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Respiratory distress and perinatal lethality in Nedd4-2-deficient mice.

Boase NA, Rychkov GY, Townley SL, Dinudom A, Candi E, Voss AK, Tsoutsman T, Semsarian C, Melino G, Koentgen F, Cook DI, Kumar S - Nat Commun (2011)

Bottom Line: This increased ENaC activity is the likely reason for premature fetal lung fluid clearance in Nedd4-2(-/-) animals, resulting in a failure to inflate lungs and perinatal lethality.A small percentage of Nedd4-2(-/-) animals survive up to 22 days, and these animals also show increased ENaC expression and develop lethal sterile inflammation of the lung.Thus, we provide critical in vivo evidence that Nedd4-2 is essential for correct regulation of ENaC expression, fetal and postnatal lung function and animal survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Haematology, Centre for Cancer Biology, SA Pathology, PO Box 14, Rundle Mall, Adelaide, South Australia 5000, Australia.

ABSTRACT
The epithelial sodium channel (ENaC) is essential for sodium homoeostasis in many epithelia. ENaC activity is required for lung fluid clearance in newborn animals and for maintenance of blood volume and blood pressure in adults. In vitro studies show that the ubiquitin ligase Nedd4-2 ubiquitinates ENaC to regulate its cell surface expression. Here we show that knockout of Nedd4-2 in mice leads to increased ENaC expression and activity in embryonic lung. This increased ENaC activity is the likely reason for premature fetal lung fluid clearance in Nedd4-2(-/-) animals, resulting in a failure to inflate lungs and perinatal lethality. A small percentage of Nedd4-2(-/-) animals survive up to 22 days, and these animals also show increased ENaC expression and develop lethal sterile inflammation of the lung. Thus, we provide critical in vivo evidence that Nedd4-2 is essential for correct regulation of ENaC expression, fetal and postnatal lung function and animal survival.

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ENaC-mediated current in lung cells.(a) A typical isolated epithelial lung cell producing ENaC current. Scale bar, 20 μm. (b) The effects of Na+ replacement with an impermeant cation (N-methyl-D-glucamine, NMDG) and the application of amiloride on the whole-cell membrane currents of lung cells isolated from wild-type and Nedd4-2−/− mice. NaCl (140 mM) was replaced with 140 mM NMDGCl in the external solution and 50 μM amiloride was applied into the bath. Each point represents amplitude of the current at −100 mV measured from responses to the voltage ramps ranging from −120 to 120 mV and applied every 2 s. Representative cells are shown. (c) Averaged I–V plots of amiloride-sensitive currents in wild-type (n=19) and Nedd4-2−/− (n=8) lung cells. Black lines represent averaged current traces obtained in response to voltage ramps ranging from −120 to 120 mV. Dashed grey lines represent s.e.m. (d) The amplitudes of the amiloride-sensitive current measured at −100 mV in wild-type (filled symbols, n=19) and Nedd4-2−/− (clear symbols, n=8) cells. Horizontal lines represent the mean±s.e.m. P<0.0001 was determined by unpaired two-tail t-test.
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f4: ENaC-mediated current in lung cells.(a) A typical isolated epithelial lung cell producing ENaC current. Scale bar, 20 μm. (b) The effects of Na+ replacement with an impermeant cation (N-methyl-D-glucamine, NMDG) and the application of amiloride on the whole-cell membrane currents of lung cells isolated from wild-type and Nedd4-2−/− mice. NaCl (140 mM) was replaced with 140 mM NMDGCl in the external solution and 50 μM amiloride was applied into the bath. Each point represents amplitude of the current at −100 mV measured from responses to the voltage ramps ranging from −120 to 120 mV and applied every 2 s. Representative cells are shown. (c) Averaged I–V plots of amiloride-sensitive currents in wild-type (n=19) and Nedd4-2−/− (n=8) lung cells. Black lines represent averaged current traces obtained in response to voltage ramps ranging from −120 to 120 mV. Dashed grey lines represent s.e.m. (d) The amplitudes of the amiloride-sensitive current measured at −100 mV in wild-type (filled symbols, n=19) and Nedd4-2−/− (clear symbols, n=8) cells. Horizontal lines represent the mean±s.e.m. P<0.0001 was determined by unpaired two-tail t-test.

Mentions: To determine whether the increased ENaC expression observed at the alveolar surface of E18.5 Nedd4-2−/− lungs correlates with increased amiloride-sensitive sodium current due to ENaC, whole-cell patch clamp recording was performed on alveolar type II lung cells (Fig. 4a). The presence of Na+ current was initially assessed by replacing 140 mM extracellular Na+ with equimolar amounts of the impermeant cation N-methyl-D-glucamine (Fig. 4b). If Na+ current was present, 50 μM amiloride was applied to the bath solution to ascertain if the current was mediated by ENaC (Fig. 4b). The I–V current/voltage plots of ENaC currents in Nedd4-2+/+ and Nedd4-2−/− cells obtained by subtracting current traces recorded before and after the application of amiloride showed strong inward rectification and reversal potential of approximately +50 mV, which is characteristic of a Na+-selective channel (Fig. 4c). The average amplitude of amiloride-sensitive Na+ current measured at −100 mV in lung cells isolated from Nedd4-2+/+ animals was −2.2±0.4 pA/pF (n=19), whereas current measured in Nedd4-2−/− cells had a significantly greater amplitude of −15.1±3.2 pA/pF (n=8; P<0.0001; Fig. 4d). These data indicate that the loss of Nedd4-2 is strongly correlated with increased ENaC current in alveolar type II cells.


