The Na/K-ATPase role as a signal transducer in lung inflammation.

Acute respiratory distress syndrome (ARDS) is marked by damage to the capillary endothelium and alveolar epithelium following edema formation and cell infiltration. Currently, there are no effective treatments for severe ARDS. Pathologies such as sepsis, pneumonia, fat embolism, and severe trauma may cause ARDS with respiratory failure. The primary mechanism of edema clearance is the epithelial cells' Na/K-ATPase (NKA) activity. NKA is an enzyme that maintains the electrochemical gradient and cell homeostasis by transporting Na+ and K+ ions across the cell membrane. Direct injury on alveolar cells or changes in ion transport caused by infections decreases the NKA activity, loosening tight junctions in epithelial cells and causing edema formation. In addition, NKA acts as a receptor triggering signal transduction in response to the binding of cardiac glycosides. The ouabain (a cardiac glycoside) and oleic acid induce lung injury by targeting NKA. Besides enzymatic inhibition, the NKA triggers intracellular signal transduction, fostering proinflammatory cytokines production and contributing to lung injury. Herein, we reviewed and discussed the crucial role of NKA in edema clearance, lung injury, and intracellular signaling pathway activation leading to lung inflammation, thus putting the NKA as a protagonist in lung injury pathology.

Acute kidney injury and renal replacement therapy: terminology standardization / Injúria renal aguda e métodos de suporte: padronização da nomenclatura

Abstract The Department of Acute Kidney Injury (IRA) of the Brazilian Society of Nephrology prepared this document for the purpose of standardizing AKI terminology and dialysis modalities in the Portuguese language for Brazil. Several terms with similar meanings have been used in AKI and its dialysis modalities, causing confusion and disparities among patients, nephrologists, health institutions, private care companies, insurance companies and government entities. These disparities can impact medical care, hospital organization and care, as well as the funding and reimbursement of AKI-related procedures. Thus, consensual nomenclature and definitions were developed, including the definitions of AKI, acute kidney disease (AKD) and chronic kidney disease (CKD). Additionally, we addressed all dialysis modalities and extracorporeal procedures related to AKI, currently approved and available in the country. The Brazilian Society of Nephrology hopes that this Consensus can standardize the terminology and provide technical support to all involved in AKI care in Brazil.

Resumo O Departamento de Injúria Renal Aguda (IRA) da Sociedade Brasileira de Nefrologia elaborou o presente documento para fins de padronização da terminologia em IRA e modalidades dialíticas na língua portuguesa para o Brasil. Diversos termos com significados semelhantes têm sido empregados em IRA e suas modalidades dialíticas, causando confusão e disparidades entre pacientes, nefrologistas, instituições de saúde, empresas privadas de assistência, seguradoras e entidades governamentais. Essas disparidades podem impactar a assistência médica, a organização e o atendimento hospitalares, assim como o financiamento e reembolso dos procedimentos relacionados com a IRA. Assim, nomenclatura e definições consensuais foram elaboradas, incluindo-se as definições de IRA, doença renal aguda (DRA) e doença renal crônica (DRC). Adicionalmente, todas as modalidades dialíticas e os procedimentos extracorpóreos relacionados a IRA, atualmente aprovados e disponíveis no país, foram abordados. A Sociedade Brasileira de Nefrologia espera que este Consenso possa padronizar a nomenclatura e prover suporte técnico para todos os atores envolvidos na assistência à IRA no Brasil.

Na/K-ATPase assay in the intact mice lung subjected to perfusion.

BACKGROUND: Among the characteristics of acute respiratory distress syndrome (ARDS) is edema formation and its resolution depends on pneumocyte Na/K-ATPase activity. Increased concentration of oleic acid (OA) in plasma induces lung injury by targeting Na/K-ATPase and, thus, interfering in sodium transport. FINDINGS: Presently, we adapted a radioactivity-free assay to detect Na/K-ATPase activity in perfused lung mice, comparing the inhibitory effect of ouabain and OA. We managed to perfuse only the lung, avoiding the systemic loss of rubidium. Rb+ incorporation into lung was measured by inductively coupled plasma optical emission spectrometry (ICP OES) technique, after lung tissue digestion. Na/K-ATPase activity was the difference between Rb+ incorporation with or without ouabain. Lung Na/K-ATPase was completely inhibited by perfusion with ouabain. However, OA caused a partial inhibition. CONCLUSIONS: In the present work the amount of incorporated Rb+ was greater than seen in our previous report, showing that the present technique is trustworthy. This new proposed assay may allow researchers to study the importance of Na/K-ATPase activity in lung pathophysiology.

