Cytosolic phospholipase A2 regulates lipid homeostasis under osmotic stress through PPARγ.

Physiologically, renal medullary cells are surrounded by a hyperosmolar interstitium. However, different pathological situations can induce abrupt changes in environmental osmolality, causing cell stress. Therefore, renal cells must adapt to survive in this new condition. We previously demonstrated that, among the mechanisms involved in osmoprotection, renal cells upregulate triglyceride biosynthesis (which helps preserve glycerophospholipid synthesis and membrane homeostasis) and cyclooxygenase-2 (which generates prostaglandins from arachidonic acid) to maintain lipid metabolism in renal tissue. Herein, we evaluated whether hyperosmolality modulates phospholipase A2 (PLA2 ) activity, leading to arachidonic acid release from membrane glycerophospholipid, and investigated its possible role in hyperosmolality-induced triglyceride synthesis and accumulation. We found that hyperosmolality induced PLA2 expression and activity in Madin-Darby canine kidney cells. Cytosolic PLA2 (cPLA2) inhibition, but not secreted or calcium-independent PLA2 (sPLA2 or iPLA2 , respectively), prevented triglyceride synthesis and reduced cell survival. Inhibition of prostaglandin synthesis with indomethacin not only failed to prevent hyperosmolality-induced triglyceride synthesis but also exacerbated it. Similar results were observed with the peroxisomal proliferator activated receptor gamma (PPARγ) agonist rosiglitazone. Furthermore, hyperosmolality increased free intracellular arachidonic acid levels, which were even higher when prostaglandin synthesis was inhibited by indomethacin. Blocking PPARγ with GW-9662 prevented the effects of both indomethacin and rosiglitazone on triglyceride synthesis and even reduced hyperosmolality-induced triglyceride synthesis, suggesting that arachidonic acid may stimulate triglyceride synthesis through PPARγ activation. These results highlight the role of cPLA2 in osmoprotection, since it is essential to provide arachidonic acid, which is involved in PPARγ-regulated triglyceride synthesis, thus guaranteeing cell survival.

Spinal cord ischemia as intraoperatory complication in shoulder surgery positioned in beach chair: case report

Abstract Spinal cord infarction is an uncommon phenomenon, which can be caused by different etiologies, constituting a real diagnostic challenge which can lead to devastating consequences. General anesthesia in beach chair positioning with intraoperative hypotension in order to avoid surgical bleeding are associated with hypoperfusion and potential neurological ischemia-related complications. We present a case of spinal cord ischemia in the context of shoulder surgery in a beach chair position.

Analysis of XBP1 Contribution to Hyperosmolarity-Induced Lipid Synthesis.

The unfolded protein response (UPR) is a complex network of intracellular pathways that transmits signals from ER lumen and/or ER bilayer to the nuclear compartment in order to activate gene transcription. UPR is activated by the loss of ER capacities, known as ER stress, and occurs to restore ER properties. In this regard, glycerolipid (GL) synthesis activation contributes to ER membrane homeostasis and IRE1α-XBP1, one UPR pathway, has a main role in lipogenic genes transcription. Herein, we describe the strategy and methodology used to evaluate whether IRE1α-XBP1 pathway regulates lipid metabolism in renal epithelial cells subjected to hyperosmolar environment. XBP1s activity was hindered by blocking IRE1α RNAse activity and by impeding its expression; under these conditions, we determined GL synthesis and lipogenic enzymes expression.

SPARC inhibition accelerates NAFLD-associated hepatocellular carcinoma development by dysregulating hepatic lipid metabolism.

