RESUMEN
BACKGROUND: The Covid pandemic and associated lockdown forced medical schools globally not only to deliver emergency remote teaching, but to consider alternative methods of high stakes assessment. Here we outline our approach to the resit virtual OSCE ("VOSCE") for final year medical students that we undertook during "lockdown" in the current pandemic. METHODS: The original 'pre Covid' examination blueprint was reviewed and modified for the virtual environment in both format and content. In anticipation of the new format delivery, a number of pre-training sessions took place for all parties, and standardised templates were developed. RESULTS: A total of 9 students undertook the VOSCE, which took the form of a two-part exam (a communication and clinical examination component, and a practical procedures component). The VOSCE was completed by all students, examiners, simulated patients and invigilators on an online digital platform with no issues with regards to technical problems. CONCLUSIONS: A total of 6 students passed the VOSCE and as such progressed to graduation. The limitation of assessing some particular types of skills across the remote format (such as practical procedures) was recognised. The training and the templates developed were helpful in case the VOSCE format needs to be adopted in future at short notice and/or expanded in future.
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Rendimiento Académico , COVID-19 , Educación a Distancia/organización & administración , Estudiantes de Medicina , Competencia Clínica , Control de Enfermedades Transmisibles , Humanos , PandemiasRESUMEN
Background: The ß2-adrenoceptor agonist dopexamine may possess anti-inflammatory actions which could reduce organ injury during endotoxemia and laparotomy. Related effects on leucocyte-endothelial adhesion remain unclear. Methods: Thirty anesthetized Wistar rats underwent laparotomy followed by induction of endotoxemia with lipopolysaccharide and peptidoglycan (n = 24) or sham (n = 6). Animals received dopexamine at 0.5 or 1 µg kg-1 min-1 (D0.5 and D1), salbutamol at 0.1 µg kg-1 min-1, or saline vehicle (Sham and Control) for 5 h. Intravital microscopy was performed in the ileum of the small intestine to assess leucocyteendothelial adhesion, arteriolar diameter, and functional capillary density. Global hemodynamics and biochemical indices of renal and hepatic function were also measured. Results: Endotoxemia was associated with an increase in adherent leucocytes in post-capillary venules, intestinal arteriolar vasoconstriction as well-reduced arterial pressure and relative cardiac index, but functional capillary density in the muscularis was not significantly altered. Dopexamine and salbutamol administration were associated with reduced leucocyte-endothelial adhesion in post-capillary venules compared to control animals. Arteriolar diameter, arterial pressure and relative cardiac index all remained similar between treated animals and controls. Functional capillary density was similar for all groups. Control group creatinine was significantly increased compared to sham and higher dose dopexamine. Conclusions: In a rodent model of laparotomy and endotoxemia, ß2-agonists were associated with reduced leucocyte-endothelial adhesion in post-capillary venules. This effect may explain some of the anti-inflammatory actions of these agents.
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Antagonistas de Receptores Adrenérgicos beta 2/uso terapéutico , Dopamina/análogos & derivados , Células Endoteliales/fisiología , Endotoxemia/tratamiento farmacológico , Leucocitos/fisiología , Vasodilatadores/uso terapéutico , Animales , Adhesión Celular , Modelos Animales de Enfermedad , Dopamina/uso terapéutico , Humanos , Laparotomía , Masculino , Microcirculación , Ratas , Ratas Wistar , Receptores Adrenérgicos beta 2/metabolismoRESUMEN
Endothelin-1 (ET-1) is involved in the pathogenesis of cardiac and renal diseases, and in the progression of tumour growth in cancer, but current diagnosis and treatment remain inadequate. Peptides derived from the 212 amino acid precursor preproendothelin-1 (ppET-1) may have utility as biomarkers, or cause biological effects that are unaffected by endothelin receptor antagonists. Here, we used specific immunoassays and LC-MS/MS to identify NT-proET-1 (ppET-1[18-50]), Endothelin-Like Domain Peptide (ELDP, ppET-1[93-166]) and CT-proET-1 (ppET-1[169-212]) in conditioned media from cultured endothelial cells. Synthesis of these peptides correlated with ET-1, and plasma ELDP and CT-proET-1 were elevated in patients with chronic heart failure. Clearance rates of NT-proET-1, ELDP and CT-proET-1 were determined after i.v. injection in anaesthetised rats. CT-proET-1 had the slowest systemic clearance, hence providing a biological basis for it being a better biomarker of ET-1 synthesis. ELDP contains the evolutionary conserved endothelin-like domain sequence, which potentially confers biological activity. On isolated arteries ELDP lacked direct vasoconstrictor effects. However, it enhanced ET-1 vasoconstriction and prolonged the increase in blood pressure in anaesthetised rats. ELDP may therefore contribute to disease pathogenesis by augmenting ET-1 responses.
