Increase In Protein Permeability Research Articles

Hypothyroidism due to nephrotic syndrome: a notable clinical entity.

Nephrotic syndrome (NS) is characterized by massive urinary protein leakage and associated hypoproteinemia due to increased protein permeability caused by impaired renal glomerular connections. Although there have been several sporadic reports regarding the relationship between NS and thyroid dysfunction, a consensus has yet to be reached. The mechanism of hypothyroidism in NS is attributed to the loss of protein-bound thyroid hormones, such as thyroxine-binding globulin, transthyretin, and albumin, into the urine. Herein, we report four adults with hypothyroidism that developed or worsened due to the onset of NS. The patients' underlying thyroid status was post-total thyroidectomy with supplemental levothyroxine (L-T4) in two patients, hypothyroidism with supplemental L-T4 due to Hashimoto's disease in one patient, and Hashimoto's disease with normal thyroid function in one patient. Our results suggest that the presence of a reduced thyroid reserve may predispose patients to hypothyroidism in NS. We conclude that NS may cause or exacerbate hypothyroidism. In such cases, an NS assessment, including a urine test, is required.

IS A HIGH HEMATOCRIT USEFUL TO REVEAL AN EARLY REEXPANSION PULMONARY EDEMA?

Introduction The Reexpansion Pulmonary Edema (RPE) is a rare condition, lethal in 20%, resulting after a fast drainage of pleural effusion, pneumothorax or a huge mediastinal mass. We report a case of RPE after treatment for pleural effusion. Beside typical symptoms as hyponatremia, hyperkalaemia, hyperglycaemia, an interesting finding was a high haematocrit value (>65%), never described. We want to stress the importance of early recognition of signs and symptoms: inadequate or delayed treatment may lead to a fatal outcome. Methods After two months from a cardiac revascularization, a 68-year-old male patient was re-admitted for respiratory failure (SpO2 Results The pulmonary edema diminished gradually and the patient was extubated after one day: the haematocrit was 42%. The radiography showed a reduction of edema (Fig.1C). At third day, the patient was discharged to the ward. Discussion Recently Meeker1 defined RPE “an uncommon complication of a common clinical scenario” with an unclear pathophysiology. Age, clinical status, rapid thoracentesis or chest drainage are important risk factors for this rare iatrogenic complication. Literature shows a removing volume of 1200-1800 ml as a safe cut-off. This process, resulting in local and systemic factors, starts with an acute lung reexpansion and consequently insurgence of pulmonary edema, related to the hydrostatic forces on pulmonary capillaries: the return of high perfusion pressure after rapid thoracentesis or chest drainage is associated with negative pressure and vessel hypoxic constriction. This reperfusion increases free oxygen radicals and anoxic stress resulted in the production of cytokines by the endothelium: selectin, TNF-a and IL-1b, generating increased protein permeability. We assume that the drop in osmotic pressure causes the leakage of liquids into the extracellular space leading to a rapid increase in the hematocrit value associated to hyponatremia: these early alerts can help the staff to avoid a rapidly worsening of respiratory function and to start a correct treatment.

A new experimental model of acid- and endotoxin-induced acute lung injury in rats.

The majority of the animal models of acute lung injury (ALI) are focused on the acute phase. This limits the studies of the mechanisms involved in later phases and the effects of long-term treatments. Thus the goal of this study was to develop an experimental ALI model of aspiration pneumonia, in which diffuse alveolar damage continues for 72 h. Rats were intratracheally instilled with one dose of HCl (0.1 mol/l) followed by another instillation of one dose of LPS (0, 10, 20, 30, or 40 μg/g body weight) 2 h later, which models aspiration of gastric contents that progresses to secondary lung injury from bacteria or bacterial products. The rats were euthanized at 24, 48, and 72 h after the last instillation. The results showed that HCl and LPS at all doses caused activation of inflammatory responses, increased protein permeability and apoptosis, and induced mild hypoxemia in rat lungs at 24 h postinstillation. However, this lung damage was present at 72 h only in rats receiving HCl and LPS at the doses of 30 and 40 μg/g body wt. Mortality (∼50%) occurred in the first 48 h and only in the rats treated with HCl and LPS at the highest dose (40 μg/g body wt). In conclusion, intratracheal instillation of HCl followed by LPS at the dose of 30 μg/g body wt results in severe diffuse alveolar damage that continues at least 72 h. This rat model of aspiration pneumonia-induced ALI will be useful for testing long-term effects of new therapeutic strategies in ALI.

