Edema Clearance Research Articles

Effects of hypercapnia on the lung.

Gases are sensed by lung cells and can activate specific intracellular signalling pathways, and thus have physiological and pathophysiological effects. Carbon dioxide (CO2 ), a primary product of oxidative metabolism, can be sensed by eukaryotic cells eliciting specific responses via recently identified signalling pathways. However, the physiological and pathophysiological effects of high CO2 (hypercapnia) on the lungs and specific lung cells, which are the primary site of CO2 elimination, are incompletely understood. In this review, we provide a physiological and mechanistic perspective on the effects of hypercapnia on the lungs and discuss the recent understanding of CO2 modulation of the alveolar epithelial function (lung oedema clearance), epithelial cell repair, innate immunity and airway function.

Acute Corneal Hydrops in Children with Primary Infantile Glaucoma: A Report of 31 Cases over 23 Years at the LVPEI.

PurposeRelatively little data exist regarding the outcomes of children with primary infantile glaucoma presenting with acute corneal hydrops. The aim of our study was to determine the surgical outcome of children of infantile glaucoma who presented with acute corneal hydrops.MethodsIn total, 38 eyes of 31 consecutive children of infantile glaucoma presented with acute corneal hydrops who underwent primary combined trabeculotomy-trabeculectomy (CTT) by a single surgeon from January 1990 to December 2012 at the LV Prasad Eye Institute (LVPEI), a tertiary eye care centre in Southern India were enrolled in this retrospective study. Primary outcome measures were intraocular pressure (IOP) control (IOP ≤ 16 mmHg under anaesthesia or IOP ≤ 21 mmHg without anaesthesia) and clearance of corneal edema. Secondary outcome measures were visual acuity (VA), corneal diameter, bleb appearance, intraoperative and postoperative complications.ResultsMean age at presentation was 6.4 months (range, 2–11 months) and seven eyes (23%) had bilateral affliction. At presentation, all eyes (100%) had moderate to severe degree of corneal edema with a mean preoperative IOP of 25.6 ±5.1 mmHg. Postoperatively, the IOP reduced to 12.0 ± 3.8 mmHg (difference = -13.6, 95% CI = -15.7 to -11.5, t = -13.18, p<0.0001), and the percentage reduction in IOP was 53.05%. Preoperatively 83% of the eyes were on antiglaucoma medication, and postoperatively 2 eyes (5.3%) required 1 antiglaucoma medication for control of IOP. Preoperatively, corneal edema was present in all eyes and postoperatively it cleared in all of them. Significant myopic astigmatism was present in 28 eyes (74%), the commonest being compound myopic astigmatism (75%) followed by simple myopic astigmatism (21%). Normal VA (best-corrected VA; BCVA ≥ 20/60) was achieved in 44.4% of the eyes and 22.2% eyes had low vision (BCVA, <20/60 to 20/400). Complete success (IOP control and clearance of corneal oedema) was obtained in 94.7% eyes. There were no significant intraoperative or postoperative complications. Two thirds of the patients showed low, elevated functional filtering bleb. No patient had any bleb leak, blebitis or bleb related endophthalmitis. The median follow-up was 36 months (range 2–228 months).ConclusionsPrimary CTT is safe and effective in controlling IOP, resulting in complete clearance of corneal edema with modest visual improvement in children of infantile glaucoma presenting with acute corneal hydrops. The outcome of the study will have a positive impact on counseling the parents preoperatively.

Macrophage-epithelial paracrine crosstalk inhibits lung edema clearance during influenza infection.