BACKGROUND AND AIMS: Non-alcoholic fatty liver (NAFLD) and its more serious form non-alcoholic steatohepatitis increase risk of hepatocellular carcinoma (HCC). Lipid metabolic alterations and its role in HCC development remain unclear. SPARC (Secreted Protein, Acidic and Rich in Cysteine) is involved in lipid metabolism, NAFLD and diabetes, but the effects on hepatic lipid metabolism and HCC development is unknown. The aim of this study was to evaluate the role of SPARC in HCC development in the context of NAFLD. METHODS: Primary hepatocyte cultures from knockout (SPARC-/- ) or wild-type (SPARC+/+ ) mice, and HepG2 cells were used to assess the effects of free fatty acids on lipid accumulation, expression of lipogenic genes and de novo triglyceride (TG) synthesis. A NAFLD-HCC model was stabilized on SPARC-/- or SPARC+/+ mice. Correlations among SPARC, lipid metabolism-related gene expression patterns and clinical prognosis were studied using HCC gene expression dataset. RESULTS: SPARC-/- mice increases hepatic lipid deposits over time. Hepatocytes from SPARC-/- mice or inhibition of SPARC by an antisense adenovirus in HepG2 cells resulted in increased TG deposit, expression of lipid-related genes and nuclear translocation of SREBP1c. Human HCC database analysis revealed that SPARC negatively correlated with genes involved in lipid metabolism, and with poor survival. In NAFLD-HCC murine model, the absence of SPARC accelerates HCC development. RNA-seq study revealed that pathways related to lipid metabolism, cellular detoxification and proliferation were upregulated in SPARC-/- tumour-bearing mice. CONCLUSIONS: The absence of SPARC is associated with an altered hepatic lipid metabolism, and an accelerated NAFLD-related HCC development.

Congenital transmission of Chagas disease in a non-endemic area, is an early diagnosis possible?

BACKGROUND: Chagas disease (CD) is an emergent disease in Europe, due to immigration. The aims of this study are to describe the epidemiological characteristics of a cohort of Chagas infected pregnant women in Spain, to assess the vertical transmission (VT) rate and evaluate the usefulness of the PCR in the diagnosis of congenital infection in the first months of life. METHODS: A descriptive, retrospective study including Chagas seropositive pregnant women who were attended at three tertiary hospitals in Madrid, from January 2012 to September 2016. Infants were examined by PCR at birth and 1 month later and serologically studied at 9 months or later. Children were considered infected when the parasite was detected by PCR at any age or when serology remained positive without decline over the age of 9 months. RESULTS: We included 122 seropositive-infected pregnant women, 81% were from Bolivia and only 8.2% had been treated before. 125 newborns were studied and finally 109 were included (12.8% lost the follow-up before performing the last serology). The VT rate was 2.75% (95% CI: 0,57-8,8%). Infected infants had positive PCR at birth and 1 month later. All of them were treated successfully with benznidazole (PCR and serology became negative later on). All non-infected children presented negative PCR. The mean age at which uninfected patients had negative serology was 10.5 months. CONCLUSIONS: The VT rate is in keeping with literature and confirms the need to carry out a screening in pregnant women coming from endemic areas. PCR seems to be a useful tool to provide early diagnosis of congenital CD.

Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 and angiopoietin-like protein 4 are associated with the increase of lipoprotein lipase activity in epicardial adipose tissue from diabetic patients.

BACKGROUND AND AIMS: Epicardial adipose tissue (EAT) is a visceral AT, surrounding myocardium and coronary arteries. Its volume is higher in Type 2 diabetic (DM2) patients, associated with cardiovascular disease risk. Lipoprotein lipase (LPL) hydrolyses triglycerides (TG) from circulating lipoproteins, supplying fatty acids to AT, contributing to its expansion. We aimed to evaluate LPL expression and activity in EAT from DM2 and no DM2 patients, and its regulators ANGPTL4, GPIHBP1 and PPARγ levels, together with VLDLR expression and EAT LPL association with VLDL characteristics. METHODS: We studied patients undergoing coronary by-pass graft (CABG) divided into CABG-DM2 (n = 21) and CABG-noDM2 (n = 29), and patients without CABG (No CABG, n = 30). During surgery, EAT and subcutaneous AT (SAT) were obtained, in which LPL activity, gene and protein expression, its regulators and VLDLR protein levels were determined. Isolated circulating VLDLs were characterized. RESULTS: EAT LPL activity was higher in CABG-DM2 compared to CABG-noDM2 and No CABG (p=0.002 and p<0.001) and in CABG-noDM2 compared to No CABG (p=0.02), without differences in its expression. ANGPTL4 levels were higher in EAT from No CABG compared to CABG-DM2 and CABG-noDM2 (p<0.001). GPIHBP1 levels were higher in EAT from CABG-DM2 and CABG-noDM2 compared to No CABG (p= 0.04). EAT from CABG-DM2 presented higher PPARγ levels than CABG-noDM2 and No CABG (p=0.02 and p=0.03). No differences were observed in VLDL composition between groups, although EAT LPL activity was inversely associated with VLDL-TG and TG/protein index (p<0.05). CONCLUSIONS: EAT LPL regulation would be mainly post-translational. The higher LPL activity in DM2 could be partly responsible for the increase in EAT volume.

