4-2 mRNA and protein, thus demonstrating that αENaC and Nedd4-2 silencing occurred in alveolar epithelial cells after ttip injection. We also looked for pDNA by PCR to determine pDNA presence in the lungs and found strong evidence for pDNA presence in both lungs. Thus we provide evidence that ENaC and Nedd4-2 are involved in the transition from lung fluid secretion to fluid absorption near term and at birth.

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Congenital diaphragmatic hernia prevents absorption of distal air space fluid in late-gestation rat fetuses

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00124.2005 ◽

2006 ◽

Vol 290 (3) ◽

pp. L478-L484 ◽

Cited By ~ 19

Author(s):

Hans G. Folkesson ◽

Cheryl J. Chapin ◽

LaMonta L. Beard ◽

Robert Ertsey ◽

Michael A. Matthay ◽

...

Keyword(s):

Congenital Diaphragmatic Hernia ◽

Diaphragmatic Hernia ◽

Fluid Secretion ◽

Alveolar Epithelial Cells ◽

Normal Lung ◽

Albumin Concentration ◽

Fluid Absorption ◽

Lung Maturation ◽

Lung Fluid ◽

Air Space

We hypothesized that congenital diaphragmatic hernia (CDH) may decrease distal air space fluid absorption due to immaturity of alveolar epithelial cells from a loss of the normal epithelial Na+ transport, as assessed by amiloride and epithelial Na+ channel (ENaC) and Na-K-ATPase expression, as well as failure to respond to endogenous epinephrine as assessed by propranolol. Timed-pregnant dams were gavage fed 100 mg of nitrofen at 9.5-day gestation to induce CDH in the fetuses, and distal air space fluid absorption experiments were carried out on 22-day gestation (term) fetuses. Controls were nitrofen-exposed fetuses without CDH. Absorption of distal air space fluid was measured from the increase in 131I-albumin concentration in an isosmolar, physiological solution instilled into the developing lungs. In controls, distal air space fluid absorption was rapid and mediated by β-adrenoceptors as demonstrated by reversal to fluid secretion after propranolol. Normal lung fluid absorption was also partially inhibited by amiloride. In contrast, CDH fetuses continued to show lung fluid secretion, and this secretion was not affected by either propranolol or amiloride. CDH lungs showed a 67% reduction in α-ENaC and β-ENaC expression, but no change in α1-Na-K-ATPase expression. These studies demonstrate: 1) CDH delays lung maturation with impaired distal air space fluid absorption secondary to inadequate Na+ uptake by the distal lung epithelium that results in fluid-filled lungs at birth with reduced capacity to establish postnatal breathing, and 2) the main stimulus to lung fluid absorption in near-term control fetuses, elevated endogenous epinephrine levels, is not functional in CDH fetuses.

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RNA interference for α-ENaC inhibits rat lung fluid absorption in vivo

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00205.2005 ◽

2006 ◽

Vol 290 (4) ◽

pp. L649-L660 ◽

Cited By ~ 53

Author(s):

Tianbo Li ◽

Hans G. Folkesson

Keyword(s):

Protein Expression ◽

Conclusive Evidence ◽

Organ Specificity ◽

Response Analysis ◽

Na Channel ◽

Fluid Absorption ◽

Alveolar Epithelial ◽

Lung Fluid ◽

Stimulation Of

We used siRNA against the α-ENaC (epithelial Na channel) subunit to investigate ENaC involvement in lung fluid absorption in rats by the impermeable tracer technique during baseline and after β-adrenoceptor stimulation by terbutaline. Terbutaline stimulation of lung fluid absorption increased fluid absorption by 165% in pSi-0-pretreated rat lungs (irrelevant siRNA-generating plasmid). Terbutaline failed to increase lung fluid absorption in rats given the specific α-ENaC siRNA-generating plasmid (pSi-4). pSi-4 pretreatment reduced baseline lung fluid absorption by ∼30%. α-ENaC was undetectable in pSi-4-pretreated lungs, regardless of condition but was normal in pSi-0-pretreated lungs. We carried out a dose-response analysis where rats were given 0–200 μg/kg body wt pSi-4, and α-ENaC mRNA and protein expressions were analyzed. To reach IC50for α-ENaC mRNA expression, 32 μg/kg body wt pSi-4 was needed, and to reach IC50for α-ENaC protein expression, 59 μg/kg body wt pSi-4 was needed. We tested for lung tissue specificity and found no changes in β-ENaC expression, at either mRNA or protein level, as well as no changes in α1-Na-K-ATPase protein expression. We isolated alveolar epithelial type II cells 24 h after in vivo pSi-4 pretreatment. In these cells, α-ENaC mRNA was undetectable, demonstrating that alveolar epithelial ENaC expression was attenuated after intratracheal α-ENaC siRNA-generating plasmid DNA instillation. We tested for organ specificity and found no changes in kidney α- and β-ENaC mRNA and protein expression. Thus we provide conclusive evidence that β-adrenoceptor stimulation of lung fluid absorption is critically ENaC dependent, whereas baseline lung fluid absorption seemed less ENaC dependent.