Influenza A viruses (IAV) can cause lung injury and acute respiratory distress syndrome (ARDS), which is characterized by accumulation of excessive fluid (edema) in the alveolar airspaces and leads to hypoxemia and death if not corrected. Clearance of excess edema fluid is driven mostly by the alveolar epithelial Na,K-ATPase and is crucial for survival of patients with ARDS. We therefore investigated whether IAV infection alters Na,K-ATPase expression and function in alveolar epithelial cells (AECs) and the ability of the lung to clear edema. IAV infection reduced Na,K-ATPase in the plasma membrane of human and murine AECs and in distal lung epithelium of infected mice. Moreover, induced Na,K-ATPase improved alveolar fluid clearance (AFC) in IAV-infected mice. We identified a paracrine cell communication network between infected and noninfected AECs and alveolar macrophages that leads to decreased alveolar epithelial Na,K-ATPase function and plasma membrane abundance and inhibition of AFC. We determined that the IAV-induced reduction of Na,K-ATPase is mediated by a host signaling pathway that involves epithelial type I IFN and an IFN-dependent elevation of macrophage TNF-related apoptosis-inducing ligand (TRAIL). Our data reveal that interruption of this cellular crosstalk improves edema resolution, which is of biologic and clinical importance to patients with IAV-induced lung injury.

With-No-Lysine Kinase 4 Mediates Alveolar Fluid Regulation in Hyperoxia-Induced Lung Injury.

To investigate mechanisms involved in the regulation of epithelial ion channels and alveolar fluid clearance in hyperoxia-induced lung injury. Laboratory animal experiments. Animal care facility procedure room in a medical center. Wild-type, STE20/SPS1-related proline/alanine-rich kinase knockout (SPAK(-/-)), and with-no-lysine kinase 4 knockin (WNK4(D561A/+)) mice. Mice were exposed to room air or 95% hyperoxia for 60 hours. Exposure to hyperoxia for 60 hours increased the lung expression of with-no-lysine kinase 4 and led to STE20/SPS1-related proline/alanine-rich kinase and sodium-potassium-chloride cotransporter phosphorylation, which resulted in the suppression of alveolar fluid clearance and increase of lung edema. WNK4(D561A/+) mice at the baseline presented an abundance of epithelium sodium channel and high levels of STE20/SPS1-related proline/alanine-rich kinase and sodium-potassium-chloride cotransporter phosphorylation. Compared with the wild-type group, hyperoxia caused greater epithelium sodium channel expression in WNK4(D561A/+) mice, but no significant difference in STE20/SPS1-related proline/alanine-rich kinase and sodium-potassium-chloride cotransporter phosphorylation. The functional inactivation of sodium-potassium-chloride cotransporter by gene knockout in SPAK(-/-) mice yielded a lower severity of lung injury and longer animal survival, whereas constitutive expression of with-no-lysine kinase 4 exacerbated the hyperoxia-induced lung injury. Pharmacologic inhibition of sodium-potassium-chloride cotransporter by inhaled furosemide improved animal survival in WNK4(D561A/+) mice. By contrast, inhibition of epithelium sodium channel exacerbated the hyperoxia-induced lung injury and animal death. With-no-lysine kinase 4 plays a crucial role in the regulation of epithelial ion channels and alveolar fluid clearance, mainly via phosphorylation and activation of STE20/SPS1-related proline/alanine-rich kinase and sodium-potassium-chloride cotransporter.

ID: 159: IFN-dependent macrophage-epithelial crosstalk induces and sustains lung injury in viral pneumonia

Influenza A viruses (IAV) may cause lung injury and acute respiratory distress syndrome (ARDS) characterized by apoptotic injury to the alveolar epithelium and accumulation of excessive fluid (edema) in the alveolar airspaces resulting in loss of barrier function, respiratory failure, and death. We recently demonstrated that inflammatory macrophages recruited from the circulation into the alveoli substantially contribute to influenza-induced lung epithelial injury by IFN-dependent expression of pro-apoptotic factors such as TNF-related apoptosis-inducing ligand (TRAIL). Following severe injury, clearance of excess edema fluid, driven by the alveolar epithelial Na, K-ATPase, is crucial for survival of patients with ARDS. We demonstrate that, apart from causing direct injury to the delicate alveolar barrier, macrophage mediators significantly impair resolution of inflammation in terms of edema clearance from the infected lungs. Using ex vivo and in vivo infection models including transgenic mice and adoptive cell transfer strategies, we identify a paracrine cell communication network between alveolar epithelial cells and alveolar macrophages leading to decreased alveolar epithelial Na, K-ATPase plasma membrane abundance and inhibition of alveolar fluid clearance. Our data furthermore reveal that therapeutic blockade of this cellular cross-talk improves edema resolution which is of clinical significance to patients with IAV-induced lung injury.

