Alejandro P. Comellas

Cortisol levels and mortality in severe sepsis

objective  Serum cortisol levels rise in response to the stress of critical illness but the optimal range of serum cortisol in such settings is not clearly defined. The objectives of this study were to determine the range of serum cortisol levels in a group of medical intensive care unit patients with severe sepsis/septic shock using uniform criteria, and to correlate serum cortisol levels to mortality.

Hypoxia-Mediated Degradation of Na,K-ATPase via Mitochondrial Reactive Oxygen Species and the Ubiquitin-Conjugating System

We set out to determine whether cellular hypoxia, via mitochondrial reactive oxygen species, promotes Na,K-ATPase degradation via the ubiquitin-conjugating system. Cells exposed to 1.5% O2 had a decrease in Na,K-ATPase activity and oxygen consumption. The total cell pool of α1 Na,K-ATPase protein decreased on exposure to 1.5% O2 for 30 hours, whereas the plasma membrane Na,K-ATPase was 50% degraded after 2 hours of hypoxia, which was prevented by lysosome and proteasome inhibitors. When Chinese hamster ovary cells that exhibit a temperature-sensitive defect in E1 ubiquitin conjugation enzyme were incubated at 40°C and 1.5% O2, the degradation of the α1 Na,K-ATPase was prevented. Exogenous reactive oxygen species increased the plasma membrane Na,K-ATPase degradation, whereas, in mitochondrial DNA deficient &rgr;0 cells and in cells transfected with small interfering RNA against Rieske iron sulfur protein, the hypoxia-mediated Na,K-ATPase degradation was prevented. The catalase/superoxide dismutase (SOD) mimetic (EUK-134) and glutathione peroxidase overexpression prevented the hypoxia-mediated Na,K-ATPase degradation and overexpression of SOD1, but not SOD2, partially inhibited the Na+ pump degradation. Accordingly, we provide evidence that during hypoxia, mitochondrial reactive oxygen species are necessary to degrade the plasma membrane Na,K-ATPase via the ubiquitin-conjugating system.

β-Agonists regulate Na,K-ATPase via novel MAPK/ERK and rapamycin-sensitive pathways

We studied whether the β‐adrenergic agonist, isoproterenol (ISO), regulates Na,K‐ATPase in alveolar epithelial cells (AEC) via a mitogen‐activated protein kinase (MAPK)/extracellular signaling related kinase (ERK) dependent pathway. ISO increased ERK activity in AEC by 10 min via a β‐adrenergic receptor, protein kinase A (PKA)‐dependent mechanism. Activation of the MAPK pathway by ISO, resulted in increased Na,K‐ATPase β1 and α1 subunit protein abundance in whole cell lysates, which resulted in functional Na,K‐ATPases at the basolateral membranes. ISO did not change the α1 or β1 mRNA steady state levels, but rapamycin, the inhibitor of the mammalian target of rapamycin, also blocked the ISO‐mediated increase in Na,K‐ATPase total protein abundance, suggesting a posttranscriptional regulation. We conclude that ISO, regulates the Na,K‐ATPase in AEC via PKA, ERK and rapamycin‐sensitive mechanisms.

Tityus discrepans venom produces a respiratory distress syndrome in rabbits through an indirect mechanism

It is well known that scorpion venom induces lung lesions and respiratory distress which are usually classified as pulmonary oedema (PO). Tityus discrepans is a scorpion that lives in the north-central area of Venezuela, is the most common source of human envenomation here and produces PO. We studied the action of the venom of Tityus discrepans on whole rabbits and on their isolated lungs perfused with Krebs saline with 1 g/l of bovine serum albumin (Krebs-BSA saline). Two milligram of venom were diluted in 250 ml of solution (approximately the rabbits total blood volume) and used to perfuse isolated lungs. Lung oedema occurred in rabbits which received 1 mg/kg of scorpion venom i.p., heparin prevented the production of this lung oedema. T. discrepans venom produced PO, in rabbits pretreated with 15 mg/kg of ajoene. Yet, Tityus venom had no effects on isolated lungs perfused with citrated or heparinized blood, and in lungs perfused with Krebs-BSA with normal Ca2+. These result show that Tityus venom does not act directly on lungs. Otherwise, we have observed that abundant microthrombi occurred in all rabbit lungs exposed to venom in vivo, suggesting that these clotting alterations are fundamental to produce PO. The presence of intravascular microthrombi is not characteristic of the usual PO hinting that scorpion venom induced pulmonary alterations are a different clinical entity. We thus propose that the use of the term pulmonary oedema in scorpionism should abandoned in favor of scorpion venom respiratory distress syndrome.