Endothelial Lipase Is an Alternative Pathway for Fatty Acid Release from Lipoproteins: Evidence from a High Fat Diet Model of Obesity in Rats.

Lipoprotein lipase (LPL) and endothelial lipase (EL) are involved in lipoprotein metabolism. In insulin-resistance, their behavior is altered. Peroxisome proliferator-activated receptors (PPAR) and apoproteins (apo)CII and CIII could be partly responsible for these alterations. To evaluate this response, we assessed Lpl and Lipg expression, protein levels, and enzyme activity in adipose tissue (AT) and heart in an obesity model. Besides, we assessed the role of PPAR and apoC. Male Wistar rats were fed with standard diet (Control, n = 14) or high-fat diet (HFD, n = 14) for 14 weeks. Glucose and lipoprotein profiles were measured. Histological studies were performed in heart and epididymal AT. Lpl and Lipg were assessed by reverse transcription polymerase chain reaction (RT-qPCR), protein levels by Western Blot, and activities by radiometric assays. Cardiac and AT PPAR expression were measured by Western Blot and hepatic Apoc2 and Apoc3 mRNA by RT-qPCR. In HFD, fat deposits were observed in hearts, whereas AT presented a higher adipocyte size. In heart and AT, no differences were found in Lipg mRNA between groups, while AT Lpl mRNA and LPL protein were decreased in HFD, without differences in heart. In both tissues, EL protein levels and activity were increased and inversely associated with decreased LPL activity, being partially responsible for the atherogenic lipoprotein profile in HFD. PPARγ expression in AT was decreased in HFD, without differences in cardiac PPARδ expression and hepatic apoC mRNA. The increase in EL activity could be an alternative pathway for fatty acid release from lipoproteins and uptake in tissues with decreased LPL activity. In AT, PPARγ could be involved in enzyme regulation.

Behavior of stroke volume variation in hemodynamic stable patients during thoracic surgery with one-lung ventilation periods / Comportamento da variação do volume sistólico em pacientes hemodinamicamente estáveis durante cirurgia torácica com períodos de ventilação monopulmonar

Abstract Introduction: In last few years, emphasis was placed in goal-directed therapy in order to optimize patient's hemodynamic status and improve their prognosis. Parameters based on the interaction between heart and lungs have been questioned in situations like low tidal volume and open chest surgery. The goal of the study was to analyze the changes that one-lung ventilation can produce over stroke volume variation and to assess the possible impact of airway pressures and lung compliance over stroke volume variation. Methods: Prospective observational study, 112 patients undergoing lung resection surgery with one-lung ventilation periods were included. Intravenous fluid therapy with crystalloids was set at 2 mL.g-1. Hypotension episodes were treated with vasoconstrictive drugs. Two-lung Ventilation was implemented with a TV of 8 mL.g-1 and one-lung ventilation was managed with a TV of 6 mL.g-1. Invasive blood pressure was monitored. We recorded the following cardiorespiratory values: heart rate, mean arterial pressure, cardiac index, stroke volume index, airway peak pressure, airway plateau pressure and static lung compliance at 3 different times during surgery: immediately after lung collapse, 30 min after initiating one-lung ventilation and after restoration of two-lung ventilation. Results: Stroke volume variation values were influenced by lung collapse (before lung collapse 14.6 (DS) vs. OLV 9.9% (DS), p < 0.0001); or after restoring two-lung ventilation (11.01 (DS), p < 0.0001). During two-lung Ventilation there was a significant correlation between airway pressures and stroke volume variation, however this correlation lacks during one-lung ventilation. Conclusion: The decrease of stroke volume variation values during one-lung ventilation with protective ventilatory strategies advices not to use the same threshold values to determine fluid responsiveness.