High respiratory rate is associated with early reduction of lung edema clearance in an experimental model of ARDS.

The independent impact of respiratory rate on ventilator-induced lung injury has not been fully elucidated. The aim of this study was to investigate the effects of two clinically relevant respiratory rates on early ventilator-induced lung injury evolution and lung edema during the protective ARDSNet strategy. We hypothesized that the use of a higher respiratory rate during a protective ARDSNet ventilation strategy increases lung inflammation and, in addition, lung edema associated to strain-induced activation of transforming growth factor beta (TGF-β) in the lung epithelium. Twelve healthy piglets were submitted to a two-hit lung injury model and randomized into two groups: LRR (20 breaths/min) and HRR (40 breaths/min). They were mechanically ventilated during 6 h according to the ARDSNet strategy. We assessed respiratory mechanics, hemodynamics, and extravascular lung water (EVLW). At the end of the experiment, the lungs were excised and wet/dry ratio, TGF-β pathway markers, regional histology, and cytokines were evaluated. No differences in oxygenation, PaCO2 levels, systemic and pulmonary arterial pressures were observed during the study. Respiratory system compliance and mean airway pressure were lower in LRR group. A decrease in EVLW over time occurred only in the LRR group (P < 0.05). Wet/dry ratio was higher in the HRR group (P < 0.05), as well as TGF-β pathway activation. Histological findings suggestive of inflammation and inflammatory tissue cytokines were higher in LRR. HRR was associated with more pulmonary edema and higher activation of the TGF-β pathway. In contrast with our hypothesis, HRR was associated with less lung inflammation.

The Role of Angiotensin II and Cyclic AMP in Alveolar Active Sodium Transport.

Active alveolar fluid clearance is important in keeping airspaces free of edema. Angiotensin II plays a role in the pathogenesis of hypertension, heart failure and others. However, little is known about its contribution to alveolar fluid clearance. Angiotensin II effects are mediated by two specific receptors; AT1 and AT2. The localization of these two receptors in the lung, specifically in alveolar epithelial cells type II, was recently reported. We hypothesize that Angiotensin II may have a role in the regulation of alveolar fluid clearance. We investigated the effect of Angiotensin II on alveolar fluid clearance in rats using the isolated perfused lung model and isolated rat alveolar epithelial cells. The rate of alveolar fluid clearance in control rats was 8.6% ± 0.1 clearance of the initial volume and decreased by 22.5%, 28.6%, 41.6%, 48.7% and 39% in rats treated with 10-10 M, 10-9 M, 10-8 M, 10-7 M or 10-6 M of Ang II respectively (P < 0.003). The inhibitory effect of Angiotensin II was restored in losartan, an AT1 specific antagonist, pretreated rats, indicating an AT1 mediated effect of Ang II on alveolar fluid clearance. The expression of Na,K-ATPase proteins and cAMP levels in alveolar epithelial cells were down-regulated following the administration of Angiotensin II; suggesting that cAMP may be involved in AngII-induced reduced Na,K-ATPase expression, though the contribution of additional factors could not be excluded. We herein suggest a novel mechanism of clinical relevance by which angiotensin adversely impairs the ability of the lungs to clear edema.

Lung Edema Clearance: Relevance to Patients with Lung Injury.