Genetic deficit of KCa 3.1 channels protects against pulmonary circulatory collapse induced by TRPV4 channel activation.

The intermediate conductance calcium/calmodulin-regulated K+ channel KCa 3.1 produces hyperpolarizing K+ currents that counteract depolarizing currents carried by transient receptor potential (TRP) channels, and provide the electrochemical driving force for Cl- and fluid movements. We investigated whether a deficiency in KCa 3.1 (KCa 3.1-/- ) protects against fatal pulmonary circulatory collapse in mice after pharmacological activation of the calcium-permeable TRP subfamily vanilloid type 4 (TRPV4) channels. An opener of TRPV4 channels, GSK1016790A, was infused in wild-type (wt) and KCa 3.1-/- mice; haemodynamic parameters, histology and pulmonary vascular reactivity were measured; and patch clamp was performed on pulmonary arterial endothelial cells (PAEC). In wt mice, GSK1016790A decreased right ventricular and systemic pressure leading to a fatal circulatory collapse that was accompanied by increased protein permeability, lung haemorrhage and fluid extravasation. In contrast, KCa 3.1-/- mice exhibited a significantly smaller drop in pressure to GSK1016790A infusion, no haemorrhage and fluid water extravasation, and the mice survived. Moreover, the GSK1016790A-induced relaxation of pulmonary arteries of KCa 3.1-/- mice was significantly less than that of wt mice. GSK1016790A induced TRPV4 currents in PAEC from wt and KCa 3.1-/- mice, which co-activated KCa 3.1 and disrupted membrane resistance in wt PAEC, but not in KCa 3.1-/- PAEC. Our findings show that a genetic deficiency of KCa 3.1 channels prevented fatal pulmonary circulatory collapse and reduced lung damage caused by pharmacological activation of calcium-permeable TRPV4 channels. Therefore, inhibition of KCa 3.1channels may have therapeutic potential in conditions characterized by abnormal high endothelial calcium signalling, barrier disruption, lung oedema and pulmonary circulatory collapse.

Latent Class Models Identify Two Subphenotypes in Respiratory Distress Syndrome with Differential Response to Positive End-Expiratory Pressure

Latent Class Models Identify Two Subphenotypes in Respiratory Distress Syndrome with Differential Response to Positive End-Expiratory Pressure

How to measure alterations in alveolar barrier function as a marker of lung injury.

The alveolar capillary membrane maintains the proper water and solute content of the epithelial lining fluid at the alveolar air-liquid interface, which is critical for adequate gas exchange in the lung. This is possible due to the alveolar fluid clearance (AFC) capacity of this membrane that assists in the removal of salt and water from the alveolar air spaces. The alveolar capillary membrane also provides a barrier that restricts the passage of proteins and water from the interstitial and vascular compartments into the alveolar air spaces. This restricted passage is due to the presence of tight junctions between adjacent alveolar epithelial cells. Severe injury to the alveolar epithelial/endothelial membrane results in increased protein permeability and impairment of AFC, which leads to the formation of protein-rich edema with the consequent deterioration of gas exchange. Many animal models of lung injury, focused on damage of the alveolar-capillary membrane, assess the AFC capacity and the barrier function. We describe a simple method to assess the AFC rate in normal and pathological conditions in mice. We also describe two complementary methods to assess the alveolar-capillary barrier function, which require measuring the concentration of endogenous plasma proteins in bronchoalveolar lavage fluid and detection of tight-junction proteins in lung tissue by immunofluorescence.

Thrombomodulin protects against lung damage created by high level of oxygen with large tidal volume mechanical ventilation in rats.