Resumo Introdução: Nos últimos anos, a importância da terapia alvo-dirigida foi enfatizada para aprimorar o estado hemodinâmico do paciente e melhorar seu prognóstico. Os parâmetros baseados na interação entre o coração e os pulmões foram questionados em situações como baixo volume corrente e cirurgia aberta do tórax. O objetivo do estudo foi analisar as alterações que a ventilação monopulmonar pode produzir na variação do volume sistólico e avaliar o possível impacto das pressões da via aérea e da complacência pulmonar sobre a variação do volume sistólico. Métodos: Estudo observacional prospectivo, no qual 112 pacientes submetidos à cirurgia de ressecção pulmonar com períodos de ventilação monopulmonar foram incluídos. A terapia de fluídos intravenosos com cristaloides foi ajustada a 2 mL.kg-1.h-1. Os episódios de hipotensão foram tratados com vasoconstritores. A ventilação dos dois pulmões (VDP) foi implantada com volume corrente de 8 mL.kg-1 e a ventilação monopulmonar foi controlada com volume corrente de 6 mL.kg-1. Foi monitorada a pressão arterial invasiva. Registramos os seguintes valores cardiorrespiratórios: frequência cardíaca, pressão arterial média, índice cardíaco, índice de volume sistólico, pressão de pico das vias aéreas, pressão de platô das vias aéreas e complacência pulmonar estática em três tempos durante a cirurgia: imediatamente após o colapso do pulmão, 30 minutos após o início da ventilação monopulmonar e após a restauração da ventilação dos dois pulmões. Resultados: Os valores de variação do volume sistólico foram influenciados pelo colapso pulmonar (antes do colapso pulmonar 14,6 [DS] vs. ventilação monopulmonar 9,9% [DS], p < 0,0001), ou após o restabelecimento da ventilação dos dois pulmões (11,01 [DS], p < 0,0001). Durante a ventilação dos dois pulmões houve uma correlação significativa entre as pressões das vias aéreas e a variação do volume sistólico, porém, essa correlação não existe durante a ventilação monopulmonar. Conclusão: A diminuição dos valores da variação do volume sistólico durante a ventilação monopulmonar com estratégias ventilatórias protetoras sugere não usar os mesmos valores de limiar para determinar a responsividade aos fluídos.

[Behaviour of stroke volume variation in hemodynamic stable patients during thoracic surgery with one-lung ventilation periods]. / Comportamento da variação do volume sistólico em pacientes hemodinamicamente estáveis durante cirurgia torácica com períodos de ventilação monopulmonar.

INTRODUCTION: In last few years, emphasis was placed in goal-directed therapy in order to optimize patient's hemodynamic status and improve their prognosis. Parameters based on the interaction between heart and lungs have been questioned in situations like low tidal volume and open chest surgery. The goal of the study was to analyze the changes that one-lung ventilation can produce over stroke volume variation and to assess the possible impact of airway pressures and lung compliance over stroke volume variation. METHODS: Prospective observational study, 112 patients undergoing lung resection surgery with one-lung ventilation periods were included. Intravenous fluid therapy with crystalloids was set at 2mL.kg-1.h-1. Hypotension episodes were treated with vasoconstrictive drugs. Two-lung ventilation was implemented with a TV of 8mL.kg-1 and one-lung ventilation was managed with a TV of 6mL.kg-1. Invasive blood pressure was monitored. We recorded the following cardiorespiratory values: heart rate, mean arterial pressure, cardiac index, stroke volume index, airway peak pressure, airway plateau pressure and static lung compliance at 3 different times during surgery: immediately after lung collapse, 30minutes after initiating one-lung ventilation and after restoration of two-lung ventilation. RESULTS: Stroke volume variation values were influenced by lung collapse (before lung collapse14.6 (DS) vs. OLV 9.9% (DS), p < 0.0001); or after restoring two-lung ventilation (11.01 (DS), p < 0.0001). During two-lung ventilation there was a significant correlation between airwaypressures and stroke volume variation, however this correlation lacks during one-lung ventilation. CONCLUSION: The decrease of stroke volume variation values during one-lung ventilation with protective ventilatory strategies advices not to use the same threshold values to determine fluid responsiveness.

Sequential and synchronized hypertonicity-induced activation of Rel-family transcription factors is required for osmoprotection in renal cells.