Pulmonary edema clearance is necessary for patients with lung injury to recover and survive. The mechanisms regulating edema clearance from the lungs are distinct from the factors contributing edema formation during injury. Edema clearance is effected via vectorial transport of Na+ out of the airspaces which generates an osmotic gradient causing water to follow the gradient out of the cells. This Na+ transport across the alveolar epithelium is mostly effected via apical Na+ and chloride channels and basolateral Na,K-ATPase. The Na,K-ATPase pumps Na+ out of the cell and K+ into the cell against their respective gradients in an ATP-consuming reaction. Two mechanisms contribute to the regulation of the Na,K-ATPase activity:recruitment of its subunits from intracellular compartments into the basolateral membrane, and transcriptional/translational regulation. Na,K-ATPase activity and edema clearance are increased by catecholamines, aldosterone, vasopressin, overexpression of the pump genes, and others. During lung injury, mechanisms regulating edema clearance are inhibited by yet unclear pathways. Better understanding of the mechanisms that regulate pulmonary edema clearance may lead to therapeutic interventions that counterbalance the inhibition of edema clearance during lung injury and improve the lungs’ ability to clear fluid, which is crucial for patient survival.

Dopamine treatment does not need speed recovery of newborns from transient tachypnea

Transient tachypnea of the newborn (TTN) results from inadequate neonatal lung fluid clearance. Low-dose dopamine induces natriuresis in the kidneys and it has been assumed that, at this low dosage, dopamine increases renal perfusion in critically ill patients. Medium doses have positive inotropic and chronotropic effects via increased β-receptor activation. Recent studies have demonstrated that dopamine stimulates the clearance of pulmonary edema. Furthermore, β-adrenergic agonists regulate Na+ channels and Na-K-ATPase activity in the pulmonary epithelium. This study investigated the effect of dopamine at different dosages on TTN treatment. A prospective controlled study examined 60 infants with TTN older than 34 weeks of gestation who required at least 24 h of O2 and nasal continuous positive airway pressure (nCPAP) treatment. The infants were randomized into three groups of 20: controls, infants treated with low-dose dopamine (3 μg/kg/min), and infants treated with a medium dose (5 μg/kg/min). The control and study groups were compared in terms of the requirement for mechanical ventilation, and the durations of nCPAP, oxygen requirement, and hospitalization. The requirement for mechanical ventilation, and durations of nCPAP, oxygen requirement, and hospitalization did not differ significantly among the three groups (P=0.54, 0.16 and 0.11, respectively). Dopamine treatment in low-moderate doses does not improve the outcome in TTN. Thus, further studies in this area are needed.

Abstract 429: HuR and microRNA-128 Competitively Regulate Vascular Endothelial Growth Factor C to Modulate Inflammatory Lymphangiogenesis

Macrophages respond to acute and chronic tissue injuries such as infection, ischemia, atherosclerosis, and tumor expansion with the production of cytokines, growth factors, and matrix remodeling factors. Many of these factors are encoded by mRNAs that, through interactions with RNA binding proteins and noncoding RNAs, are subject to signal-dependent post-transcriptional regulation. Macrophage recruitment and adhesion-triggered signaling, specifically by β2 integrin engagement, promotes stabilization of some mRNAs by inducing the nuclear-to-cytoplasmic translocation of the RNA binding protein HuR. HuR binds and stabilizes mRNAs bearing AU-rich elements that, when unbound by HuR, confer short mRNA half-life and limit protein production. By promoting stability and translation, HuR functionally opposes RNA degradation and translational repression by microRNAs. Lymphangiogenesis, largely mediated by VEGF-C’s engagement of its lymphatic endothelial cell receptor VEGFR3, plays a role in tissue edema clearance, cholesterol homeostasis, tumor metastasis, hypertension, and atherosclerosis. Here, we report competitive post-transcriptional regulation of VEGF-C in macrophages by HuR and miR-128. Translation of VEGF-C is repressed by the binding of miR-128 to its 3’-UTR. This binding and repression is abrogated by β2 integrin engagement in an HuR-dependent manner. Using an HuR conditional allele and a transgenic fluorescent marker of lymphatic endothelium, we show that macrophage-specific HuR deletion in mice leads to a reduction in lymphatic vessel proliferation and VEGF-C protein expression in an in vivo model of inflammatory lymphangiogenesis. Our data implicate the loss of HuR and consequent unopposed miR-128 activity in impaired VEGF-C production and lymphatic expansion in vivo. Our investigation includes transcriptome and RISC footprint profiling of activated primary macrophages to identify additional HuR regulated factors that act in concert with VEGF-C to drive lymphangiogenesis. In conclusion, this study describes a novel regulatory mechanism in which adhesion signaling in macrophages promotes growth factor expression necessary for lymphatic vascular expansion in response to inflammatory stimuli.