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Células Endoteliales/citología , Endotelina-1/metabolismo , Insuficiencia Cardíaca/diagnóstico , Fragmentos de Péptidos/administración & dosificación , Precursores de Proteínas/química , Células A549 , Biomarcadores/sangre , Línea Celular , Cromatografía Liquida , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Células Endoteliales/metabolismo , Endotelina-1/química , Insuficiencia Cardíaca/metabolismo , Humanos , Inyecciones Intravenosas , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacocinética , Espectrometría de Masas en TándemRESUMEN
BACKGROUND: Acute kidney injury (AKI) is a major risk factor for the development of chronic kidney disease. Nuclear factor-κB is a nuclear transcription factor activated post-ischemia, responsible for the transcription of proinflammatory proteins. The role of nuclear factor-κB in the renal fibrosis post-AKI is unknown. METHODS AND RESULTS: We used a rat model of AKI caused by unilateral nephrectomy plus contralateral ischemia (30 minutes) and reperfusion injury (up to 28 days) to show impairment of renal function (peak: 24 hours), activation of nuclear factor-κB (peak: 48 hours), and fibrosis (28 days). In humans, AKI is diagnosed by a rise in serum creatinine. We have discovered that the IκB kinase inhibitor IKK16 (even when given at peak serum creatinine) still improved functional and structural recovery and reduced myofibroblast formation, macrophage infiltration, transforming growth factor-ß expression, and Smad2/3 phosphorylation. AKI resulted in fibrosis within 28 days (Sirius red staining, expression of fibronectin), which was abolished by IKK16. To confirm the efficacy of IKK16 in a more severe model of fibrosis, animals were subject to 14 days of unilateral ureteral obstruction, resulting in tubulointerstitial fibrosis, myofibroblast formation, and macrophage infiltration, all of which were attenuated by IKK16. CONCLUSIONS: Inhibition of IκB kinase at peak creatinine improves functional recovery, reduces further injury, and prevents fibrosis.
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Lesión Renal Aguda/tratamiento farmacológico , Quinasa I-kappa B/antagonistas & inhibidores , Riñón/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Actinas/metabolismo , Lesión Renal Aguda/enzimología , Lesión Renal Aguda/patología , Lesión Renal Aguda/fisiopatología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Creatinina/sangre , Modelos Animales de Enfermedad , Fibrosis , Quinasa I-kappa B/metabolismo , Riñón/enzimología , Riñón/patología , Riñón/fisiopatología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patología , FN-kappa B/metabolismo , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
OBJECTIVE: To evaluate the effects of artesunate on organ injury and dysfunction associated with hemorrhagic shock (HS) in the rat. BACKGROUND: HS is still a common cause of death in severely injured patients and is characterized by impairment of organ perfusion, systemic inflammatory response, and multiple organ failure. There is no specific therapy that reduces organ injury/dysfunction. Artesunate exhibits pharmacological actions beyond its antimalarial activity, such as anticancer, antiviral, and anti-inflammatory effects. METHODS: Rats were submitted to HS. Mean arterial pressure was reduced to 30âmm Hg for 90âminutes, followed by resuscitation. Rats were randomly treated with artesunate (2.4 or 4.8âmg/kg i.v.) or vehicle upon resuscitation. Four hours later, parameters of organ injury and dysfunction were assessed. RESULTS: Artesunate attenuated the multiple organ injury and dysfunction caused by HS. Pathway analysis of RNA sequencing provided good evidence to support an effect of artesunate on the Akt-survival pathway, leading to downregulation of interleukin-1 receptor-associated kinase 1. Using Western blot analysis, we confirmed that treatment of HS rats with artesunate enhanced the phosphorylation (activation) of Protein kinase B (Akt) and endothelial nitric oxide synthase and the phosphorylation (inhibition) of glycogen synthase kinase-3ß (GSK-3ß). Moreover, artesunate attenuated the HS-induced activation of nuclear factor kappa B and reduced the expression of proinflammatory proteins (inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin 6). CONCLUSIONS: Artesunate attenuated the organ injury/dysfunction associated with HS by a mechanism that involves the activation of the Akt-endothelial nitric oxide synthase survival pathway, and the inhibition of glycogen synthase kinase-3ß and nuclear factor kappa B. A phase II clinical trial evaluating the effects of good manufacturing practice-artesunate in patients with trauma and severe hemorrhage is planned.