BackgroundVentilator-induced lung injury (VILI) is associated with inflammatory responses in the lung. Thrombomodulin (TM), a component of the coagulation system, has anticoagulant and anti-inflammatory effects. We hypothesized that the administration of recombinant human soluble TM (rhsTM) would block the development of lung injury.MethodsLung injury was induced by high tidal volume ventilation for 2 h with 100% oxygen in rats. Rats were ventilated with a tidal volume of 35 ml/kg with pretreatment via a subcutaneous injection of 3 mg/kg rhsTM (HV (high tidal volume)/TM) or saline (HV/SAL) 12 h before mechanical ventilation. Rats ventilated with a tidal volume of 6 ml/kg under 100% oxygen with rhsTM (LV (low tidal volume)/TM) or saline (LV/SAL) were used as controls. Lung protein permeability was determined by Evans blue dye (EBD) extravasation.ResultsLung injury was successfully induced in the HV/SAL group compared with the LV/SAL group, as shown by the significant decrease in arterial oxygen pressure (PaO2), increased protein permeability, and increase in mean pulmonary artery pressure (mPAP) and ratio of mean pulmonary artery pressure to mean artery pressure (Pp/Ps). Treatment of rats with lung injury with rhsTM (HV/TM) significantly attenuated the decrease in PaO2 and the increase in both mPAP and Pp/Ps, which was associated with a decrease in the lung protein permeability. Lung tissue mRNA expressions of interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor-α, and macrophage inflammatory protein (MIP)-2 were significantly higher in HV than in LV rats. Rats with VILI treated with rhsTM (HV/TM) had significantly lower mRNA expressions of IL-1α, IL-1β, IL-6, and MIP-2 than those expressions in HV/SAL rats.ConclusionsAdministration of rhsTM may prevent the development of lung injury created by high level of oxygen with large tidal volume mechanical ventilation, which has concomitant decrease in proinflammatory cytokine and chemokine expression in the lung.

Tobacco smoke induces epithelial barrier dysfunction via receptor EphA2 signaling

Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors are the largest family of receptor tyrosine kinases (RTKs) that mediate various cellular and developmental processes. The degrees of expression of these key molecules control the cell-cell interactions. Although the role of Eph receptors and their ligand Ephrins is well studied in developmental processes, their function in tobacco smoke (TS)-induced epithelial barrier dysfunction is unknown. We hypothesized that TS may induce permeability in bronchial airway epithelial cell (BAEpC) monolayer by modulating receptor EphA2 expression, actin cytoskeleton, adherens junction, and focal adhesion proteins. Here we report that in BAEpCs, acute TS exposure significantly upregulated EphA2 and EphrinA1 expression, disrupted the actin filaments, decreased E-cadherin expression, and increased protein permeability, whereas the focal adhesion protein paxillin was unaffected. Silencing the receptor EphA2 expression with silencing interference RNA (siRNA) significantly attenuated TS-induced hyperpermeability in BAEpCs. In addition, when BAEpC monolayer was transfected with EphA2-expressing plasmid and treated with recombinant EphrinA1, the transepithelial electrical resistance decreased significantly. Furthermore, TS downregulated E-cadherin expression and induced hyperpermeability across BAEpC monolayer in a Erk1/Erk2, p38, and JNK MAPK-dependent manner. TS induced hyperpermeability in BAEpC monolayer by targeting cell-cell adhesions, and interestingly cell-matrix adhesions were unaffected. The present data suggest that TS causes significant damage to the BAEpCs via induction of EphA2 and downregulation of E-cadherin. Induction of EphA2 in the BAEpCs exposed to TS may be an important signaling event in the pathogenesis of TS-induced epithelial injury.

Conditioned media from mesenchymal stromal cells restore sodium transport and preserve epithelial permeability in an in vitro model of acute alveolar injury

Mesenchymal stromal cells (MSCs) or their media (MSC-M) were reported to reverse acute lung injury (ALI)-induced decrease of alveolar fluid clearance. To determine the mechanisms by which MSC-M exert their beneficial effects, an in vitro model of alveolar epithelial injury was created by exposing primary rat alveolar epithelial cells (AECs) to hypoxia (3% O2) plus cytomix, a combination of IL-1β, TNF-α, and IFN-γ. MSC-M were collected from human MSCs exposed for 12 h to either normoxia (MSC-M) or to hypoxia plus cytomix (HCYT-MSC-M). This latter condition was used to model the effect of alveolar inflammation and hypoxia on paracrine secretion of MSCs in the injured lung. Comparison of paracrine soluble factors in MSC media showed that the IL-1 receptor antagonist and prostaglandin E2 were markedly increased while keratinocyte growth factor (KGF) was twofold lower in HCYT-MSC-M compared with MSC-M. In AECs, hypoxia plus cytomix increased protein permeability, reduced amiloride-sensitive short-circuit current (AS-Isc), and also decreased the number of α-epithelial sodium channel (α-ENaC) subunits in the apical membrane. To test the effects of MSC media, MSC-M and HCYT-MSC-M were added for an additional 12 h to AECs exposed to hypoxia plus cytomix. MSC-M and HCYT-MSC-M completely restored epithelial permeability to normal. MSC-M, but not HCYT-MSC-M, significantly prevented the hypoxia plus cytomix-induced decrease of ENaC activity and restored apical α-ENaC channels. Interestingly, KGF-deprived MSC-M were unable to restore amiloride-sensitive sodium transport, indicating a possible role for KGF in the beneficial effect of MSC-M. These results indicate that MSC-M may be a preferable therapeutic option for ALI.