Electroporation-mediated gene delivery of Na+,K+ -ATPase, and ENaC subunits to the lung attenuates acute respiratory distress syndrome in a two-hit porcine model.

Acute respiratory distress syndrome (ARDS) is a common cause of organ failure with an associated mortality rate of 40%. The initiating event is disruption of alveolar-capillary interface causing leakage of edema into alveoli. Electroporation-mediated gene delivery of epithelial sodium channel (ENaC) and Na+,K+ -ATPase into alveolar cells would improve alveolar clearance of edema and attenuate ARDS. Pigs were anesthetized and instrumented, and the superior mesenteric artery was clamped to cause gut ischemia/reperfusion injury and peritoneal sepsis by fecal clot implantation. Animals were ventilated according to ARDSnet protocol. Four hours after injury, animals were randomized into groups: (i) treatment: Na+,K+ -ATPase/ENaC plasmid (n = 5) and (ii) control: empty plasmid (n = 5). Plasmids were delivered to the lung using bronchoscope. Electroporation was delivered using eight-square-wave electric pulses across the chest. Following electroporation, pigs were monitored 48 h. The Pao2/Fio2 ratio and lung compliance were higher in the treatment group. Lung wet/dry ratio was lower in the treatment group. Relative expression of the Na+,K+ -ATPase transgene was higher throughout lungs receiving treatment plasmids. Quantitative histopathology revealed a reduction in intra-alveolar fibrin in the treatment group. Bronchoalveolar lavage showed increased surfactant protein B in the treatment group. Survival was improved in the treatment group. Electroporation-mediated transfer of Na+,K+ -ATPase/ENaC plasmids improved lung function, reduced fibrin deposits, decreased lung edema, and improved survival in a translational porcine model of ARDS. Gene therapy can attenuate ARDS pathophysiology in a high-fidelity animal model, suggesting a potential new therapy for patients.

Pleomorphic xanthoastrocytoma with anaplastic features: one case report and review of literature

Objective To investigate the clinicopathological features of pleomorphic xanthoastrocytoma with anaplastic features (PXA-A). Methods The clinical manifestations, imaging, histopathological features, and immunophenotype were analyzed in one case of PXA-A, and relevant literatures were reviewed. Results The patient was a 58-year-old woman. MRI examination revealed a parenchyma mass with irregularly long T1 and long T2 signal in right temporal lobe and basal ganglia region. The border was clear and peritumoral edema was inconspicuous. The mesocephalon and right ventricle were compressed, and the midline was shifted to left. Enhanced MRI showed multiple flaky and nodular enhancement. Histologically, tumor cells showed remarkable cellular pleomorphism, and they were composed of mononuclear cells, multinuclear giant tumor cells, frothy tumor cells and spindle cells. Eosinophilic granular bodies and intranuclear inclusions were seen. Tumor cells in partial regions were intensively arranged, with obvious atypia. Immunohistochemical analysis showed immunoreactivity of the cells to glial fibrillary acidic protein (GFAP), Vimentin (Vim), S-100 protein (S-100), neuronal nuclei (NeuN) and P53. The cells showed a negative reaction for synaptophysin (Syn), chromogranin A (CgA), neurofilament protein (NF), CD34 and isocitrate dehydrogenase 1 (IDH1). The Ki-67 label index was 8.20% . Conclusions PXA-A is a rare tumor. The imaging features can offer a few diagnostic cues. However, a definite diagnosis depends on the histological and immunohistochemical features. doi: 10.3969/j.issn.1672-6731.2014.12.013

