ISM1 promotes alveolar fluid clearance in acute lung injury by targeting cell-surface GRP78 and inhibiting PI3K/AKT/mTOR signaling pathway

Introduction Impaired alveolar fluid clearance (AFC) is a major pathological feature of acute lung injury (ALI). The epithelial sodium channel (ENaC) and Na + /K + -ATPase are the principal ion transport proteins that mediate AFC. Isthmin-1 (ISM1), a secreted protein with diverse biological functions, has been reported to protect against ALI; however, whether this protective effect is mediated through modulation of AFC remains unknown. This study aimed to elucidate the role and mechanism of ISM1 in regulating AFC in lipopolysaccharide (LPS)-induced ALI. Methods In vivo , ISM1 was overexpressed in mice using an adeno-associated virus serotype 5 (AAV5) vector, and an anti-GRP78 blocking antibody was administered to neutralize cell-surface GRP78 function. In vitro , LPS-stimulated A549 cells were treated with recombinant ISM1 protein, anti-GRP78 antibody, or the PI3K activator 740Y-P. The severity of ALI was assessed by hematoxylin-eosin staining, lung wet/dry weight ratio, bronchoalveolar lavage fluid total protein concentration, and inflammatory cytokine levels. α-ENaC and Na + /K + -ATPase α1 expression were evaluated by Western blot, RT-qPCR, and immunofluorescence, and PI3K-AKT-mTOR pathway activity was analyzed in both models. Results ISM1 overexpression attenuated LPS-induced ALI, as shown by reduced lung injury scores, lung wet/dry weight ratios, BALF total protein concentrations, and inflammatory cytokine levels, together with upregulated α-ENaC and Na + /K + -ATPase α1 expression. Mechanistically, ISM1 promoted α-ENaC and Na + /K + -ATPase α1 expression by suppressing the PI3K-AKT-mTOR signaling pathway, and these effects were significantly attenuated by GRP78 blockade. Conclusions These findings suggest that ISM1 promotes alveolar fluid clearance and protects against ALI through a GRP78-dependent mechanism involving inhibition of the PI3K-AKT-mTOR pathway, suggesting that ISM1 may have protective effects against ALI.