Restoration of Megalin-Mediated Clearance of Alveolar Protein as a Novel Therapeutic Approach for Acute Lung Injury.

Acute respiratory distress syndrome constitutes a significant disease burden with regard to both morbidity and mortality. Current therapies are mostly supportive and do not address the underlying pathophysiologic mechanisms. Removal of protein-rich alveolar edema-a clinical hallmark of acute respiratory distress syndrome-is critical for survival. Here, we describe a transforming growth factor (TGF)-ß-triggered mechanism, in which megalin, the primary mediator of alveolar protein transport, is negatively regulated by glycogen synthase kinase (GSK) 3ß, with protein phosphatase 1 and nuclear inhibitor of protein phosphatase 1 being involved in the signaling cascade. Inhibition of GSK3ß rescued transepithelial protein clearance in primary alveolar epithelial cells after TGF-ß treatment. Moreover, in a bleomycin-based model of acute lung injury, megalin+/- animals (the megalin-/- variant is lethal due to postnatal respiratory failure) showed a marked increase in intra-alveolar protein and more severe lung injury compared with wild-type littermates. In contrast, wild-type mice treated with the clinically relevant GSK3ß inhibitors, tideglusib and valproate, exhibited significantly decreased alveolar protein concentrations, which was associated with improved lung function and histopathology. Together, we discovered that the TGF-ß-GSK3ß-megalin axis is centrally involved in disturbances of alveolar protein clearance in acute lung injury and provide preclinical evidence for therapeutic efficacy of GSK3ß inhibition.

Megalin mediates transepithelial albumin clearance from the alveolar space of intact rabbit lungs.

The alveolo-capillary barrier is effectively impermeable to large solutes such as proteins. A hallmark of acute lung injury/acute respiratory distress syndrome is the accumulation of protein-rich oedema fluid in the distal airspaces. Excess protein must be cleared from the alveolar space for recovery; however, the mechanisms of protein clearance remain incompletely understood. In intact rabbit lungs 29.8 ± 2.2% of the radio-labelled alveolar albumin was transported to the vascular compartment at 37°C within 120 min, as assessed by real-time measurement of 125I-albumin clearance from the alveolar space. At 4°C or 22°C significantly lower albumin clearance (3.7 ± 0.4 or 16.2 ± 1.1%, respectively) was observed. Deposition of a 1000-fold molar excess of unlabelled albumin into the alveolar space or inhibition of cytoskeletal rearrangement or clathrin-dependent endocytosis largely inhibited the transport of 125I-albumin to the vasculature, while administration of unlabelled albumin to the vascular space had no effect on albumin clearance. Furthermore, albumin uptake capacity was measured as about 0.37 mg ml−1 in cultured rat lung epithelial monolayers, further highlighting the (patho)physiological relevance of active alveolar epithelial protein transport. Moreover, gene silencing and pharmacological inhibition of the multi-ligand receptor megalin resulted in significantly decreased albumin binding and uptake in monolayers of primary alveolar type II and type I-like and cultured lung epithelial cells. Our data indicate that clearance of albumin from the distal air spaces is facilitated by an active, high-capacity, megalin-mediated transport process across the alveolar epithelium. Further understanding of this mechanism is of clinical importance, since an inability to clear excess protein from the alveolar space is associated with poor outcome in patients with acute lung injury/acute respiratory distress syndrome.

Carbon monoxide rapidly impairs alveolar fluid clearance by inhibiting epithelial sodium channels.

Carbon monoxide (CO) is currently being evaluated as a therapeutic modality in the treatment of patients with acute lung injury and acute respiratory distress syndrome. No study has assessed the effects of CO on transepithelial ion transport and alveolar fluid reabsorption, two key aspects of alveolocapillary barrier function that are perturbed in acute lung injury/acute respiratory distress syndrome. Both CO gas (250 ppm) and CO donated by the CO donor, CO-releasing molecule (CORM)-3 (100 microM in epithelial lining fluid), applied to healthy, isolated, ventilated, and perfused rabbit lungs, significantly blocked (22)Na(+) clearance from the alveolar compartment, and blocked alveolar fluid reabsorption after fluid challenge. Apical application of two CO donors, CORM-3 or CORM-A1 (100 microM), irreversibly inhibited amiloride-sensitive short-circuit currents in H441 human bronchiolar epithelial cells and primary rat alveolar type II cells by up to 40%. Using a nystatin permabilization approach, the CO effect was localized to amiloride-sensitive channels on the apical surface. This effect was abolished by hemoglobin, a scavenger of CO, and was not observed when inactive forms of CO donors were employed. The effects of CO were not blocked by 8-bromoguanosine-3',5'-cyclic guanosine monophosphate, soluble guanylate cyclase inhibitors (methylene blue and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), or inhibitors of trafficking events (phalloidin oleate, MG-132, and brefeldin A), but the amiloride affinity of H441 cells was reduced after CO exposure. These data indicate that CO rapidly inhibits sodium absorption across the airway epithelium by cyclic guanosine monophosphate- and trafficking-independent mechanisms, which may rely on critical histidine residues in amiloride-sensitive channels or associated regulatory proteins on the apical surface of lung epithelial cells.