NF-κB and TonEBP belong to the Rel-superfamily of transcription factors. Several specific stimuli, including hypertonicity which is a key factor for renal physiology, are able to activate them. It has been reported that, after hypertonic challenge, NF-κB activity can be modulated by TonEBP, considered as the master regulator of transcriptional activity in the presence of changes in environmental tonicity. In the present work we evaluated whether hypertonicity-induced gene transcription mediated by p65/RelA and TonEBP occurs by an independent action of each transcription factor or by acting together. To do this, we evaluated the expression of their specific target genes and cyclooxygenase-2 (COX-2), a common target of both transcription factors, in the renal epithelial cell line Madin-Darby canine kidney (MDCK) subjected to hypertonic environment. The results herein indicate that hypertonicity activates the Rel-family transcription factors p65/RelA and TonEBP in MDCK cells, and that both are required for hypertonic induction of COX-2 and of their specific target genes. In addition, present data show that p65/RelA modulates TonEBP expression and both colocalize in nuclei of hypertonic cultures of MDCK cells. Thus, a sequential and synchronized action p65/RelA → TonEBP would be necessary for the expression of hypertonicity-induced protective genes.

Risk of coronary artery involvement in Kawasaki disease.

INTRODUCTION: Kawasaki disease refers to systemic vasculitis with risk of coronary artery disease. Our objective is to identify risk factors associated with coronary artery disease in patients with complete and incomplete Kawasaki disease. MATERIAL AND METHODS: Descriptive, retrospective study conducted in patients diagnosed with Kawasaki disease in a tertiary-care hospital between 2008 and 2014. The American Heart Association diagnostic criteria were used to define complete and incomplete Kawasaki disease. RESULTS: Thirty-one children were diagnosed with Kawasaki disease; 24 met the criteria for the complete form, and 7, for the incomplete form of this condition. Five had coronary artery disease. One of them had incomplete Kawasaki disease (1/7= 14.3%), and the remaining four had the complete form (4/24= 16.7%). No significant differences were found between both groups (p= 1.0). Patients with coronary artery involvement had a higher C-reactive protein level (median: 16.2 mg/dL versus 8.4 mg/dL, p= 0.047) and lower albuminemia (median: 3.2 mg/dL versus 3.99 mg/dL, p= 0.002). CONCLUSIONS: The risk of coronary artery involvement in incomplete Kawasaki disease is similar to that in complete Kawasaki disease; therefore, in patients with the incomplete form, immunoglobulin therapy should not be delayed. In our population, C-reactive protein and albumin levels were related to a higher risk of coronary artery involvement.

INTRODUCCIÓN: La enfermedad de Kawasaki es una vasculitis sistémica con riesgo de afectación coronaria. Nuestro objetivo es identificar los factores de riesgo asociados a la afectación coronaria en pacientes con enfermedad de Kawasaki completa e incompleta. MATERIAL AND MÉTODOS: Estudio descriptivo retrospectivo de los pacientes diagnosticados con enfermedad de Kawasaki en un hospital terciario entre 2008 y 2014. Se utilizaron los criterios diagnósticos de la Asociación Americana de Cardiología para definir la enfermedad de Kawasaki en su forma completa e incompleta. RESULTADOS: Treinta y un niños fueron diagnosticados con enfermedad de Kawasaki; 24 cumplían criterios para la forma completa y 7, para la incompleta. Cinco presentaron afectación coronaria. Uno de ellos presentaba enfermedad de Kawasaki incompleta (1/7= 14,3%), y los 4 restantes, enfermedad de Kawasaki completa (4/24= 16,7%). No se encontraron diferencias significativas en el riesgo de afectación coronaria entre ambos grupos (p= 1,0). Los pacientes con afectación coronaria tenían una proteína C reactiva mayor (mediana: 16,2 mg/dl vs. 8,4 mg/dl; p= 0,047) y una menor albuminemia (mediana: 3,2 mg/dl vs. 3,99 mg/dl; p= 0,002). CONCLUSIONES: El riesgo de afectación coronaria de la enfermedad de Kawasaki incompleta es similar al de la enfermedad de Kawasaki complet por lo que, en pacientes con la forma incompleta de la enfermedad, no se debería demorar el tratamiento con inmunoglobulina. En nuestra población, los valores de proteína C reactiva y de albúmina se relacionan con un mayor riesgo de afectación coronaria.