Respiratory distress and perinatal lethality in Nedd4-2-deficient mice.

Boase NA, Rychkov GY, Townley SL, Dinudom A, Candi E, Voss AK, Tsoutsman T, Semsarian C, Melino G, Koentgen F, Cook DI, Kumar S - Nat Commun (2011)

ENaC-mediated current in lung cells.(a) A typical isolated epithelial lung cell producing ENaC current. Scale bar, 20 μm. (b) The effects of Na+ replacement with an impermeant cation (N-methyl-D-glucamine, NMDG) and the application of amiloride on the whole-cell membrane currents of lung cells isolated from wild-type and Nedd4-2−/− mice. NaCl (140 mM) was replaced with 140 mM NMDGCl in the external solution and 50 μM amiloride was applied into the bath. Each point represents amplitude of the current at −100 mV measured from responses to the voltage ramps ranging from −120 to 120 mV and applied every 2 s. Representative cells are shown. (c) Averaged I–V plots of amiloride-sensitive currents in wild-type (n=19) and Nedd4-2−/− (n=8) lung cells. Black lines represent averaged current traces obtained in response to voltage ramps ranging from −120 to 120 mV. Dashed grey lines represent s.e.m. (d) The amplitudes of the amiloride-sensitive current measured at −100 mV in wild-type (filled symbols, n=19) and Nedd4-2−/− (clear symbols, n=8) cells. Horizontal lines represent the mean±s.e.m. P<0.0001 was determined by unpaired two-tail t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104547&req=5

f4: ENaC-mediated current in lung cells.(a) A typical isolated epithelial lung cell producing ENaC current. Scale bar, 20 μm. (b) The effects of Na+ replacement with an impermeant cation (N-methyl-D-glucamine, NMDG) and the application of amiloride on the whole-cell membrane currents of lung cells isolated from wild-type and Nedd4-2−/− mice. NaCl (140 mM) was replaced with 140 mM NMDGCl in the external solution and 50 μM amiloride was applied into the bath. Each point represents amplitude of the current at −100 mV measured from responses to the voltage ramps ranging from −120 to 120 mV and applied every 2 s. Representative cells are shown. (c) Averaged I–V plots of amiloride-sensitive currents in wild-type (n=19) and Nedd4-2−/− (n=8) lung cells. Black lines represent averaged current traces obtained in response to voltage ramps ranging from −120 to 120 mV. Dashed grey lines represent s.e.m. (d) The amplitudes of the amiloride-sensitive current measured at −100 mV in wild-type (filled symbols, n=19) and Nedd4-2−/− (clear symbols, n=8) cells. Horizontal lines represent the mean±s.e.m. P<0.0001 was determined by unpaired two-tail t-test.
Mentions: To determine whether the increased ENaC expression observed at the alveolar surface of E18.5 Nedd4-2−/− lungs correlates with increased amiloride-sensitive sodium current due to ENaC, whole-cell patch clamp recording was performed on alveolar type II lung cells (Fig. 4a). The presence of Na+ current was initially assessed by replacing 140 mM extracellular Na+ with equimolar amounts of the impermeant cation N-methyl-D-glucamine (Fig. 4b). If Na+ current was present, 50 μM amiloride was applied to the bath solution to ascertain if the current was mediated by ENaC (Fig. 4b). The I–V current/voltage plots of ENaC currents in Nedd4-2+/+ and Nedd4-2−/− cells obtained by subtracting current traces recorded before and after the application of amiloride showed strong inward rectification and reversal potential of approximately +50 mV, which is characteristic of a Na+-selective channel (Fig. 4c). The average amplitude of amiloride-sensitive Na+ current measured at −100 mV in lung cells isolated from Nedd4-2+/+ animals was −2.2±0.4 pA/pF (n=19), whereas current measured in Nedd4-2−/− cells had a significantly greater amplitude of −15.1±3.2 pA/pF (n=8; P<0.0001; Fig. 4d). These data indicate that the loss of Nedd4-2 is strongly correlated with increased ENaC current in alveolar type II cells.

Bottom Line: This increased ENaC activity is the likely reason for premature fetal lung fluid clearance in Nedd4-2(-/-) animals, resulting in a failure to inflate lungs and perinatal lethality.A small percentage of Nedd4-2(-/-) animals survive up to 22 days, and these animals also show increased ENaC expression and develop lethal sterile inflammation of the lung.Thus, we provide critical in vivo evidence that Nedd4-2 is essential for correct regulation of ENaC expression, fetal and postnatal lung function and animal survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Haematology, Centre for Cancer Biology, SA Pathology, PO Box 14, Rundle Mall, Adelaide, South Australia 5000, Australia.

ABSTRACT
The epithelial sodium channel (ENaC) is essential for sodium homoeostasis in many epithelia. ENaC activity is required for lung fluid clearance in newborn animals and for maintenance of blood volume and blood pressure in adults. In vitro studies show that the ubiquitin ligase Nedd4-2 ubiquitinates ENaC to regulate its cell surface expression. Here we show that knockout of Nedd4-2 in mice leads to increased ENaC expression and activity in embryonic lung. This increased ENaC activity is the likely reason for premature fetal lung fluid clearance in Nedd4-2(-/-) animals, resulting in a failure to inflate lungs and perinatal lethality. A small percentage of Nedd4-2(-/-) animals survive up to 22 days, and these animals also show increased ENaC expression and develop lethal sterile inflammation of the lung. Thus, we provide critical in vivo evidence that Nedd4-2 is essential for correct regulation of ENaC expression, fetal and postnatal lung function and animal survival.

Show MeSH
Related in: MedlinePlus