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Regulation of ENaC-mediated alveolar fluid clearance by insulin via PI3K/Akt pathway in LPS-induced acute lung injury.

Deng W, Li CY, Tong J, Zhang W, Wang DX - Respir. Res. (2012)

Bottom Line: In vivo, insulin decreased TLW, enchanced AFC, increased the expressions of α-,β-, and γ-ENaC and the level of phosphorylated Akt, attenuated lung injury and improved the survival rate in LPS-induced ALI, the effects of which were blocked by wortmannin.Amiloride, a sodium channel inhibitor, significantly reduced insulin-induced increase in AFC.Immunoprecipitation studies showed that levels of Nedd4-2 binding to ENaC were decreased by insulin via PI3K/Akt pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Respiratory Medicine, Second Affiliated Hospital of Chongqing Medical University, Yuzhong District, Chongqing, China.

ABSTRACT

Background: Stimulation of epithelial sodium channel (ENaC) increases Na(+) transport, a driving force of alveolar fluid clearance (AFC) to keep alveolar spaces free of edema fluid that is beneficial for acute lung injury (ALI). It is well recognized that regulation of ENaC by insulin via PI3K pathway, but the mechanism of this signaling pathway to regulate AFC and ENaC in ALI remains unclear. The aim of this study was to investigate the effect of insulin on AFC in ALI and clarify the pathway in which insulin regulates the expression of ENaC in vitro and in vivo.

Methods: A model of ALI (LPS at a dose of 5.0 mg/kg) with non-hyperglycemia was established in Sprague-Dawley rats receiving continuous exogenous insulin by micro-osmotic pumps and wortmannin. The lungs were isolated for measurement of bronchoalveolar lavage fluid(BALF), total lung water content(TLW), and AFC after ALI for 8 hours. Alveolar epithelial type II cells were pre-incubated with LY294002, Akt inhibitor and SGK1 inhibitor 30 minutes before insulin treatment for 2 hours. The expressions of α-,β-, and γ-ENaC were detected by immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting.

Results: In vivo, insulin decreased TLW, enchanced AFC, increased the expressions of α-,β-, and γ-ENaC and the level of phosphorylated Akt, attenuated lung injury and improved the survival rate in LPS-induced ALI, the effects of which were blocked by wortmannin. Amiloride, a sodium channel inhibitor, significantly reduced insulin-induced increase in AFC. In vitro, insulin increased the expressions of α-,β-, and γ-ENaC as well as the level of phosphorylated Akt but LY294002 and Akt inhibitor significantly prevented insulin-induced increase in the expression of ENaC and the level of phosphorylated Akt respectively. Immunoprecipitation studies showed that levels of Nedd4-2 binding to ENaC were decreased by insulin via PI3K/Akt pathway.

Conclusions: Our study demonstrated that insulin alleviated pulmonary edema and enhanced AFC by increasing the expression of ENaC that dependent upon PI3K/Akt pathway by inhibition of Nedd4-2.

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Related in: MedlinePlus

Expressions of alveolar epithelial sodium channel(α-ENaC, β-ENaC and γ-ENaC) in rat lung were measured by RT-PCR(A) and western blotting(B) 8 hours after LPS-induced actue lung injury or saline treatment (n = 5 per group). Proteins using the same antibodies against α-ENaC, β-ENaC and γ-ENaC plus blocking peptides specific for these antibodies were re-blotted(C). Data are presented as mean ± S.E.M.ΔP < 0.05 vs Control group;*P < 0.05 vs LPS group;# P < 0.05 vs LPS + Insulin group.
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Figure 6: Expressions of alveolar epithelial sodium channel(α-ENaC, β-ENaC and γ-ENaC) in rat lung were measured by RT-PCR(A) and western blotting(B) 8 hours after LPS-induced actue lung injury or saline treatment (n = 5 per group). Proteins using the same antibodies against α-ENaC, β-ENaC and γ-ENaC plus blocking peptides specific for these antibodies were re-blotted(C). Data are presented as mean ± S.E.M.ΔP < 0.05 vs Control group;*P < 0.05 vs LPS group;# P < 0.05 vs LPS + Insulin group.