Cytoprotective-Selective Activated Protein C Attenuates Pseudomonas aeruginosa–Induced Lung Injury in Mice

Inhibition of the small GTPase RhoA attenuates the development of pulmonary edema and restores positive alveolar fluid clearance in a murine model of Pseudomonas aeruginosa pneumonia. Activated protein C (aPC) blocks the development of an unfavorably low ratio of small GTPase Rac1/RhoA activity in lung endothelium through endothelial protein C receptor (EPCR)/protease-activated receptor-1 (PAR-1)-dependent signaling mechanisms that include transactivating the sphingosine-1-phosphate (S1P) pathway. However, whether aPC's cytoprotective effects can attenuate the development of pulmonary edema and death associated with P. aeruginosa pneumonia in mice remains unknown. Thus, we determined whether the normalization of a depressed ratio of activated Rac1/RhoA by aPC would attenuate the P. aeruginosa-mediated increase in protein permeability across lung endothelial and alveolar epithelial barriers. Pretreatment with aPC significantly reduced P. aeruginosa-induced increases in paracellular permeability across pulmonary endothelial cell and alveolar epithelial monolayers via an inhibition of RhoA activation and a promotion of Rac1 activation that required the EPCR-PAR-1 and S1P pathways. Furthermore, pretreatment with aPC attenuated the development of pulmonary edema in a murine model of P. aeruginosa pneumonia. Finally, a cytoprotective-selective aPC mutant, aPC-5A, which lacks most of aPC's anticoagulant activity, reproduced the protective effect of wild-type aPC by attenuating the development of pulmonary edema and decreasing mortality in a murine model of P. aeruginosa pneumonia. Taken together, these results demonstrate a critical role for the cytoprotective activities of aPC in attenuating P. aeruginosa-induced lung vascular permeability and mortality, suggesting that cytoprotective-selective aPC-5A with diminished bleeding risks could attenuate the lung damage caused by P. aeruginosa in critically ill patients.

Critical Role of the Small GTPase RhoA in the Development of Pulmonary Edema Induced by Pseudomonas aeruginosa in Mice

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe pneumonia in critically ill patients. We have reported previously that P. aeruginosa exotoxins S and T mediate in vitro the increase in protein permeability across lung endothelial cell monolayers via a RhoA-dependent mechanism. However, whether inhibition of RhoA would significantly attenuate P. aeruginosa-mediated lung injury in mice is unknown. P. aeruginosa-induced paracellular protein permeability was measured across bovine lung endothelial and rat alveolar epithelial type II cell monolayers with I-albumin. Some cell monolayers were pretreated with RhoA inhibitor CGX0287 1 h before P. aeruginosa exposure. At 4 h after exposure, lung endothelial and epithelial permeability, bacterial counts, bronchoalveolar lavage fluid levels of keratinocyte-derived chemokine, myeloperoxidase activity, and alveolar fluid clearance were measured. Some mice were treated intraperitoneally with CGX0287 1 h before or after airspace instillation of P. aeruginosa. RhoA inhibition attenuated in vitro P. aeruginosa-mediated increase in lung endothelial and epithelial permeability to protein and in vivo the development of pulmonary edema and inhibition of alveolar fluid clearance associated with P. aeruginosa pneumonia. Furthermore, RhoA inhibition decreased the systemic dissemination of P. aeruginosa and neutrophil activity in the lung tissue observed after airspace instillation of these bacteria. The small GTPase RhoA plays a critical role in mediating lung injury associated with P. aeruginosa pneumonia in mice. Thus, transient blockade of RhoA could attenuate lung damage caused by P. aeruginosa in critically ill patients.