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Artemisininas/uso terapéutico , Insuficiencia Multiorgánica/prevención & control , Sustancias Protectoras/uso terapéutico , Resucitación/efectos adversos , Choque Hemorrágico/terapia , Animales , Artesunato , Biomarcadores/metabolismo , Terapia Combinada , Masculino , Insuficiencia Multiorgánica/etiología , Insuficiencia Multiorgánica/metabolismo , Distribución Aleatoria , Ratas , Ratas Wistar , Choque Hemorrágico/metabolismo , Resultado del TratamientoRESUMEN
Patients with CKD requiring dialysis have a higher risk of sepsis and a 100-fold higher mortality rate than the general population with sepsis. The severity of cardiac dysfunction predicts mortality in patients with sepsis. Here, we investigated the effect of preexisting CKD on cardiac function in mice with sepsis and whether inhibition of IκB kinase (IKK) reduces the cardiac dysfunction in CKD sepsis. Male C57BL/6 mice underwent 5/6 nephrectomy, and 8 weeks later, they were subjected to LPS (2 mg/kg) or sepsis by cecal ligation and puncture (CLP). Compared with sham operation, nephrectomy resulted in significant increases in urea and creatinine levels, a small (P<0.05) reduction in ejection fraction (echocardiography), and increases in the cardiac levels of phosphorylated IκBα, Akt, and extracellular signal-regulated kinase 1/2; nuclear translocation of the NF-κB subunit p65; and inducible nitric oxide synthase (iNOS) expression. When subjected to LPS or CLP, compared with sham-operated controls, CKD mice exhibited exacerbation of cardiac dysfunction and lung inflammation, greater increases in levels of plasma cytokines (TNF-α, IL-1ß, IL-6, and IL-10), and greater increases in the cardiac levels of phosphorylated IKKα/ß and IκBα, nuclear translocation of p65, and iNOS expression. Treatment of CKD mice with an IKK inhibitor (IKK 16; 1 mg/kg) 1 hour after CLP or LPS administration attenuated these effects. Thus, preexisting CKD aggravates the cardiac dysfunction caused by sepsis or endotoxemia in mice; this effect may be caused by increased cardiac NF-κB activation and iNOS expression.
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Cardiopatías/enzimología , Cardiopatías/prevención & control , Quinasa I-kappa B/antagonistas & inhibidores , Insuficiencia Renal Crónica/enzimología , Sepsis/complicaciones , Animales , Cardiopatías/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Insuficiencia Renal Crónica/complicacionesRESUMEN
BACKGROUND: Tissue infiltration by neutrophils during acute inflammatory states causes substantial tissue injury. While the magnitude of tissue neutrophil accumulation in innate immune responses is profoundly greater in males than females, fundamental aspects of the molecular mechanisms underlying these sex differences remain largely unknown. METHODS: We investigated sex differences in neutrophil stimulation and recruitment in ischemia/reperfusion (I/R; mesenteric or renal) or carrageenan pleurisy in rats or mice, as well as skin injury in human volunteers. The induction of potent chemoattractive mediators (chemokines) and neutrophil adhesion molecules were measured by real-time PCR, flow cytometry, and protein assays. RESULTS: Mesenteric I/R in age-matched Wistar rats resulted in substantially more neutrophil accumulation and tissue injury at 2 h reperfusion in males than females. Using intravital microscopy, we show that the immediate (<30 min) neutrophil response to I/R is similar in males and females but that prolonged neutrophil recruitment occurs in males at sites local and distal to inflammatory insult partly due to an increase in circulating neutrophil populations with elevated surface expression of adhesion molecules. Sex differences in neutrophil kinetics were correlated with sustained induction of chemokine Cxcl5 in the tissue, circulation, and bone marrow of males but not females. Furthermore, blockade of Cxcl5 in males prior to ischemia resulted in neutrophil responses that were similar in magnitude to those in females. Conversely, administration of Cxcl5 to males in the absence of I/R was sufficient to increase levels of systemic neutrophils. Cxcl5 treatment of bone marrow neutrophils in vitro caused substantial induction of neutrophil-mobilizing cytokine granulocyte colony-stimulating factor (GCSF) and expression of ß2 integrin that accounts for sexual dimorphism in circulating neutrophil populations in I/R. Moreover, male Cxcl5-stimulated bone marrow neutrophils had an increased capacity to adhere to ß2 integrin ligand ICAM-1, implicating a greater sensitivity of male leukocytes to Cxcl5-mediated activation. Differential induction of Cxcl5 (human CXCL6) between the sexes was also evident in murine renal I/R, rat pleurisy, and human skin blisters and correlated with the magnitude of neutrophil accumulation in tissues. CONCLUSIONS: Our study reveals that sex-specific induction of chemokine Cxcl5/CXCL6 contributes to sexual dimorphism in neutrophil recruitment in diverse acute inflammatory responses partly due to increased stimulation and trafficking of bone marrow neutrophils in males.