Mentions: To clarify the effect of insulin on AFC mediated by ENaC, the expressions of α-, β- and γ-ENaC were measured by RT-PCR and western blotting respectively. Two forms (90 kDa and 65 kDa) of α-ENaC were detected by western blotting (Figure 6 B; Figure 7 B). In vivo, the mRNA and protein expression levels of α-, β- and γ-ENaC in rat lung showed significant increases by insulin treatment 8 hours after LPS-induced ALI (P < 0.05, Figure 6 A, B), but the mRNA and protein expression levels of three ENaC subunits were significantly decreased with the administration of wortmannin compared with those by insulin treatment (P < 0.05, Figure 6 A, B). In vitro, the mRNA and protein expression levels of α-, β- and γ-ENaC were significantly increased by insulin treatment for 2 hours in ATII cells (P < 0.05, Figure 7A, B), but pretreatment with LY294002 and Akt inhibitor prevented the insulin-induced increase in the mRNA and protein expression levels of α-, β- and γ-ENaC in ATII cells respectively (P < 0.05, Figure 7A, B). In addtion, the mRNA and protein expression levels of α-, β- and γ-ENaC in ATII cells with co- administration of Akt inhibitor and SGK1inhibitor showed the similar changes compared with those by LY294002 treatment(P > 0.05, Figure 7A, B), and were significantly decreased compared with those by Akt inhibitor treatment (P < 0.05, Figure 7A, B). The bands were absent when proteins were blotted with the α-ENaC, β-ENaC and γ-ENaC antibodies in the presence of the blocking peptide both in vivo(Figure 6C) and in vitro(Figure 7 C). These results indicated that insulin-induced expression of ENaC by Akt phosphorylation via activating PI3K pathway.


Regulation of ENaC-mediated alveolar fluid clearance by insulin via PI3K/Akt pathway in LPS-induced acute lung injury.

Deng W, Li CY, Tong J, Zhang W, Wang DX - Respir. Res. (2012)

Expressions of alveolar epithelial sodium channel(α-ENaC, β-ENaC and γ-ENaC) in rat lung were measured by RT-PCR(A) and western blotting(B) 8 hours after LPS-induced actue lung injury or saline treatment (n = 5 per group). Proteins using the same antibodies against α-ENaC, β-ENaC and γ-ENaC plus blocking peptides specific for these antibodies were re-blotted(C). Data are presented as mean ± S.E.M.ΔP < 0.05 vs Control group;*P < 0.05 vs LPS group;# P < 0.05 vs LPS + Insulin group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3362779&req=5

Figure 6: Expressions of alveolar epithelial sodium channel(α-ENaC, β-ENaC and γ-ENaC) in rat lung were measured by RT-PCR(A) and western blotting(B) 8 hours after LPS-induced actue lung injury or saline treatment (n = 5 per group). Proteins using the same antibodies against α-ENaC, β-ENaC and γ-ENaC plus blocking peptides specific for these antibodies were re-blotted(C). Data are presented as mean ± S.E.M.ΔP < 0.05 vs Control group;*P < 0.05 vs LPS group;# P < 0.05 vs LPS + Insulin group.
Mentions: To clarify the effect of insulin on AFC mediated by ENaC, the expressions of α-, β- and γ-ENaC were measured by RT-PCR and western blotting respectively. Two forms (90 kDa and 65 kDa) of α-ENaC were detected by western blotting (Figure 6 B; Figure 7 B). In vivo, the mRNA and protein expression levels of α-, β- and γ-ENaC in rat lung showed significant increases by insulin treatment 8 hours after LPS-induced ALI (P < 0.05, Figure 6 A, B), but the mRNA and protein expression levels of three ENaC subunits were significantly decreased with the administration of wortmannin compared with those by insulin treatment (P < 0.05, Figure 6 A, B). In vitro, the mRNA and protein expression levels of α-, β- and γ-ENaC were significantly increased by insulin treatment for 2 hours in ATII cells (P < 0.05, Figure 7A, B), but pretreatment with LY294002 and Akt inhibitor prevented the insulin-induced increase in the mRNA and protein expression levels of α-, β- and γ-ENaC in ATII cells respectively (P < 0.05, Figure 7A, B). In addtion, the mRNA and protein expression levels of α-, β- and γ-ENaC in ATII cells with co- administration of Akt inhibitor and SGK1inhibitor showed the similar changes compared with those by LY294002 treatment(P > 0.05, Figure 7A, B), and were significantly decreased compared with those by Akt inhibitor treatment (P < 0.05, Figure 7A, B). The bands were absent when proteins were blotted with the α-ENaC, β-ENaC and γ-ENaC antibodies in the presence of the blocking peptide both in vivo(Figure 6C) and in vitro(Figure 7 C). These results indicated that insulin-induced expression of ENaC by Akt phosphorylation via activating PI3K pathway.