Claudin‐18 May Contribute to the Increase of Protein Permeability in Cultured Human Alveolar Epithelial Type II Cells Exposed to Proinflammatory Cytokines

Our previous study demonstrated that proinflammatory cytokines increase protein permeability across human alveolar type II (AT II) cell monolayers (J Biol Chem 2007). However, the mechanisms are not fully understood. The family of claudin transmembrane proteins had been implicated in the regulation of epithelial paracellular permeability. In this study we determined the gene profile of cultured human AT II cells exposed to cytomix (TNF‐α, IL‐1β and IFN‐γ) to identify candidate genes that may contribute to the increase in protein permeability. Human AT II cells were isolated and cultured on collagen‐coated wells. After 48 hr, the cells were exposed to serum‐free medium (control) or cytomix for 72hr. cDNA was prepared and hybridized to Affymetrix U133A microarray chips. Western blot was used to test for protein expression of candidate genes. mRNA was detected at a significant level for 50% of the probes, representing approximately 7200 genes. Although mRNAs for claudin 1, 3, 4, 6, 7, 8, 9, 10, 15, 18 were detected, only claudin 18 mRNA was significantly decreased (95%) by cytomix exposure. Western blot confirmed that protein expression of claudin‐18 was significantly reduced by cytomix. The reduction of claudin 18 expression was restored by the NF‐kB inhibitor, BMS‐345541. We conclude that claudin 18 is regulated by cytokines and may play a role in protein permeability change across human ATII cell monolayers.

Role of Small GTPases and αvβ5 Integrin in Pseudomonas aeruginosa–Induced Increase in Lung Endothelial Permeability

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe pneumonia associated with airspace flooding with protein-rich edema in critically ill patients. The type III secretion system is a major virulence factor and contributes to dissemination of P. aeruginosa. However, it is still unknown which particular bacterial toxin and which cellular pathways are responsible for the increase in lung endothelial permeability induced by P. aeruginosa. Thus, the first objective of this study was to determine the mechanisms by which this species causes an increase in lung endothelial permeability. The results showed that ExoS and ExoT, two of the four known P. aeruginosa type III cytotoxins, were primarily responsible for bacterium-induced increases in protein permeability across the lung endothelium via an inhibition of Rac1 and an activation of the RhoA signaling pathway. In addition, inhibition of the alphavbeta5 integrin, a central regulator of lung vascular permeability, prevented these P. aeruginosa-mediated increases in albumin flux due to endothelial permeability. Finally, prior activation of the stress protein response or adenoviral gene transfer of the inducible heat shock protein Hsp72 also inhibited the damaging effects of P. aeruginosa on the barrier function of lung endothelium. Taken together, these results demonstrate the critical role of the RhoA/alphavbeta5 integrin pathway in mediating P. aeruginosa-induced lung vascular permeability. In addition, activation of the stress protein response with pharmacologic inhibitors of Hsp90 may protect lungs against P. aeruginosa-induced permeability changes.

Angiopoietin‐1 reverses protein permeability in an injury model of cultured human alveolar epithelial type II cells

OBJECTIVE: We recently reported that acute lung injury (ALI) pulmonary edema and cytomix (IL1β, TNFα and IFNγ) increase epithelial protein permeability (JBC, 2007). Angiopoietin‐1 (Ang‐1) maintains vascular integrity and reduces endothelial activation in a model of ALI (AJRCCM 2007). In this study, we hypothesized that Ang‐1 could reverse increased protein permeability in cultured human alveolar epithelial type II cells (ATII) exposed to cytomix.METHODS: Isolated human ATII cells were cultured in an air‐liquid interface and protein permeability was measured. Cells were exposed to control medium, cytomix (50 ng/ml) and cytomix with recombinant human Ang‐1 (10,000 pg/ml).RESULTS: 1) Cytomix increased protein permeability in cultured ATII cells. 2) Recombinant human Ang‐1 decreased ATII protein permeability significantly after exposure to cytomix (Figure 1).CONCLUSIONS: Ang‐1 can reverse protein permeability across ATII cells exposed to cytomix. These results are the first results to show a potential role for Ang‐1 in regulation of alveolar epithelial permeability. The data support a further role of Ang‐1 in the treatment of ALI.Supported by NIH HL51856 and HL51854