Riesgo de afectación coronaria en la enfermedad de Kawasaki / Risk of coronary artery involvement in Kawasaki disease

Introducción: La enfermedad de Kawasaki es una vasculitis sistémica con riesgo de afectación coronaria. Nuestro objetivo es identificar los factores de riesgo asociados a la afectación coronaria en pacientes con enfermedad de Kawasaki completa e incompleta. Material y métodos: Estudio descriptivo retrospectivo de los pacientes diagnosticados con enfermedad de Kawasaki en un hospital terciario entre 2008 y 2014. Se utilizaron los criterios diagnósticos de la Asociación Americana de Cardiología para definir la enfermedad de Kawasaki en su forma completa e incompleta. Resultados: Treinta y un niños fueron diagnosticados con enfermedad de Kawasaki; 24 cumplían criterios para la forma completa y 7, para la incompleta. Cinco presentaron afectación coronaria. Uno de ellos presentaba enfermedad de Kawasaki incompleta (1/7= 14,3%), y los 4 restantes, enfermedad de Kawasaki completa (4/24= 16,7%). No se encontraron diferencias significativas en el riesgo de afectación coronaria entre ambos grupos (p= 1,0). Los pacientes con afectación coronaria tenían una proteína C reactiva mayor (mediana: 16,2 mg/dl vs. 8,4 mg/dl; p= 0,047) y una menor albuminemia (mediana: 3,2 mg/dl vs. 3,99 mg/dl; p= 0,002). Conclusiones: El riesgo de afectación coronaria de la enfermedad de Kawasaki incompleta es similar al de la enfermedad de Kawasaki completa, por lo que, en pacientes con la forma incompleta de la enfermedad, no se debería demorar el tratamiento con inmunoglobulina. En nuestra población, los valores de proteína C reactiva y de albúmina se relacionan con un mayor riesgo de afectación coronaria.

Introduction: Kawasaki disease refers to systemic vasculitis with risk of coronary artery disease. Our objective is to identify risk factors associated with coronary artery disease in patients with complete and incomplete Kawasaki disease. Material and methods: Descriptive, retrospective study conducted in patients diagnosed with Kawasaki disease in a tertiary-care hospital between 2008 and 2014. The American Heart Association diagnostic criteria were used to define complete and incomplete Kawasaki disease. Results: Thirty-one children were diagnosed with Kawasaki disease; 24 met the criteria for the complete form, and 7, for the incomplete form of this condition. Five had coronary artery disease. One of them had incomplete Kawasaki disease (1/7= 14.3%), and the remaining four had the complete form (4/24= 16.7%). No significant differences were found between both groups (p= 1.0). Patients with coronary artery involvement had a higher C-reactive protein level (median: 16.2 mg/dL versus 8.4 mg/dL, p= 0.047) and lower albuminemia (median: 3.2 mg/dL versus 3.99 mg/dL, p= 0.002). Conclusions: The risk of coronary artery involvement in incomplete Kawasaki disease is similar to that in complete Kawasaki disease; therefore, in patients with the incomplete form, immunoglobulin therapy should not be delayed. In our population, C-reactive protein and albumin levels were related to a higher risk of coronary artery involvement.

Vasopresores en resucitación cardiopulmonar: la controversia continúa / Vasopressors for cardiopulmonary resuscitation

The Advanced Cardiac Life Support proposes the use of vasopressor drugs cardiopulmonary resuscitation, with the primary purpose of effectively and early restoration of spontaneous circulation. However, the increased return of spontaneous circulation with vasopressors has not improved neurological outcome at hospital discharge. Adrenaline has traditionally been the main tool of the cardiopulmonary resuscitation guidelines despite the lack of conclusive scientific evidence. Therefore other alternatives were considered at experimental level that has have failed to overcome the clinical results of this drug. In contrast to progress on the standardized management of cardiopulmonary resuscitation, the controversy regarding the effectiveness of vasopressor therapy remains open in the last years due to lack of clinical data to support their usefulness.