TSH Regulates Pendrin Membrane Abundance and Enhances Iodide Efflux in Thyroid Cells

Thyroid hormones are essential for normal development and metabolism. Their synthesis requires transport of iodide into thyroid follicles. The mechanisms involving the apical efflux of iodide into the follicular lumen are poorly elucidated. The discovery of mutations in the SLC26A4 gene in patients with Pendred syndrome (congenital deafness, goiter, and defective iodide organification) suggested a possible role for the encoded protein, pendrin, as an apical iodide transporter. We determined whether TSH regulates pendrin abundance at the plasma membrane and whether this influences iodide efflux. Results of immunoblot and immunofluorescence experiments reveal that TSH and forskolin rapidly increase pendrin abundance at the plasma membrane through the protein kinase A pathway in PCCL-3 rat thyroid cells. The increase in pendrin membrane abundance correlates with a decrease in intracellular iodide as determined by measuring intracellular (125)iodide and can be inhibited by specific blocking of pendrin. Elimination of the putative protein kinase A phosphorylation site T717A results in a diminished translocation to the membrane in response to forskolin. These results demonstrate that pendrin translocates to the membrane in response to TSH and suggest that it may have a physiological role in apical iodide transport and thyroid hormone synthesis.

Hypoxia increases transepithelial electrical conductance and reduces occludin at the plasma membrane in alveolar epithelial cells via PKC-ζ and PP2A pathway

During pulmonary edema, the alveolar space is exposed to a hypoxic environment. The integrity of the alveolar epithelial barrier is required for the reabsorption of alveolar fluid. Tight junctions (TJ) maintain the integrity of this barrier. We set out to determine whether hypoxia creates a dysfunctional alveolar epithelial barrier, evidenced by an increase in transepithelial electrical conductance (G(t)), due to a decrease in the abundance of TJ proteins at the plasma membrane. Alveolar epithelial cells (AEC) exposed to mild hypoxia (Po(2) = 50 mmHg) for 30 and 60 min decreased occludin abundance at the plasma membrane and significantly increased G(t). Other cell adhesion molecules such as E-cadherin and claudins were not affected by hypoxia. AEC exposed to hypoxia increased superoxide, but not hydrogen peroxide (H(2)O(2)). Overexpression of superoxide dismutase 1 (SOD1) but not SOD2 prevented the hypoxia-induced G(t) increase and occludin reduction in AEC. Also, overexpression of catalase had a similar effect as SOD1, despite not detecting any increase in H(2)O(2) during hypoxia. Blocking PKC-ζ and protein phosphatase 2A (PP2A) prevented the hypoxia-induced occludin reduction at the plasma membrane and increase in G(t). In summary, we show that superoxide, PKC-ζ, and PP2A are involved in the hypoxia-induced increase in G(t) and occludin reduction at the plasma membrane in AEC.