Insulin upregulates the expression of epithelial sodium channel in vitro and in a mouse model of acute lung injury: Role of mTORC2/SGK1 pathway

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by proteinaceous pulmonary edema and severe arterial hypoxemia with a mortality of approximately 40%. Stimulation of epithelial sodium channel (ENaC) promotes Na+ transport, a rate-limiting step for pulmonary edema reabsorption. Insulin is known to participate in the ion transport; however, its role in pulmonary edema clearance and the regulatory mechanism involved have not been fully elucidated. In the current study, in a lipopolysaccharide-based mouse model of ALI, we found that insulin alleviated pulmonary edema by promoting ENaC-mediated alveolar fluid clearance through serum and glucocorticoid induced kinase-1 (SGK1). In alveolar epithelial cells, insulin increased the expression of α-, β-, and γ-ENaC, which was blocked by the mammalian target of rapamycin complex 2 (mTORC2) inhibitor or knockdown of Rictor (a necessary component of mTORC2), and SGK1 inhibitor, respectively. In addition, an immunoprecipitation study demonstrated that SGK1(Ser422) phosphorylation, the key step for complete SGK1 activation by insulin, was conducted through PI3K/mTORC2 pathway. Finally, we testified the role of mTORC2 in vivo by demonstrating that PP242 prevented insulin-stimulated SGK1 activation and ENaC increase during ALI. The data revealed that during ALI, insulin stimulates alveolar fluid clearance by upregulating the expression of α-, β-, and γ-ENaC at the cell surface, which was, at least, partially through activating mTROC2/SGK1 signaling pathway.

Murine lung injury caused by Leptospira interrogans glycolipoprotein, a specific Na/K-ATPase inhibitor.

BackgroundLeptospiral glycolipoprotein (GLP) is a potent and specific Na/K-ATPase inhibitor. Severe pulmonary form of leptospirosis is characterized by edema, inflammation and intra-alveolar hemorrhage having a dismal prognosis. Resolution of edema and inflammation determines the outcome of lung injury. Na/K-ATPase activity is responsible for edema clearance. This enzyme works as a cell receptor that triggers activation of mitogen-activated protein kinase (MAPK) intracellular signaling pathway. Therefore, injection of GLP into lungs induces injury by triggering inflammation.MethodsWe injected GLP and ouabain, into mice lungs and compared their effects. Bronchoalveolar lavage fluid (BALF) was collected for cell and lipid body counting and measurement of protein and lipid mediators (PGE2 and LTB4). The levels of the IL-6, TNFα, IL-1B and MIP-1α were also quantified. Lung images illustrate the injury and whole-body plethysmography was performed to assay lung function. We used Toll-like receptor 4 (TLR4) knockout mice to evaluate leptospiral GLP-induced lung injury. Na/K-ATPase activity was determined in lung cells by nonradioactive rubidium incorporation. We analyzed MAPK p38 activation in lung and in epithelial and endothelial cells.ResultsLeptospiral GLP and ouabain induced lung edema, cell migration and activation, production of lipid mediators and cytokines and hemorrhage. They induced lung function alterations and inhibited rubidium incorporation. Using TLR4 knockout mice, we showed that the GLP action was not dependent on TLR4 activation. GLP activated of p38 and enhanced cytokine production in cell cultures which was reversed by a selective p38 inhibitor.ConclusionsGLP and ouabain induced lung injury, as evidenced by increased lung inflammation and hemorrhage. To our knowledge, this is the first report showing GLP induces lung injury. GLP and ouabain are Na/K-ATPase targets, triggering intracellular signaling pathways. We showed p38 activation by GLP-induced lung injury, which was may be linked to Na/K-ATPase inhibition. Lung inflammation induced by GLP was not dependent on TLR4 activation.

