Claudin-4 Levels Are Associated with Intact Alveolar Fluid Clearance in Human Lungs

ABSTRACTBackground and PurposeLung oedema in association with suppressed fibrinolysis is a hallmark of lung injury. We aimed to test whether plasmin cleaves epithelial sodium channels (ENaC) to resolve lung oedema fluid.Experimental ApproachesHuman lungs and airway acid-instilled mice were used for analysing fluid resolution. In silico prediction, mutagenesis, Xenopus oocytes, immunoblotting, voltage clamp, mass spectrometry, protein docking, and alveolar fluid clearance were combined for identifying plasmin specific cleavage sites and benefits.Key ResultsPlasmin led to a marked increment in lung fluid resolution in both human lungs ex vivo and injured mice. Plasmin specifically activated αβγENaC channels in oocytes in a time-dependent manner. Deletion of four consensus proteolysis tracts (αΔ432-444, γΔ131-138, γΔ178-193, and γΔ410-422) eliminated plasmin-induced activation significantly. Further, immunoblotting assays identified 7 cleavage sites (K126, R135, K136, R153, K168, R178, K179) for plasmin to trim both furin-cleaved C-terminal fragments and full-length human γENaC proteins. In addition to confirming the 7 cleavage sites, 9 new sites (R122, R137, R138, K150, K170, R172, R180, K181, K189) in synthesized peptides were found to be cleaved by plasmin with mass spectrometry. These cleavage sites were located in the finger and the thumb, particularly the GRIP domain of human ENaC 3D model composed of two proteolytic centres for plasmin. Novel uncleaved sites beyond the GRIP domain in both α and γ subunits were identified to interrupt the plasmin cleavage-induced conformational change in ENaC channel complexes. Additionally, plasmin could regulate ENaC activity via the G protein signal.Conclusion and ImplicationsWe demonstrate that plasmin could cleave ENaC to benefit the blood-gas exchange by resolving oedema fluid as a potent fibrinolytic therapy for oedematous pulmonary diseases.Bullet point summaryWhat is already knowSerine proteases proteolytically cleave epithelial sodium channels, including plasmin and uPA acutely.Activity of epithelial sodium channels is increased post proteolysis.What this study addsPlasmin cleaves up to 16 sites composed of two proteolytic centres in both full-length and furin-cleaved human γ subunit of epithelial sodium channels in hours.Non-proteolytic sites in both α and γ subunits interrupt the plasmin cleavage-induced channel gating.Intratracheally instilled plasmin facilitates alveolar fluid clearance in normal human and injured mouse lungs.Clinical significanceActivation of human lung epithelial sodium channels by plasmin may benefit lung oedema resolution as a novel therapy for ARDS.

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Clinical grade allogeneic human mesenchymal stem cells restore alveolar fluid clearance in human lungs rejected for transplantation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00358.2013 ◽

2014 ◽

Vol 306 (9) ◽

pp. L809-L815 ◽

Cited By ~ 86

Author(s):

D. F. McAuley ◽

G. F. Curley ◽

U. I. Hamid ◽

J. G. Laffey ◽

J. Abbott ◽

...

Keyword(s):

Pulmonary Edema ◽

Ex Vivo ◽

Solid Organ ◽

Clinical Grade ◽

Alveolar Fluid Clearance ◽

Potential Donor ◽

Ex Vivo Lung Perfusion ◽

Ischemic Time ◽

Lung Lobe ◽

Human Lungs

The lack of suitable donors for all solid-organ transplant programs is exacerbated in lung transplantation by the low utilization of potential donor lungs, due primarily to donor lung injury and dysfunction, including pulmonary edema. The current studies were designed to determine if intravenous clinical-grade human mesenchymal stem (stromal) cells (hMSCs) would be effective in restoring alveolar fluid clearance (AFC) in the human ex vivo lung perfusion model, using lungs that had been deemed unsuitable for transplantation and had been subjected to prolonged ischemic time. The human lungs were perfused with 5% albumin in a balanced electrolyte solution and oxygenated with continuous positive airway pressure. Baseline AFC was measured in the control lobe and if AFC was impaired (defined as <10%/h), the lungs received either hMSC (5 × 106cells) added to the perfusate or perfusion only as a control. AFC was measured in a different lung lobe at 4 h. Intravenous hMSC restored AFC in the injured lungs to a normal level. In contrast, perfusion only did not increase AFC. This positive effect on AFC was reduced by intrabronchial administration of a neutralizing antibody to keratinocyte growth factor (KGF). Thus, intravenous allogeneic hMSCs are effective in restoring the capacity of the alveolar epithelium to remove alveolar fluid at a normal rate, suggesting that this therapy may be effective in enhancing the resolution of pulmonary edema in human lungs deemed clinically unsuitable for transplantation.

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Preservation of alveolar epithelial fluid transport mechanisms in rewarmed human lung after severe hypothermia

Journal of Applied Physiology ◽

10.1152/jappl.1996.80.5.1681 ◽

1996 ◽

Vol 80 (5) ◽

pp. 1681-1686 ◽

Cited By ~ 40

Author(s):

T. Sakuma ◽

S. Suzuki ◽

K. Usuda ◽

M. Handa ◽

G. Okaniwa ◽

...

Keyword(s):

Fluid Transport ◽

Human Lung ◽

Ex Vivo ◽

Transport Mechanisms ◽

Alveolar Fluid Clearance ◽

Alveolar Epithelial ◽

Severe Hypothermia ◽

Human Lungs ◽

Channel Transport

Although hypothermia abolishes alveolar fluid clearance in the in situ goat lung and in the ex vivo human lung, it is unknown whether alveolar fluid clearance resumes in lungs that are rewarmed after severe hypothermia. An isosmolar albumin solution was instilled into resected human lungs that were rewarmed to 37 degrees C after hypothermia (7 +/- 3 degrees C), and then alveolar fluid clearance was measured by the concentration of albumin in the alveolar fluid sample after 4 h. In control experiments in lungs that had not been cooled and rewarmed, alveolar fluid clearance was 11 +/- 2% over 4 h. In separate experiments, hypothermia completely abolished alveolar fluid clearance. However, alveolar fluid clearance resumed to a normal level of 12 +/- 1% over 4 h in the lungs that were rewarmed after hypothermia. Amiloride decreased alveolar fluid clearance by 47% in the rewarmed lungs. Terbutaline increased alveolar fluid clearance by nearly 300% in 2-h experiments in the rewarmed lungs (P < 0.05). The results of this study indicate that alveolar sodium-channel transport mechanisms are preserved in resected human lungs that are exposed to rewarming after hypothermia.

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