El soporte vital avanzado propone el uso de vasopresores durante la resucitación cardiopulmonar, con la finalidad primordial de reestablecer de manera precoz y efectiva la circulación espontánea. Sin embargo, el aumento del retorno a la circulación espontánea asociada a los vasopresores no ha demostrado una mejoría en el pronóstico neurológico al alta hospitalaria. La adrenalina, ha sido tradicionalmente la herramienta principal de las guías de resucitación cardiopulmonar pese a la falta de evidencia científica concluyente. Por ello se han planteado otras alternativas a nivel experimental que no han logrado superar los resultados de este fármaco a nivel clínico. En contraste con los avances respecto al manejo estandarizado de la resucitación cardiopulmonar, el debate con respecto a la efectividad de la terapia vasopresora se mantiene abierto en los últimos años dada la falta de datos clínicos que corroboren su real utilidad.

Coordinate regulation between the nuclear receptor peroxisome proliferator-activated receptor-γ and cyclooxygenase-2 in renal epithelial cells.

The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors involved in lipid metabolism and glucose utilization, in cell growth, differentiation and apoptosis, and in the regulation of pro-inflammatory genes expression such as cyclooxygenase-2 (COX-2). PPARγ is the main isoform in the renal inner medulla where it is believed to possess nephroprotective actions. In this kidney zone, COX-2 acts as an osmoprotective gene and its expression is modulated by changes in interstitial osmolarity. In the present work we evaluated whether hyperosmolar-induced COX-2 expression is modulated by PPARγ in renal epithelial cells MDCK subjected to high NaCl medium. The results presented herein show that ligand-activated PPARγ repressed COX-2 expression. But more important, the present findings show that hyperosmolar medium decreased PPARγ protein and increases the PPARγ phosphorylated form, which is inactive. ERK1/2 and p38 activation precedes PPARγ disappearance and induced-COX-2 expression. Therefore, the decrease in PPARγ expression is required for hyperosmotic induction of COX-2. We also found that PGE2, the main product of COX-2 in MDCK cells, induced these changes in PPARγ protein. Our results may alert on the long term use of thiazolidinediones (TZD) since they could affect renal medullary function that depends on COX-2 for cellular protection against osmotic stress.

Environmental hyperosmolality regulates phospholipid biosynthesis in the renal epithelial cell line MDCK.

Hyperosmolality is a key signal for renal physiology. On the one hand, it contributes to the differentiation of renal medullary structures and to the development of the urinary concentrating mechanism. On the other, it is a stress factor. In both cases, hyperosmolality activates processes that require an adequate extension of cellular membranes. In the present work, we examined whether hyperosmolality regulates phospholipid biosynthesis, which is needed for the membrane biogenesis in the renal epithelial cell line Madin-Darby canine kidney (MDCK). Because phospholipids are the structural determinants of all cell membranes, we evaluated their content, synthesis, and regulation in MDCK cultures subjected to different hyperosmotic concentrations of NaCl, urea, or both. Hyperosmolality increased phospholipid content in a concentration-dependent manner. Such an effect was exclusively due to changes in NaCl concentration and occurred at the initial stage of hyperosmolar treatment concomitantly with the expression of the osmoprotective protein COX-2. The hypertonic upregulation of phosphatidylcholine (PC) synthesis, the main constituent of all cell membranes, involved the transcriptional activation of two main regulatory enzymes, choline kinase (CK) and cytidylyltransferase α (CCTα) and required ERK1/2 activation. Considering that physiologically, renal medullary cells are constantly exposed to high and variable NaCl, these findings could contribute to explaining how renal cells could maintain cellular integrity even in a nonfavorable environment.

Monoclonal B-cell lymphocytosis (MBL, CD4+/CD8 weak T-cell large granular lymphocytic leukemia (T-LGL leukemia) and monoclonal gammopathy of unknown significance (MGUS): molecular and flow cytometry characterization of three concomitant hematological disorders.

The diagnosis of T-cell large granular lymphocytic leukemia in association with other B-cell disorders is uncommon but not unknown. However, the concomitant presence of three hematological diseases is extraordinarily rare. We report an 88-year-old male patient with three simultaneous clonal disorders, that is, CD4+/CD8(weak) T-cell large granular lymphocytic leukemia, monoclonal gammopathy of unknown significance and monoclonal B-cell lymphocytosis. The patient has only minimal complaints and has no anemia, neutropenia or thrombocytopenia. Lymphadenopathy and hepatosplenomegaly were not present. The three disorders were characterized by flow cytometry analysis, and the clonality of the T-cell large granular lymphocytic leukemia was confirmed by polymerase chain reaction. Interestingly, the patient has different B-cell clones, given that plasma cells of monoclonal gammopathy of unknown significance exhibited a kappa light-chain restriction population and, on the other hand, B-lymphocytes of monoclonal B-cell lymphocytosis exhibited a lambda light-chain restriction population. This finding does not support the antigen-driven hypothesis for the development of multi-compartment diseases, but suggests that T-cell large granular lymphocytic expansion might represent a direct antitumor immunological response to both B-cell and plasma-cell aberrant populations, as part of the immune surveillance against malignant neoplasms.