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Mechanisms and sequelae of increased alveolar fluid clearance in hyperoxic rats

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.272.3.l407 ◽

1997 ◽

Vol 272 (3) ◽

pp. L407-L412 ◽

Cited By ~ 35

Author(s):

G. Yue ◽

S. Matalon

Keyword(s):

Alveolar Epithelial Cells ◽

Na Channels ◽

Alveolar Fluid Clearance ◽

Na Transport ◽

Alveolar Epithelial ◽

Lung Fluid ◽

Isotonic Fluid ◽

Epithelial Na Channels ◽

Alveolar Edema

We instilled 4 ml isotonic fluid containing trace amounts of fluorescently labeled dextran (molecular mass 150 kDa) in the lungs of rats exposed to either 85% O(2) for 7 days or to 85% O(2) for 7 days and 100% O(2) for 3 days. We withdrew the fluid every hour for a 3-h period and calculated alveolar fluid clearance (AFC) from changes in dextran concentration. Postinstillation (3 h), AFC values in the control and the two hyperoxic groups were 51 +/- 1, 63 +/- 2, and 62 +/- 3 (SE), respectively (%instilled volume; n > or = 5; P < 0.05). Addition of either 1 mM amiloride or N-ethyl-N-isopropyl amiloride (EIPA) in the instillate decreased the AFC values in all groups 3 h later to approximately 30% of instilled volume. Instillation of phenamil, an irreversible blocker of epithelial Na+ channels into the lungs of rats exposed to 85% O(2) for 7 days and 100% O(2) for 2 days, resulted in a significant increase of their extravascular lung fluid volumes 24 h later. These results demonstrate the existence of EIPA-inhibitable Na+ channels in alveolar epithelial cells in vivo and indicate that an increase in Na+ transport plays an important role in limiting the amount of alveolar edema in O(2)-damaged lungs.

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Indicator dilution using a fluorescent indicator

Journal of Applied Physiology ◽

10.1152/jappl.1982.52.5.1368 ◽

1982 ◽

Vol 52 (5) ◽

pp. 1368-1374 ◽

Cited By ~ 6

Author(s):

V. B. Elings ◽

F. R. Lewis ◽

J. Briggs

Keyword(s):

Extravascular Lung Water ◽

Detection System ◽

Indicator Dilution ◽

Fluorescent Light ◽

Left Heart ◽

Lung Water ◽

Fluorescent Indicator ◽

Double Indicator Dilution

In vitro and in vivo indicator-dilution measurements are made with a fluorescent indicator and a novel detection system using a catheter containing a single optical fiber that carries both the exciting and returning fluorescent light. These fluorescent-dilution measurements are compared with simultaneous green dye-dilution measurements. The double-indicator-dilution measurement of extravascular lung water using heat and fluorescence is compared with gravimetric measurements. Also investigated is the sensitivity of the fluorescent measurement to changes in O2 saturation and hematocrit of the blood. An example of the measurement of a right-to-left heart shunt with this new indicator is given.

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Regulation of sgk by aldosterone and its effects on the epithelial Na+ channel

AJP Renal Physiology ◽

10.1152/ajprenal.2000.278.4.f613 ◽

2000 ◽

Vol 278 (4) ◽

pp. F613-F619 ◽

Cited By ~ 95

Author(s):

Alexander Shigaev ◽

Carol Asher ◽

Hedva Latter ◽

Haim Garty ◽

Eitan Reuveny

Keyword(s):

De Novo ◽

Rat Kidney ◽

Kidney Cortex ◽

Na Channel ◽

Xenopus Laevis Oocytes ◽

Fourfold Increase ◽

Tight Epithelia ◽

High Level

Aldosterone is the major corticosteroid regulating Na+ absorption in tight epithelia and acts primarily by activating the epithelial Na+ channel (ENaC) through unknown induced proteins. Recently, it has been reported that aldosterone induces the serum- and glucocorticoid-dependent kinase sgk and that coexpressing ENaC with this kinase in Xenopus laevis oocytes increases the amiloride-sensitive Na+current (Chen SY, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Proc Natl Acad Sci USA 96: 2514–2519, 1999). The present study was done to further characterize regulation of sgk by aldosterone in native mammalian epithelia and to examine its effect on ENaC. With both in vivo and in vitro protocols, an almost fivefold increase in the abundance of sgk mRNA has been demonstrated in rat kidney and colon but not in lung. Induction of sgk by aldosterone was detected in kidney cortex and medulla, whereas the papilla expressed a constitutively high level of the kinase. The increase in sgkmRNA was detected as early as 30 min after the hormonal application and was independent of de novo protein synthesis. The observed aldosterone dose-response relationships suggest that the response is mediated, at least in part, by occupancy of the mineralocorticoid receptor. Coexpressing sgk and ENaC in Xenopus oocytes evoked a fourfold increase in the amiloride-blockable Na+ channel activity. A point mutation in the β-subunit known to impair regulation of the channel by Nedd4 (Y618A) had no significant effect on the response to sgk.