RESUMEN
Nuclear factor-kappa B (NF-κB) activation is widely implicated in multiple organ failure (MOF); however, a direct inhibitor of IκB kinase (IKK), which plays a pivotal role in the activation of NF-κB, has not been investigated in shock. Thus, the aim of the present work was to investigate the effects of an IKK inhibitor on the MOF associated with hemorrhagic shock (HS). Therefore, rats were subjected to HS and were resuscitated with the shed blood. Rats were treated with the inhibitor of IKK or vehicle at resuscitation. Four hours later, blood and organs were assessed for organ injury and signaling events involved in the activation of NF-κB. Additionally, survival following serum deprivation was assessed in HK-2 cells treated with the inhibitor of IKK. HS resulted in renal dysfunction, lung, liver and muscular injury, and increases in serum inflammatory cytokines. Kidney and liver tissue from HS rats revealed increases in phosphorylation of IKKαß and IκBα, nuclear translocation of NF-κB and expression of inducible isoform of nitric oxide synthase (iNOS). IKK16 treatment upon resuscitation attenuated NF-κB activation and activated the Akt survival pathway, leading to a significant attenuation of all of the above parameters. Furthermore, IKK16 exhibited cytoprotective effects in human kidney cells. In conclusion, the inhibitor of IKK complex attenuated the MOF associated with HS. This effect may be due to the inhibition of the NF-κB pathway and activation of the survival kinase Akt. Thus, the inhibition of the IKK complex might be an effective strategy for the prevention of MOF associated with HS.
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Proteínas I-kappa B/genética , Insuficiencia Multiorgánica/genética , FN-kappa B/genética , Choque Hemorrágico/genética , Animales , Humanos , Proteínas I-kappa B/antagonistas & inhibidores , Riñón/lesiones , Riñón/metabolismo , Riñón/patología , Lipopolisacáridos/toxicidad , Hígado/lesiones , Hígado/metabolismo , Hígado/patología , Lesión Pulmonar/genética , Insuficiencia Multiorgánica/complicaciones , Insuficiencia Multiorgánica/patología , Insuficiencia Multiorgánica/terapia , Inhibidor NF-kappaB alfa , Fosforilación , Ratas , Choque Hemorrágico/complicaciones , Choque Hemorrágico/patología , Choque Hemorrágico/terapia , Transducción de SeñalRESUMEN
METHODS: Male rats were 'pretreated' with phosphate-buffered saline (PBS; i.p.) or LPS (1 mg/kg; i.p.) 24 h prior to HS. Mean arterial pressure (MAP) was maintained at 30 ± 2 mmHg for 90 min or until 25% of the shed blood had to be re-injected to sustain MAP. This was followed by resuscitation with the remaining shed blood. Four hours after resuscitation, parameters of organ dysfunction and systemic inflammation were assessed. RESULTS: HS resulted in renal dysfunction, and liver and muscular injury. At a first glance, LPS preconditioning attenuated organ dysfunction. However, we discovered that HS-rats that had been preconditioned with LPS (a) were not able to sustain a MAP at 30 mmHg for more than 50 min and (b) the volume of blood withdrawn in these animals was significantly less than in the PBS-control group. This effect was associated with an enhanced formation of the nitric oxide (NO) derived from inducible NO synthase (iNOS). Thus, a further control group in which all animals were resuscitated after 50 min of hemorrhage was performed. Then, LPS preconditioning aggravated both circulatory failure and organ dysfunction. Most notably, HS-rats pretreated with LPS exhibited a dramatic increase in NF-κB activation and pro-inflammatory cytokines. CONCLUSION: In conclusion, LPS preconditioning predisposed animals to an earlier vascular decompensation, which may be mediated by an excess of NO production secondary to induction of iNOS and activation of NF-κB. Moreover, LPS preconditioning increased the formation of pro-inflammatory cytokines, which is likely to have contributed to the observed aggravation of organ injury/dysfunction caused by HS.