Role of endothelin-1 in acute lung injury

The alveolar-capillary membrane serves as a barrier that prevents the accumulation of fluid in the alveolar space and restricts the diffusion of large solutes while facilitating an efficient gas exchange. When this barrier becomes dysfunctional, patients develop acute lung injury (ALI), which is characterized by pulmonary edema and increased lung inflammation that leads to a life-threatening impairment of gas exchange. In addition to the increase of inflammatory cytokines, plasma levels of endothelin-1 (ET-1), which is a primarily endothelium-derived vasoconstrictor, are increased in patients with ALI. As patients recover, ET-1 levels decrease, which suggests that ET-1 may not only be a marker of endothelial dysfunction but may have a role in the pathogenesis of ALI. While pulmonary edema accumulates, alveolar fluid clearance (AFC) is of critical importance, as failure to return to normal clearance is associated with poor prognosis in patients with pulmonary edema. AFC involves active transport mechanisms where sodium (Na(+)) is actively transported from the alveolar airspaces, across the alveolar epithelium, and into the pulmonary circulation, which creates an osmotic gradient that is responsible for the clearance of lung edema. In this article, we review the relevance of ET-1 in the development of ALI, not only as a vasoconstrictor molecule but also by inhibiting AFC via the activation of endothelial ET-B receptors and generation. Furthermore, this review highlights the therapeutic role of drugs such as beta-adrenergic agonists and, in particular, of endothelin receptor antagonists in patients with ALI.

Frequency of exacerbations in patients with chronic obstructive pulmonary disease: an analysis of the SPIROMICS cohort

BACKGROUND Present treatment strategies to stratify exacerbation risk in patients with chronic obstructive pulmonary disease (COPD) rely on a history of two or more events in the previous year. We aimed to understand year to year variability in exacerbations and factors associated with consistent exacerbations over time. METHODS In this longitudinal, prospective analysis of exacerbations in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) cohort, we analysed patients aged 40-80 years with COPD for whom 3 years of prospective data were available, identified through various means including care at academic and non-academic medical centres, word of mouth, and existing patient registries. Participants were enrolled in the study between Nov 12, 2010, and July 31, 2015. We classified patients according to yearly exacerbation frequency: no exacerbations in any year; one exacerbation in every year during 3 years of follow-up; and those with inconsistent exacerbations (individuals who had both years with exacerbations and years without during the 3 years of follow-up). Participants were characterised by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) spirometric category (1-4) on the basis of post-bronchodilator FEV1. Stepwise logistic regression was used to compare factors associated with one or more acute exacerbations of COPD every year for 3 years versus no exacerbations in the same timeframe. Additionally, a stepwise zero-inflated negative binomial model was used to assess predictors of exacerbation count during follow-up in all patients with available data. Baseline symptom burden was assessed with the COPD assessment test. This trial is registered with ClinicalTrials.gov, number NCT01969344. FINDINGS 2981 patients were enrolled during the study. 1843 patients had COPD, of which 1105 patients had 3 years of complete, prospective follow-up data. 538 (49%) of 1105 patients had at least one acute exacerbation during the 3 years of follow-up, whereas 567 (51%) had none. 82 (7%) of 1105 patients had at least one acute exacerbation each year, whereas only 23 (2%) had two or more acute exacerbations in each year. An inconsistent pattern (both years with and without acute exacerbations) was common (456 [41%] of the group), particularly among GOLD stages 3 and 4 patients (256 [56%] of 456). In logistic regression, consistent acute exacerbations (≥1 event per year for 3 years) were associated with higher baseline symptom burden, previous exacerbations, greater evidence of small airway abnormality on CT, lower interleukin-15 concentrations, and higher interleukin-8 concentrations, than were no acute exacerbations. INTERPRETATION Although acute exacerbations are common, the exacerbation status of most individuals varies markedly from year to year. Among patients who had any acute exacerbation over 3 years, very few repeatedly had two or more events per year. In addition to symptoms and history of exacerbations in the year before study enrolment, we identified several novel biomarkers associated with consistent exacerbations, including CT-defined small airway abnormality, and interleukin-15 and interleukin-8 concentrations. FUNDING National Institutes of Health, and National Heart, Lung, and Blood Institute.Background Current treatment strategies to stratify exacerbation risk rely on history of ≥2 events in the previous year. To understand year-to-year variability and factors associated with consistent exacerbations over time, we present a prospective analysis of the SPIROMICS cohort. Methods We analyzed SPIROMICS participants with COPD and three years of prospective data (n=1,105). We classified participants according to yearly exacerbation frequency. Stepwise logistic regression compared factors associated with individuals experiencing ≥1 AECOPD in every year for three years versus none. Results During three years follow-up, 48·7% of participants experienced at least one AECOPD, while the majority (51·3%) experienced none. Only 2·1% had ≥2 AECOPD in each year. An inconsistent pattern (both years with and years without AECOPD) was common (41·3% of the group), particularly among GOLD stages 3 and 4 subjects (56·1%). In logistic regression, consistent AECOPD (≥1 event per year for three years) as compared to no AECOPD were associated with higher baseline symptom burden assessed with the COPD Assessment Test, previous exacerbations, greater evidence of small airway abnormality by computed tomography, lower Interleukin-15 (IL-15) and elevated Interleukin-8 (IL-8). Conclusions Although AECOPD are common, the exacerbation status of most individuals varies markedly from year to year. Among participants who experienced any AECOPD over three years, very few repeatedly experienced ≥2 events/year. In addition to symptoms and history of exacerbations in the prior year, we identified several novel biomarkers associated with consistent exacerbations, including CT-defined small airway abnormality, IL-15 and IL-8.

