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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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Pulmonary abnormalities in embryonic lungs from Nedd4-2−/− mice.(a) Haematoxylin and eosin-stained lung sections. Scale bars, 50 μm. (b) Graphical representation of E18.5 lung flotation rates. Error bars represent the standard deviation. (c) Wet/dry ratio of E17.5 lung tissues. (d) Wet/dry ratio of E18.5 lung tissues. The horizontal lines in red represent the mean value. *For comparison between wild type and Nedd4-2−/− standard two-tailed unpaired t-test were performed. P-values are as follows (b) P=0.0005, (c) P=0.0027 and (d) P=0.0004.
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f2: Pulmonary abnormalities in embryonic lungs from Nedd4-2−/− mice.(a) Haematoxylin and eosin-stained lung sections. Scale bars, 50 μm. (b) Graphical representation of E18.5 lung flotation rates. Error bars represent the standard deviation. (c) Wet/dry ratio of E17.5 lung tissues. (d) Wet/dry ratio of E18.5 lung tissues. The horizontal lines in red represent the mean value. *For comparison between wild type and Nedd4-2−/− standard two-tailed unpaired t-test were performed. P-values are as follows (b) P=0.0005, (c) P=0.0027 and (d) P=0.0004.

Mentions: Histological examination of lungs at E15.5 and E17.5 revealed that lung architecture and development was grossly normal in Nedd4-2−/− animals (Fig. 2a). However, at E18.5 the lung architecture was significantly altered in Nedd4-2−/− animals compared with wild type, with collapsed alveolar spaces resulting in increased tissue density (Fig. 2a and Supplementary Fig. S2a–d). To further analyse this phenotype, lungs dissected from Nedd4-2−/−, Nedd4-2+/− and Nedd4-2+/+ caesarian-derived pups at E18.5 were placed in water and observed for flotation to indicate whether respiration had taken place. Whole lungs (including trachea), heart and thymus from Nedd4-2−/−, Nedd4-2+/− and Nedd4-2+/+ floated. However when lungs were dissected into smaller pieces, all individual pieces of Nedd4-2+/+ and Nedd4-2+/− lungs floated, whereas approximately half of the individual Nedd4-2−/− lung pieces sank, indicating that no aeration had occurred in these tissue pieces (Fig. 2b). The inability of Nedd4-2−/− pups to fully aerate their lungs was consistent with their physical signs of respiratory distress. In addition, Nedd4-2−/− lungs showed a reduced amount of embryonic lung fluid compared with wild type in utero at both E17.5 and E18.5 (Fig. 2c,d), presumably leading to the smaller alveolar spaces observed. These observations indicate that loss of Nedd4-2 results in premature lung fluid clearance, leading to a failure to inflate the lungs and subsequent respiratory distress and perinatal lethality.


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)

Pulmonary abnormalities in embryonic lungs from Nedd4-2−/− mice.(a) Haematoxylin and eosin-stained lung sections. Scale bars, 50 μm. (b) Graphical representation of E18.5 lung flotation rates. Error bars represent the standard deviation. (c) Wet/dry ratio of E17.5 lung tissues. (d) Wet/dry ratio of E18.5 lung tissues. The horizontal lines in red represent the mean value. *For comparison between wild type and Nedd4-2−/− standard two-tailed unpaired t-test were performed. P-values are as follows (b) P=0.0005, (c) P=0.0027 and (d) P=0.0004.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Pulmonary abnormalities in embryonic lungs from Nedd4-2−/− mice.(a) Haematoxylin and eosin-stained lung sections. Scale bars, 50 μm. (b) Graphical representation of E18.5 lung flotation rates. Error bars represent the standard deviation. (c) Wet/dry ratio of E17.5 lung tissues. (d) Wet/dry ratio of E18.5 lung tissues. The horizontal lines in red represent the mean value. *For comparison between wild type and Nedd4-2−/− standard two-tailed unpaired t-test were performed. P-values are as follows (b) P=0.0005, (c) P=0.0027 and (d) P=0.0004.
Mentions: Histological examination of lungs at E15.5 and E17.5 revealed that lung architecture and development was grossly normal in Nedd4-2−/− animals (Fig. 2a). However, at E18.5 the lung architecture was significantly altered in Nedd4-2−/− animals compared with wild type, with collapsed alveolar spaces resulting in increased tissue density (Fig. 2a and Supplementary Fig. S2a–d). To further analyse this phenotype, lungs dissected from Nedd4-2−/−, Nedd4-2+/− and Nedd4-2+/+ caesarian-derived pups at E18.5 were placed in water and observed for flotation to indicate whether respiration had taken place. Whole lungs (including trachea), heart and thymus from Nedd4-2−/−, Nedd4-2+/− and Nedd4-2+/+ floated. However when lungs were dissected into smaller pieces, all individual pieces of Nedd4-2+/+ and Nedd4-2+/− lungs floated, whereas approximately half of the individual Nedd4-2−/− lung pieces sank, indicating that no aeration had occurred in these tissue pieces (Fig. 2b). The inability of Nedd4-2−/− pups to fully aerate their lungs was consistent with their physical signs of respiratory distress. In addition, Nedd4-2−/− lungs showed a reduced amount of embryonic lung fluid compared with wild type in utero at both E17.5 and E18.5 (Fig. 2c,d), presumably leading to the smaller alveolar spaces observed. These observations indicate that loss of Nedd4-2 results in premature lung fluid clearance, leading to a failure to inflate the lungs and subsequent respiratory distress and perinatal lethality.

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