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In vivo analysis of fluid transport in cystic fibrosis airway epithelia of bronchial xenografts

AJP Cell Physiology ◽

10.1152/ajpcell.1996.270.5.c1326 ◽

1996 ◽

Vol 270 (5) ◽

pp. C1326-C1335 ◽

Cited By ~ 61

Author(s):

Y. Zhang ◽

J. Yankaskas ◽

J. Wilson ◽

J. F. Engelhardt

Keyword(s):

Cystic Fibrosis ◽

Fluid Transport ◽

Xenograft Model ◽

Na Channel ◽

Transmembrane Conductance Regulator ◽

Fluid Absorption ◽

Cystic Fibrosis Airway ◽

In Vivo Analysis ◽

Cf Transmembrane Conductance Regulator

An in vivo human bronchial xenograft model system was used to simultaneously analyze electrolyte and fluid transport defects in fully differentiated human cystic fibrosis (CF) and non-CF proximal airways. CF airways demonstrated three discernible defects when compared with non-CF, including 1) a lack of adenosine 3',5'-cylic monophosphate (cAMP)-inducible Cl- secretion, 2) a fourfold higher basal fluid absorption rate, and 3) an altered regulation of fluid absorption in response to amiloride-stimulated changes in Na+ transport. A unique finding in this study demonstrated that treatment of epithelia with amiloride led to a greater than threefold decrease in the rate of fluid absorption in CF tissues as contrasted to a greater than threefold increase in the rate of fluid absorption in non-CF tissues. The removal of apical Na+ from amiloride-treated non-CF xenografts was capable of ablating this amiloride-induced increase in fluid absorption. In light of the recent interactions demonstrated between CF transmembrane conductance regulator (CFTR) and the rat epithelial, amiloride-sensitive Na+ channel, these findings implicate additional complexities between the Na+ conductance pathways and fluid transport in normal and CF proximal airways. Such findings suggest that CFTR may also regulate amiloride-insensitive Na+ channels.

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β-Adrenergic agonists differentially regulate highly selective and nonselective epithelial sodium channels to promote alveolar fluid clearance in vivo

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00038.2012 ◽

2012 ◽

Vol 302 (11) ◽

pp. L1167-L1178 ◽

Cited By ~ 32

Author(s):

Charles A. Downs ◽

Lisa H. Kriener ◽

Ling Yu ◽

Douglas C. Eaton ◽

Lucky Jain ◽

...

Keyword(s):

Single Channel ◽

Selective Inhibition ◽

Alveolar Epithelial Cells ◽

Alveolar Type ◽

Channel Measurements ◽

Open Probability ◽

Alveolar Epithelial ◽

Lung Fluid ◽

T1 And T2

β-Adrenergic receptors (β-AR) increase epithelial sodium channel (ENaC) activity to promote lung fluid clearance. However, the effect of selective β-AR agonist on highly selective cation (HSC) channels or nonselective cation (NSC) channels in alveolar type 1 (T1) and type 2 (T2) cells is unknown. We hypothesized that stimulation with β1-AR agonist (denopamine) or β2-AR agonist (terbutaline) would increase HSC and/or NSC channel activity in alveolar epithelial cells. We performed single-channel measurements from T1 and T2 cells accessed from rat lung slices. Terbutaline (20 μM) increased HSC ENaC activity (open probability, NPo) in T1 (from 0.96 ± 0.61 to 1.25 ± 0.71, n = 5, P <0.05) and T2 cells (from 0.28 ± 0.14 to 1.0 ± 0.30, n = 8, P = 0.02). Denopamine (20 μM) increased NSC NPo in T1 cells (from 0.34 ± 0.09 to 0.63 ± 0.14, n = 7, P = 0.02) and in T2 cells (from 0.47 ± 0.09 to 0.68 ± 0.10, P = 0.004). In vivo X-ray imaging of lung fluid clearance and ICI 118,551 selective inhibition of β2-ARs confirmed patch-clamp findings. cAMP concentrations increased following treatment with denopamine or terbutaline ( n = 3, P < 0.002). The effects of systemic (intraperitoneal, IP) and local (intratracheal, IT) modes of delivery on lung fluid clearance were assessed. IT delivery of denopamine promoted alveolar flooding, whereas IP delivery promoted delayed fluid clearance. In summary, β-AR agonists differentially regulate HSC and NSC in T1 and T2 cells to promote lung fluid clearance in vivo, and the mode of drug delivery is critical for maximizing β-AR agonist efficacy.

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