Bottom Line: In vivo, insulin decreased TLW, enchanced AFC, increased the expressions of α-,β-, and γ-ENaC and the level of phosphorylated Akt, attenuated lung injury and improved the survival rate in LPS-induced ALI, the effects of which were blocked by wortmannin.Amiloride, a sodium channel inhibitor, significantly reduced insulin-induced increase in AFC.Immunoprecipitation studies showed that levels of Nedd4-2 binding to ENaC were decreased by insulin via PI3K/Akt pathway.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Respiratory Medicine, Second Affiliated Hospital of Chongqing Medical University, Yuzhong District, Chongqing, China.

ABSTRACT

Background: Stimulation of epithelial sodium channel (ENaC) increases Na(+) transport, a driving force of alveolar fluid clearance (AFC) to keep alveolar spaces free of edema fluid that is beneficial for acute lung injury (ALI). It is well recognized that regulation of ENaC by insulin via PI3K pathway, but the mechanism of this signaling pathway to regulate AFC and ENaC in ALI remains unclear. The aim of this study was to investigate the effect of insulin on AFC in ALI and clarify the pathway in which insulin regulates the expression of ENaC in vitro and in vivo.

Methods: A model of ALI (LPS at a dose of 5.0 mg/kg) with non-hyperglycemia was established in Sprague-Dawley rats receiving continuous exogenous insulin by micro-osmotic pumps and wortmannin. The lungs were isolated for measurement of bronchoalveolar lavage fluid(BALF), total lung water content(TLW), and AFC after ALI for 8 hours. Alveolar epithelial type II cells were pre-incubated with LY294002, Akt inhibitor and SGK1 inhibitor 30 minutes before insulin treatment for 2 hours. The expressions of α-,β-, and γ-ENaC were detected by immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting.

Results: In vivo, insulin decreased TLW, enchanced AFC, increased the expressions of α-,β-, and γ-ENaC and the level of phosphorylated Akt, attenuated lung injury and improved the survival rate in LPS-induced ALI, the effects of which were blocked by wortmannin. Amiloride, a sodium channel inhibitor, significantly reduced insulin-induced increase in AFC. In vitro, insulin increased the expressions of α-,β-, and γ-ENaC as well as the level of phosphorylated Akt but LY294002 and Akt inhibitor significantly prevented insulin-induced increase in the expression of ENaC and the level of phosphorylated Akt respectively. Immunoprecipitation studies showed that levels of Nedd4-2 binding to ENaC were decreased by insulin via PI3K/Akt pathway.

Conclusions: Our study demonstrated that insulin alleviated pulmonary edema and enhanced AFC by increasing the expression of ENaC that dependent upon PI3K/Akt pathway by inhibition of Nedd4-2.

Show MeSH
Related in: MedlinePlus