The rate-limiting enzyme in phosphatidylcholine synthesis is associated with nuclear speckles under stress conditions.

Phosphatidylcholine (PtdCho) is the most abundant phospholipid in eukaryotic membranes and its biosynthetic pathway is generally controlled by CTP:Phosphocholine Cytidylyltransferase (CCT), which is considered the rate-limiting enzyme. CCT is an amphitropic protein, whose enzymatic activity is commonly associated with endoplasmic reticulum (ER) translocation; however, most of the enzyme is intranuclearly located. Here we demonstrate that CCTα is concentrated in the nucleoplasm of MDCK cells. Confocal immunofluorescence revealed that extracellular hypertonicity shifted the diffuse intranuclear distribution of the enzyme to intranuclear domains in a foci pattern. One population of CCTα foci colocalised and interacted with lamin A/C speckles, which also contained the pre-mRNA processing factor SC-35, and was resistant to detergent and salt extraction. The lamin A/C silencing allowed us to visualise a second more labile population of CCTα foci that consisted of lamin A/C-independent foci non-resistant to extraction. We demonstrated that CCTα translocation is not restricted to its redistribution from the nucleus to the ER and that intranuclear redistribution must thus be considered. We suggest that the intranuclear organelle distribution of CCTα is a novel mechanism for the regulation of enzyme activity.

Bradykinin induces formation of vesicle-like structures containing vinculin and PtdIns(4,5)P2 in renal papillary collecting duct cells.

Focal adhesions (FAs) are structures of cell attachment to the extracellular matrix. We previously demonstrated that the intrarenal hormone bradykinin (BK) induces the restructuring of FAs in papillary collecting duct cells by dissipation of vinculin, but not talin, from FAs through a mechanism that involves PLCbeta activation, and that it also induces actin cytoskeleton reorganization. In the present study we investigated the mechanism by which BK induces the dissipation of vinculin-stained FAs in collecting duct cells. We found that BK induces the internalization of vinculin by a noncaveolar and independent pinocytic pathway and that at least a fraction of this protein is delivered to the recycling endosomal compartment, where it colocalizes with the transferrin receptor. Regarding the reassembly of vinculin-stained FAs, we found that BK induces the formation of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-enriched vinculin-containing vesicles, which, by following a polarized exocytic route, transport vinculin to the site of FA assembly, an action that depends on actin filaments. The present study, which was carried out with cells that were not genetically manipulated, shows for the first time that BK induces the formation of vesicle-like structures containing vinculin and PtdIns(4,5)P2, which transport vinculin to the site of FA assembly. Therefore, the modulation of the formation of these vesicle-like structures could be a physiological mechanism through which the cell can reuse the BK-induced internalized vinculin to be delivered for newly forming FAs in renal papillary collecting duct cells.

Membrane lipid composition plays a central role in the maintenance of epithelial cell adhesion to the extracellular matrix.

Focal contacts (FC) are membrane-associated multi-protein complexes that mediate cell-extracellular matrix (ECM) adhesion. FC complexes are inserted in detergent-resistant membrane microdomains enriched in phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2); however, the influence of membrane lipid composition in the preservation of FC structures has not been extensively addressed. In the present work, we studied the contribution of membrane lipids to the preservation of renal epithelial cell adhesion structures. We biochemically characterized the lipid composition of membrane-containing FC complexes. By using cholesterol and PtdIns(4,5)P2)affecting agents, we demonstrated that such agents did not affect any particular type of lipid but induced the formation of new FC-containing domains of completely different lipid composition. By using both biochemical approaches and fluorescence microscopy we demonstrated that phospholipid composition plays an essential role in the in vivo maintenance of FC structures involved in cell-ECM adhesion.