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

BACKGROUND: Leptospiral 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. METHODS: We 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. RESULTS: Leptospiral 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. CONCLUSIONS: GLP 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.

Oleic acid induces lung injury in mice through activation of the ERK pathway.

Oleic acid (OA) can induce acute lung injury in experimental models. In the present work, we used intratracheal OA injection to show augmented oedema formation, cell migration and activation, lipid mediator, and cytokine productions in the bronchoalveolar fluids of Swiss Webster mice. We also demonstrated that OA-induced pulmonary injury is dependent on ERK1/2 activation, since U0126, an inhibitor of ERK1/2 phosphorylation, blocked neutrophil migration, oedema, and lipid body formation as well as IL-6, but not IL-1ß production. Using a mice strain carrying a null mutation for the TLR4 receptor, we proved that increased inflammatory parameters after OA challenges were not due to the activation of the TLR4 receptor. With OA being a Na/K-ATPase inhibitor, we suggest the possible involvement of this enzyme as an OA target triggering lung inflammation.

Na/K-ATPase assay in the intact guinea pig liver submitted to in situ perfusion.

We describe an assay for the enzyme Na/K-ATPase in intact guinea pig livers perfused through the portal vein with modified Hank's solution. The model uses the measurement of non-radioactive rubidium ion incorporation by liver cells, both in the absence and in the presence of the specific Na/K-ATPase inhibitor ouabain, followed by a rinsing procedure with cold saline. The concentration of Rb+ in acid-digested liver lobes was measured by atomic emission spectrometry and Na/K pump activity was calculated by the difference between the incorporation of Rb+ in the absence and in the presence of ouabain. The optimal conditions for Rb+ incorporation were: perfusion flow rate, 3 ml/min per liver; perfusion time at 37 degrees C, 60 min; rinsing time with cold saline, 5-10 min; and concentration of ouabain, 3 mM. The calculated ouabain IC(50) was 100 microM. The major advantage of this model is the possibility of testing experimental drugs affecting this enzyme in conditions close to those in the intact organ.

Role of nonesterified unsaturated fatty acids in the pathophysiological processes of leptospiral infection.

Organ malfunctions in patients with leptospirosis have been associated with the bacterial glycolipoprotein endotoxin and with its nonesterified unsaturated fatty acid (NEUFA) components. We examined the involvement of NEUFAs in the pathophysiological processes of leptospirosis. Patients showed a moderate increase in serum concentrations of oleic and linoleic acids but an important decrease in serum concentrations of albumin. A highly significant correlation between serum concentrations of creatinine or total bilirubin and the oleic-plus-linoleic acid : albumin ratio was revealed. We used the Na(+),K(+)-ATPase inhibitory property of NEUFAs to test the capacity of serum to prevent the cytotoxic effects of NEUFAs in vitro. Albumin solutions and serum samples from healthy volunteers, but not serum samples from severely affected patients, were able to revert the Na(+),K(+)-ATPase inhibition by oleic acid. On the basis of these data, we defined a "serum protection factor" that can be helpful in predicting NEUFA toxicity. Our data support the concept that the administration of human albumin to patients may be helpful in severe leptospirosis cases.

Differential regulation of collecting duct Na+, K+-ATPase and K+ excretion by furosemide and piretanide: role of bradykinin.

