Netrin-1 Promotes Epithelial Sodium Channel-Mediated Alveolar Fluid Clearance via Activation of the Adenosine 2B Receptor in Lipopolysaccharide-Induced Acute Lung Injury

Abstract

Background: The epithelial sodium channel (ENaC) is the driving force for pulmonary edema absorption in acute lung injury (ALI). Netrin-1 is a newly found anti-inflammatory factor that works by activating the adenosine 2B receptor (A2BAR). Meanwhile, activated A2BAR has the potential to enhance ENaC-dependent alveolar fluid clearance (AFC). However, whether netrin-1 can increase ENaC-mediated AFC by activating A2BAR remains unclear. Objectives: To investigate the effect of netrin-1 on AFC in ALI and clarify the pathway via which netrin-1 regulates the expression of ENaC in vivo and in vitro. Methods: An ALI model was established by intratracheal instillation of lipopolysaccharide (LPS; 5 mg/kg) in C57BL/J mice, followed by netrin-1 with or without pretreatment with PSB1115, via the caudal vein. Twenty-four hours later, the lungs were isolated for determination of the bronchoalveolar lavage fluid, the lung wet/dry weight (W/D) ratio, AFC, the expressions of α-, β-, and γ-ENaC, and cyclic adenosine monophosphate (cAMP) levels. LPS-stimulated MLE-12 cells were incubated with netrin-1 with or without preincubation with PSB1115. Twenty-four hours later, the expressions of α-, β-, and γ-ENaC were detected. Results: In vivo, netrin-1 expression was significantly decreased during ALI. Substituted netrin-1 significantly dampened the lung injury, decreased the W/D ratio, and enhanced AFC, the expressions of α-, β-, and γ-ENaC, and cAMP levels in ALI, which were abolished by specific A2BAR inhibitor PSB1115. In vitro, netrin-1 increased the expressions of α-, β-, and γ-ENaC, which were prevented by PSB1115. Conclusion: These results indicate that netrin-1 dampens pulmonary inflammation and increases ENaC-mediated AFC to alleviate pulmonary edema in LPS-induced ALI by enhancing cAMP levels through the activation of A2BAR.