Interleukin-1β Causes Acute Lung Injury via αvβ5 and αvβ6 Integrin–Dependent Mechanisms

Interleukin (IL)-1beta has previously been shown to be among the most biologically active cytokines in the lungs of patients with acute lung injury (ALI). Furthermore, there is experimental evidence that lung vascular permeability increases after short-term exposure to IL-1 protein, although the exact mechanism is unknown. Therefore, the objective of this study was to determine the mechanisms of IL-1beta-mediated increase in lung vascular permeability and pulmonary edema following transient overexpression of this cytokine in the lungs by adenoviral gene transfer. Lung vascular permeability increased with intrapulmonary IL-1beta production with a maximal effect 7 days after instillation of the adenovirus. Furthermore, inhibition of the alphavbeta6 integrin and/or transforming growth factor-beta attenuated the IL-1beta-induced ALI. The results of in vitro studies indicated that IL-1beta caused the activation of transforming growth factor-beta via RhoA/alphavbeta6 integrin-dependent mechanisms and the inhibition of the alphavbeta6 integrin and/or transforming growth factor-beta signaling completely blocked the IL-1beta-mediated protein permeability across alveolar epithelial cell monolayers. In addition, IL-1beta increased protein permeability across lung endothelial cell monolayers via RhoA- and alphavbeta5 integrin-dependent mechanisms. The final series of in vivo experiments demonstrated that pretreatment with blocking antibodies to both the alphavbeta5 and alphavbeta6 integrins had an additive protective effect against IL-1beta-induced ALI. In summary, these results demonstrate a critical role for the alphavbeta5/beta6 integrins in mediating the IL-1beta-induced ALI and indicate that these integrins could be a potentially attractive therapeutic target in ALI.

A Role for the Endothelial Glycocalyx in Regulating Microvascular Permeability in Diabetes Mellitus

Diabetic angiopathy is a major cause of morbidity and mortality in diabetes mellitus. Endothelial dysfunction and associated alterations in blood flow, pressure and permeability are widely accepted phenomena in the diabetic milieu and are understood to lead to microangiopathy. Despite the clinical importance of diabetic microangiopathy, the mechanisms of pathogenesis remain elusive. In particular, much is yet to be understood about the nature of the putative increased permeability with respect to diabetes. Microvessel permeability is intrinsically difficult to measure and a surrogate (solute or solvent flux) is usually reported, the measurement of which is hampered by haemodynamic factors, such as flow rate, hydrostatic pressure gradient, solute concentration and surface area available for exchange. Very few studies describing the measurement of permeability with respect to diabetes have controlled for all these factors. As a result, the nature of the increased microvessel permeability in diabetes mellitus and indeed its causes are poorly understood. Recent studies have shown that hyperglycaemia can alter the glycocalyx structure, and parallel findings have shown that the apparent increase in permeability demonstrated in hyperglycaemia may be due to an increase in the permeability of the vessels to water, and not an increase in protein permeability, an effect attributable to altered glycocalyx. This review focuses on the current understanding of microvascular permeability in terms of the endothelial glycocalyx-fibre-matrix theory, those methods used to determine permeability in the context of diabetes, and the more recent developments in our understanding of elevated microvascular permeability in the diabetic circulation.

Shigatoxin-Induced Endothelin-1 Expression in Cultured Podocytes Autocrinally Mediates Actin Remodeling