Fluids in ARDS: from onset through recovery.

Early acute respiratory distress syndrome (ARDS) is characterized by protein-rich inflammatory lung edema often associated with a hydrostatic component. Mechanical ventilation with positive intrathoracic pressure further induces salt and water retention, while impairing the pathways designed for edema clearance. In this framework, we will review the recent findings on fluid strategy and edema clearance in ARDS. Consistently, conservative strategies lead to better oxygenation and reduce the length of mechanical ventilation. A possible drawback associated with conservative strategy is the impaired cognitive function. Echography may be used for safer use of furosemide or hemofiltration therapy during edema clearance. Albumin and furosemide techniques may accelerate edema clearance, particularly when pulmonary capillary permeability is restored. Beta-2 agonist therapy does not accelerate edema clearance and is potentially dangerous. Lung edema is likely the single pathogenic factor more relevant for ARDS severity and outcome. Fluid overload must be avoided. Several monitoring techniques are available to reach this target. No specific studies are available to recommend a given fluid composition in ARDS. In our opinion, the general recommendations for fluid composition suggested for severe sepsis and septic shock should be applied to ARDS that may be considered an organ-confined sepsis.

Mechanisms of astrocyte-mediated cerebral edema.

Cerebral edema formation stems from disruption of blood brain barrier (BBB) integrity and occurs after injury to the CNS. Due to the restrictive skull, relatively small increases in brain volume can translate into impaired tissue perfusion and brain herniation. In excess, cerebral edema can be gravely harmful. Astrocytes are key participants in cerebral edema by virtue of their relationship with the cerebral vasculature, their unique compliment of solute and water transport proteins, and their general role in brain volume homeostasis. Following the discovery of aquaporins, passive conduits of water flow, aquaporin 4 (AQP4) was identified as the predominant astrocyte water channel. Normally, AQP4 is highly enriched at perivascular endfeet, the outermost layer of the BBB, whereas after injury, AQP4 expression disseminates to the entire astrocytic plasmalemma, a phenomenon termed dysregulation. Arguably, the most important role of AQP4 is to rapidly neutralize osmotic gradients generated by ionic transporters. In pathological conditions, AQP4 is believed to be intimately involved in the formation and clearance of cerebral edema. In this review, we discuss aquaporin function and localization in the BBB during health and injury, and we examine post-injury ionic events that modulate AQP4-dependent edema formation.

Molecular mechanism of lung oedema clearance by AP301: dependence of ENaC pore forming subunits (LB781)

Pulmonary oedema is a life threatening condition, caused by acute lung injury. Although failure of gas exchange in the alveolar spaces causes most of the morbidity and mortality, it remained uncertain how epithelial sodium channel lose their liquid clearance ability. Furthermore, the subunit composition of native ENaC in diseased pulmonary epithelium is still unknown. AP301, a lectin‐like domain derived peptide of TNF‐α, increases amiloride‐sensitive current in A549 cells as well as in heterologously expressed hENaC, an effect which is lost after deglycosylation. Consequently, the present study was undertaken to determine the interaction of AP301 with hENaC glycosylation sites and subunit compositions of hENaC. We substituted asparagine to glutamine in α‐hENaC, and studied the AP301 induced increase in sodium current in different subunit compositions. The effect of AP301 in HEK and CHO cells heterologously expressing human ENaC WT and mutants in addition to different subunit compositions (α, ß, γ and δ) was measured in patch clamp experiments. Indeed, AP301 weakly activated single as well as multiple asparagine mutants of α‐hENaC. For maximal AP301 activity αβγ‐ or δβγ‐hENaC co‐expression was required. Conclusively, αβγδ‐hENaC elicited a robust AP301 response at very low EC50 values. These findings suggest that specific interaction of AP301 ENaC requires precedent binding to glycosylated extracellular loop and pore‐forming α or δ delta subunits.Grant Funding Source: Supported by Austrian Research Promotion Agency (FFG).