In response to chronic treatment with furosemide, collecting ducts adapt their function to the initial loss of Na+ to prevent further Na+ loss and extracellular volume decrease. This adaptation, which includes the overexpression of Na+, K+-ATPase, is thought to account for most of the kaliuretic effect of furosemide. Because piretanide is reported to be less kaliuretic than equidiuretic doses of furosemide, the authors compared the effects of 1-wk treatment with the two loop diuretics on urinary potassium excretion and on Na+, K+-ATPase activity in the collecting duct. At equidiuretic and equinatriuretic doses, furosemide increased urinary potassium excretion as well as collecting duct Na+, K+-ATPase activity, whereas piretanide had no effect on either parameter. These effects of furosemide were curtailed by concomitant administration of the angiotensin-converting enzyme inhibitor enalapril, but they were not altered either by clamping changes in plasma aldosterone or by blocking type I angiotensin receptors. Treatment with the antagonist of bradykinin B2 receptors Hoe140 mimicked the two effects of furosemide. In addition, the effects of Hoe140 and furosemide were not additive. Finally, piretanide increased urinary bradykinin excretion, whereas furosemide did not. These results suggest that induction of collecting duct Na+, K+-ATPase (a) accounts for the kaliuretic effect of furosemide, (b) is independent of the renin/angiotensin/aldosterone system, (c) results from increased Na+ delivery to the collecting duct and enhanced intracellular Na+ concentration, and (d) is prevented in piretanide treated rats by increased bradykinin production that may limit apical Na+ entry in collecting duct principal cells.

Técnica simplificada de dosagem de K+ e NA+ intra-eritrocitários / A simple method for measurement of intraerythrocytic K+ and NA+

Os autores desenvolveram uma técnica minituarizada e simples para a dosagem de sódio (Nal) e de potássio (KI) intra-eritrocitário, baseada na medida da concentraçao destes íons em uma solução hemolisada, preparada a partir de uma massa conhecida de eritrócitos. O estudo foi realizado, em quadruplicatas, com sangue venoso obtido de dois grupos de indivíduos: grupo I - 55 pacientes hospitalizados e portadores de hipocalemia diagnosticada até 12h antes da coleta; grupo II - 13 indivíduos saudáveis. Os resultados obtidos mostram que a técnica é de boa reprodutividade, com coeficiente de variaçao de 4,7 por cento entre as replicatas de um mesmo indivíduo. O grupo I apresentou níveis de KI inferiores aos do grupo II (87,70 ñ 7,0mEq/l vs 101,56 ñ 3,86mEq/l), o que coincidiu com a tendência apresentada pelos níveis de potássio sérico dos dois grupos (3,66 ñ 0,79mEq/l vs 4,16 ñ 0,31mEq/l). Por sua vez, os valores médios de Nal não foram significativamente diferentes entre os dois grupos (9,40 ñ 2,20 vs 9,27 ñ 1,0mEq/l). Em conclusao, a técnica proposta mostrou-se adequada, com a reprodução de valores determinados previamente na literatura por outros métodos. Por representar um método simples, rápido e de fácil execuçao, esta técnica é passivel de ser empregada quatiadianamente em qualquer laboratório de análises clínicas

Fisiopatologia da leptospirose - hipótese para a insuficiência renal aguda peculiar / Physiopathology of leptospirosis - hypothesis for peculiar acute renal failure

Sao tecidas consideraçoes gerais sobre a insuficiência renal aguda (IRA) da leptospirose e acentuadas as principais características observadas numa série de 109 pacientes, destacando a tendência a hipopotassemia, verificada na metade dos casos, oligúricos ou nao oligúricos. Sobre tal tendência, um dos autores (MY-I) realizou investigaçoes (89-96) que indicaram: baixo potássio intra e extracelular, principalmente nos casos de IRA; presença de um fator plasmático capaz de evitar "in vitro" a ensima Na-K-ATPase de uma preparaçao purificada; a presença de uma endotoxina glicolipoprotéica da Leptospira interrogans cujo alvo parece ser a Na-K-ATPase, que é especificamente inibida no rim de coelhos. Sao feitos comentários clínicos sobre os envolvimentos cardio-vascular, pulmonar, renal, hepático e neuromuscular e sobre as anormalidades eletrolíticas, além de consideraçoes terapêuticas. Por fim é oferecida uma hipótese fisiopatológica para explicar a hipopotassemia na IRA da leptospirose, fundamentada na inibiçao da Na-K-ATPase nos diversos órgaos, provocada pela lise das leptospiras.