Shigatoxin (Stx) is the offending agent of post-diarrheal hemolytic uremic syndrome, characterized by glomerular ischemic changes preceding microvascular thrombosis. Because podocytes are highly sensitive to Stx cytotoxicity and represent a source of vasoactive molecules, we studied whether Stx-2 modulated the production of endothelin-1 (ET-1), taken as candidate mediator of podocyte dysfunction. Stx-2 enhanced ET-1 mRNA and protein expression via activation of nuclear factor kappaB (NF-kappaB) and activator protein-1 (Ap-1) to the extent that transfection with the dominant-negative mutant of IkappaB-kinase 2 or with Ap-1 decoy oligodeoxynucleotides reduced ET-1 mRNA levels. We propose a role for p38 and p42/44 mitogen-activated protein kinases (MAPKs) in mediating NF-kappaB-dependent gene transcription induced by Stx-2, based on data that Stx-2 phosphorylated p38 and p42/44 MAPKs and that MAPK inhibitors reduced transcription of NF-kappaB promoter/luciferase reporter gene construct induced by Stx-2. Stx-2 caused F-actin redistribution and intercellular gaps via production of ET-1 acting on ETA receptor, because cytoskeleton changes were prevented by ETA receptor blockade. Exogenous ET-1 induced cytoskeleton rearrangement and intercellular gaps via phosphatidylinositol-3 kinase and Rho-kinase pathway and increased protein permeability across the podocyte monolayer. These data suggest that the podocyte is a target of Stx, a novel stimulus for the synthesis of ET-1, which may control cytoskeleton remodeling and glomerular permeability in an autocrine fashion.

Protein permeability in lung injury: now in real time again?

the formation and the resolution of pulmonary edema is determined by several mechanisms, including lung vascular pressure, lung endothelial and epithelial permeability to protein, and the capacity of the alveolar epithelium to effectively remove alveolar edema fluid. Impaired alveolar fluid

Activation of the stress protein response prevents the development of pulmonary edema by inhibiting VEGF cell signaling in a model of lung ischemia‐reperfusion injury in rats

Lung endothelial damage is a characteristic morphological feature of ischemia-reperfusion (I/R) injury, although the molecular steps involved in the loss of endothelial integrity are still poorly understood. We tested the hypothesis that the activation of vascular endothelial growth factor (VEGF) cell signaling would be responsible for the increase in lung vascular permeability seen early after the onset of I/R in rats. Furthermore, we hypothesized that the I/R-induced pulmonary edema would be significantly attenuated in rats by the activation of the stress protein response. Pretreatment with Ad Flk-1, an adenovirus encoding for the soluble VEGF receptor type II, prevented I/R-mediated increase in lung vascular permeability in rats. Furthermore, the I/R-induced lung injury was significantly decreased by prior activation of the stress protein response with geldanamycin or pyrrolidine dithiocarbamate. In vitro studies demonstrated that VEGF caused an increase in protein permeability across primary cultures of bovine macro- and microvascular lung endothelial cell monolayers that were associated with a phosphorylation of VE- and E-cadherin and the formation of actin stress fibers. Activation of the stress protein response prevented the VEGF-mediated changes in protein permeability across these cell monolayers and reduced the phosphorylation of VE-and E-cadherins, as well as the formation of actin stress fibers in these cells.

Extravascular lung water to blood volume ratios as measures of permeability in sepsis-induced ALI/ARDS

We studied the relationship, and the effect of fluid loading on this, between the ratio of extravascular lung water (EVLW) to intrathoracic/pulmonary blood volumes (ITBV, PBV) and the radionuclide pulmonary leak index (PLI) to protein during sepsis-induced acute lung injury/acute respiratory distress syndrome (ALI/ARDS). A prospective observational study, in the intensive care unit of a university hospital. Twenty-two consecutive mechanically ventilated patients with sepsis-related ALI/ARDS from pneumonia (n = 12) or extrapulmonary sources (n = 10), without elevated cardiac filling pressures. Crystalloid (1700-1800 ml) or colloid (1000-1800 ml) fluid loading until target filling pressures. Protein permeability was assessed noninvasively over the lungs with help of 67Ga-labeled transferrin and 99mTc-labeled red blood cells (Pulmonary leak index, upper limit normal 14.1 x 10(-3)/min) and EVLW and blood volumes by the thermal-dye transpulmonary dilution technique before and after fluid loading. Prior to fluids the pulmonary leak index related to the ratio of EVLW/ITBV and EVLW/PBV (r(s) = 0.46) particularly when the pulmonary leak index was below 100 x 10(-3)/min and in extrapulmonary sepsis (PLI vs. EVLW/PBV r(s) = 0.71). Fluid loading did not alter EVLW, EVLW/ITBV, or EVLW/PBV or the relationship to PLI. The data demonstrate that EVLW/ITBV or EVLW/PBV are imperfect measures of increased protein permeability in mechanically ventilated patients with sepsis-induced ALI/ARDS particularly when the PLI is severely increased and during pneumonia, independent of fluid status.