β2-Agonists and Acute Respiratory Distress Syndrome

β₂-Agonists and Acute Respiratory Distress Syndrome

Aquaporin-4 Mitigates Retrograde Degeneration of Rubrospinal Neurons by Facilitating Edema Clearance and Glial Scar Formation After Spinal Cord Injury in Mice

Atrophy of upper motor neurons hampers axonal regeneration and functional recovery following spinal cord injury (SCI). Apart from the severity of primary injury, a series of secondary pathological damages including spinal cord edema and glial scar formation affect the fate of injured upper motor neurons. The aquaporin-4 (AQP4) water channel plays a critical role in water homeostasis and migration of astrocytes in the central nervous system, probably offering a new therapeutic target for protecting against upper motor neuron degeneration after SCI. To test this hypothesis, we examined the effect of AQP4 deficiency on atrophy of rubrospinal neurons after unilateral rubrospinal tract transection at the fourth cervical level in mice. AQP4 gene knockout (AQP4-/-) mice exhibited high extent of spinal cord edema at 72 h after lesion compared with wild-type littermates. AQP4-/- mice showed impairments in astrocyte migration toward the transected site with a greater lesion volume at 1 week after surgery and glial scar formation with a larger cyst volume at 6 weeks. More severe atrophy and loss of axotomized rubrospinal neurons as well as axonal degeneration in the rubrospinal tract rostral to the lesion were observed in AQP4-/- mice at 6 weeks after SCI. AQP4 expression was downregulated at the lesioned spinal segment at 3 days and 1 week after injury, but upregulated at 6 weeks. These results demonstrated that AQP4 not only mitigates spinal cord damage but also ameliorates retrograde degeneration of rubrospinal neurons by promoting edema clearance and glial scar formation after laceration SCI. This finding supports the notion that AQP4 may be a promising therapeutic target for SCI.

Molecular Mechanism of Lung Oedema Clearance by AP301: Dependence of ENaC Pore Forming Subunits

Sodium absorption by epithial sodium channel (ENaC) is main driving force of lung liquid clearance from the alveolar space. Despite many years of intense research, the subunit composition of native ENaC in pulmonary epithelium is unknown. Different subunits (α, s , γ and δ) containing ENaC have been identified. It is not clear that α subunit containing ENaC channels are responsible for ENaC-mediated Na+ currents and subsequent fluid flux. AP301, a cyclic peptide, comprising the human tumour necrosis factor lectin like domain (TIP domain) sequence, has been shown to reduce extravascular lung water and improve lung function in a mouse, rat and pig model. Consequently, the present study was undertaken to determine: First, whether ENaC is the specific target of AP301. Second, what is the subunit combination of ENaC for maximum AP301-induced activation. Furthermore, this study explores the potential of AP301 to discriminate between different ENaC channel populations. The effect of AP301 in A549 cells and in HEK and CHO cells heterologously expressing human ENaC subunits (α, s, γ and δ) was measured in patch clamp experiments. For maximal AP301 activity αβγ- or δβγ-ENaC co-expression was required. Conclusively, AP301 increased current in proteolytically activated (cleaved) but not near silent (uncleaved) ENaC in a reversible manner. After deglycosylation of extracellular domains of ENaC, no increase in current was observed. Thus, our data suggest that specific interaction of AP301 with both endogenously and heterologously expressed ENaC requires precedent binding to glycosylated extracellular loop(s) and pore-forming α or δ delta subunits.