Insulin regulates alveolar epithelial function by inducing Na+/K+-ATPase translocation to the plasma membrane in a process mediated by the action of Akt

Stimulation of Na+/K+-ATPase translocation to the cell surface increases active Na+ transport, which is the driving force of alveolar fluid reabsorption, a process necessary to keep the lungs free of edema and to allow normal gas exchange. Here, we provide evidence that insulin increases alveolar fluid reabsorption and Na+/K+-ATPase activity by increasing its translocation to the plasma membrane in alveolar epithelial cells. Insulin-induced Akt activation is necessary and sufficient to promote Na+/K+-ATPase translocation to the plasma membrane. Phosphorylation of AS160 by Akt is also required in this process, whereas inactivation of the Rab GTPase-activating protein domain of AS160 promotes partial Na+/K+-ATPase translocation in the absence of insulin. We found that Rab10 functions as a downstream target of AS160 in insulin-induced Na+/K+-ATPase translocation. Collectively, these results suggest that Akt plays a major role in Na+/K+-ATPase intracellular translocation and thus in alveolar fluid reabsorption.

Endothelin-1 Impairs Alveolar Epithelial Function via Endothelial ETB Receptor

RATIONALE Endothelin-1 (ET-1) is increased in patients with high-altitude pulmonary edema and acute respiratory distress syndrome, and these patients have decreased alveolar fluid reabsorption (AFR). OBJECTIVES To determine whether ET-1 impairs AFR via activation of endothelial cells and nitric oxide (NO) generation. METHODS Isolated perfused rat lung, transgenic rats deficient in ETB receptors, coincubation of lung human microvascular endothelial cells (HMVEC-L) with rat alveolar epithelial type II cells or A549 cells, ouabain-sensitive 86Rb+ uptake. MEASUREMENTS AND MAIN RESULTS The ET-1-induced decrease in AFR was prevented by blocking the endothelin receptor ETB, but not ETA. Endothelial-epithelial cell interaction is required, as direct exposure of alveolar epithelial cells (AECs) to ET-1 did not affect Na,K-ATPase function or protein abundance at the plasma membrane, whereas coincubation of HMVEC-L and AECs with ET-1 decreased Na,K-ATPase activity and protein abundance at the plasma membrane. Exposing transgenic rats deficient in ETB receptors in the pulmonary vasculature (ET-B(-/-)) to ET-1 did not decrease AFR or Na,K-ATPase protein abundance at the plasma membrane of AECs. Exposing HMVEC-L to ET-1 led to increased NO, and the ET-1-induced down-regulation of Na,K-ATPase was prevented by the NO synthase inhibitor l-NAME, but not by a guanylate cyclase inhibitor. CONCLUSIONS We provide the first evidence that ET-1, via an endothelial-epithelial interaction, leads to decreased AFR by a mechanism involving activation of endothelial ETB receptors and NO generation leading to alveolar epithelial Na,K-ATPase down-regulation in a cGMP-independent manner.