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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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Loss of Nedd4-2 results in perinatal lethality.(a) Targeting vector design. Top, Nedd4-2 wild-type allele exons 13–19 (solid bars). Middle, targeting construct for homologous recombination, with *indicating exonic regions deleted. A stop codon was introduced at exon 15. Bottom, disrupted allele. (b) Nedd4-2 expression in E18.5 lung from wild-type (+/+); heterozygous (+/−) and homozygous (−/−) animals. (c) Gross morphology of E18.5 pups. (d) Nedd4 expression in E18.5 tissues. (e) Survival curve for Nedd4-2−/− animals that overcome neonatal death (n=28).
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f1: Loss of Nedd4-2 results in perinatal lethality.(a) Targeting vector design. Top, Nedd4-2 wild-type allele exons 13–19 (solid bars). Middle, targeting construct for homologous recombination, with *indicating exonic regions deleted. A stop codon was introduced at exon 15. Bottom, disrupted allele. (b) Nedd4-2 expression in E18.5 lung from wild-type (+/+); heterozygous (+/−) and homozygous (−/−) animals. (c) Gross morphology of E18.5 pups. (d) Nedd4 expression in E18.5 tissues. (e) Survival curve for Nedd4-2−/− animals that overcome neonatal death (n=28).

Mentions: To study the physiological function of Nedd4-2, knockout mice were generated in Bruce4 C57BL/6 embryonic stem (ES) cells to introduce a stop codon in exon 15 of Nedd4-2 (Fig. 1a)23. We validated that we had created a allele of Nedd4-2 by immunoblotting E18.5 lung tissue protein lysates that confirmed the absence of Nedd4-2 protein in Nedd4-2 homozygous knockout mice (Fig. 1b). Furthermore, we did not detect any Nedd4-2 transcripts in the knockout mice (Supplementary Fig. S1), further validating that our strategy has resulted in a Nedd4-2 allele (referred to as Nedd4-2−/− hereafter). Both the Nedd4-2 isoforms present in the Nedd4-2+/− lung showed approximately half the protein levels of wild-type mice (Fig. 1b). The gross morphology of Nedd4-2−/− pups appeared the same as wild type (Fig. 1c). Nedd4 and Nedd4-2 are the two most closely related members of the Nedd4 family and may be able to substitute for each other, but there was no compensatory increase in the expression of Nedd4 observed in the absence of Nedd4-2 (Fig. 1d).


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)

Loss of Nedd4-2 results in perinatal lethality.(a) Targeting vector design. Top, Nedd4-2 wild-type allele exons 13–19 (solid bars). Middle, targeting construct for homologous recombination, with *indicating exonic regions deleted. A stop codon was introduced at exon 15. Bottom, disrupted allele. (b) Nedd4-2 expression in E18.5 lung from wild-type (+/+); heterozygous (+/−) and homozygous (−/−) animals. (c) Gross morphology of E18.5 pups. (d) Nedd4 expression in E18.5 tissues. (e) Survival curve for Nedd4-2−/− animals that overcome neonatal death (n=28).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Loss of Nedd4-2 results in perinatal lethality.(a) Targeting vector design. Top, Nedd4-2 wild-type allele exons 13–19 (solid bars). Middle, targeting construct for homologous recombination, with *indicating exonic regions deleted. A stop codon was introduced at exon 15. Bottom, disrupted allele. (b) Nedd4-2 expression in E18.5 lung from wild-type (+/+); heterozygous (+/−) and homozygous (−/−) animals. (c) Gross morphology of E18.5 pups. (d) Nedd4 expression in E18.5 tissues. (e) Survival curve for Nedd4-2−/− animals that overcome neonatal death (n=28).
Mentions: To study the physiological function of Nedd4-2, knockout mice were generated in Bruce4 C57BL/6 embryonic stem (ES) cells to introduce a stop codon in exon 15 of Nedd4-2 (Fig. 1a)23. We validated that we had created a allele of Nedd4-2 by immunoblotting E18.5 lung tissue protein lysates that confirmed the absence of Nedd4-2 protein in Nedd4-2 homozygous knockout mice (Fig. 1b). Furthermore, we did not detect any Nedd4-2 transcripts in the knockout mice (Supplementary Fig. S1), further validating that our strategy has resulted in a Nedd4-2 allele (referred to as Nedd4-2−/− hereafter). Both the Nedd4-2 isoforms present in the Nedd4-2+/− lung showed approximately half the protein levels of wild-type mice (Fig. 1b). The gross morphology of Nedd4-2−/− pups appeared the same as wild type (Fig. 1c). Nedd4 and Nedd4-2 are the two most closely related members of the Nedd4 family and may be able to substitute for each other, but there was no compensatory increase in the expression of Nedd4 observed in the absence of Nedd4-2 (Fig. 1d).

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