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Lipopolisacáridos/farmacología , Choque Hemorrágico/inmunología , Animales , Citocinas/sangre , Riñón/metabolismo , Riñón/patología , Hígado/metabolismo , Hígado/patología , Masculino , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ratas Wistar , Choque Hemorrágico/sangre , Choque Hemorrágico/patologíaRESUMEN
Development of cardiac dysfunction is associated with increased morbidity and mortality in patients with sepsis. Increasing evidence shows that gender determines the degree of inflammatory response of the host and that females tolerate sepsis better than males. It is unknown whether gender affects the cardiac dysfunction in animals or patients with sepsis. To investigate this, male or female C57BL/6 mice were subjected to either lipopolysaccharide (LPS)/peptidoglycan (PepG) co-administration or cecal ligation and puncture (CLP). At 18 hours after LPS/PepG injection or 24 hours after CLP, cardiac function was evaluated by echocardiography. The septic insult caused a significant cardiac dysfunction in both genders. However, the cardiac dysfunction was significantly less pronounced in females in comparison with males subjected to LPS (3 mg/kg)/PepG (0.1 mg/kg) or CLP. Compared with males injected with LPS (3 mg/kg)/PepG (0.1 mg/kg), western blotting analysis of the myocardium from females injected with LPS/PepG revealed i) profound increases in phosphorylation of Akt and eNOS; ii) significant decreases in phosphorylation of IκBα, nuclear translocation of the NF-κB subunit p65, decreased expression of iNOS and decreased synthesis of TNF-α and IL-6 in the heart. However, the gender dimorphism of the cardiac dysfunction secondary to LPS/PepG was not observed when higher doses of LPS (9 mg/kg)/PepG (1 mg/kg) were used. In conclusion, the cardiac dysfunction caused by sepsis was less pronounced in female than in male mice. The protection of female hearts against the dysfunction associated with sepsis is (at least in part) attributable to cardiac activation of the Akt/eNOS survival pathway, decreased activation of NF-κB, and decreased expression of iNOS, TNF-α and IL-6. It should be noted that the observed gender dimorphism of the cardiac dysfunction in sepsis was not seen when a very severe stimulus (high dose of LPS/PepG co-administration) was used to cause cardiac dysfunction.
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Insuficiencia Cardíaca/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sepsis/metabolismo , Factores de Edad , Animales , Femenino , Regulación de la Expresión Génica , Insuficiencia Cardíaca/inducido químicamente , Insuficiencia Cardíaca/complicaciones , Insuficiencia Cardíaca/genética , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolisacáridos , Masculino , Ratones , Ratones Endogámicos C57BL , Inhibidor NF-kappaB alfa , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Peptidoglicano , Fosforilación , Proteínas Proto-Oncogénicas c-akt/genética , Sepsis/inducido químicamente , Sepsis/complicaciones , Sepsis/genética , Factores Sexuales , Transducción de Señal , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The combination of obesity and type 2 diabetes is a serious health problem, which is projected to afflict 300 million people worldwide by 2020. Both clinical and translational laboratory studies have demonstrated that chronic inflammation is associated with obesity and obesity-related conditions such as insulin resistance. However, the precise etiopathogenetic mechanisms linking obesity to diabetes remain to be elucidated, and the pathways that mediate this phenomenon are not fully characterized. One of the most recently identified signaling pathways, whose activation seems to affect many metabolic disorders, is the "inflammasome," a multiprotein complex composed of NLRP3 (nucleotide-binding domain and leucine-rich repeat protein 3), ASC (apoptosis-associated speck-like protein containing a CARD), and procaspase-1. NLRP3 inflammasome activation leads to the processing and secretion of the proinflammatory cytokines interleukin- (IL-) 1 ß and IL-18. The goal of this paper is to review new insights on the effects of the NLRP3 inflammasome activation in the complex mechanisms of crosstalk between different organs, for a better understanding of the role of chronic inflammation in metabolic disease pathogenesis. We will provide here a perspective on the current research on NLRP3 inflammasome, which may represent an innovative therapeutic target to reverse the detrimental metabolic consequences of the metabolic inflammation.
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Inflamasomas/metabolismo , Inflamación/metabolismo , Enfermedades Metabólicas/metabolismo , Tejido Adiposo/embriología , Animales , Biopsia , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Mucosa Intestinal/metabolismo , Riñón/metabolismo , Proteínas Repetidas Ricas en Leucina , Hígado/metabolismo , Músculo Esquelético/metabolismo , Obesidad/metabolismo , Pronóstico , Proteínas/metabolismo , Especies Reactivas de Oxígeno , Transducción de SeñalRESUMEN
Nuclear factor κB (NF-κB) plays a pivotal role in sepsis. Activation of NF-κB is initiated by the signal-induced ubiquitylation and subsequent degradation of inhibitors of kappa B (IκBs) primarily via activation of the IκB kinase (IKK). This study was designed to investigate the effects of IKK inhibition on sepsis-associated multiple organ dysfunction and/or injury (MOD) and to elucidate underlying signaling mechanisms in two different in vivo models: male C57BL/6 mice were subjected to either bacterial cell wall components [lipopolysaccharide and peptidoglycan (LPS/PepG)] or underwent cecal ligation and puncture (CLP) to induce sepsis-associated MOD. At 1 hour after LPS/PepG or CLP, mice were treated with the IKK inhibitor IKK 16 (1 mg/kg body weight). At 24 hours, parameters of organ dysfunction and/or injury were assessed in both models. Mice developed a significant impairment in systolic contractility (echocardiography), and significant increases in serum creatinine, serum alanine aminotransferase and lung myeloperoxidase activity, thus indicating cardiac dysfunction, renal dysfunction, hepatocellular injury and lung inflammation, respectively. Treatment with IKK 16 attenuated the impairment in systolic contractility, renal dysfunction, hepatocellular injury and lung inflammation in LPS/PepG-induced MOD and in polymicrobial sepsis. Compared with mice that were injected with LPS/PepG or underwent CLP, immunoblot analyses of heart and liver tissues from mice that were injected with LPS/PepG or underwent CLP and were also treated with IKK 16 revealed: (1) significant attenuation of the increased phosphorylation of IκBα; (2) significant attenuation of the increased nuclear translocation of the NF-κB subunit p65; (3) significant attenuation of the increase in inducible nitric oxide synthase (iNOS) expression; and (4) a significant increase in the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). Here, we report for the first time that delayed IKK inhibition reduces MOD in experimental sepsis. We suggest that this protective effect is (at least in part) attributable to inhibition of inflammation through NF-κB, the subsequent decrease in iNOS expression and the activation of the Akt-eNOS survival pathway.
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Quinasa I-kappa B/antagonistas & inhibidores , Insuficiencia Multiorgánica/complicaciones , Insuficiencia Multiorgánica/enzimología , Sepsis/complicaciones , Sepsis/enzimología , Animales , Ciego/efectos de los fármacos , Ciego/patología , Ciego/fisiopatología , Pruebas de Función Cardíaca/efectos de los fármacos , Quinasa I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Riñón/efectos de los fármacos , Riñón/patología , Riñón/fisiopatología , Ligadura , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Insuficiencia Multiorgánica/patología , Insuficiencia Multiorgánica/fisiopatología , Miocardio/enzimología , Miocardio/patología , Inhibidor NF-kappaB alfa , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Peptidoglicano/administración & dosificación , Peptidoglicano/farmacología , Neumonía/complicaciones , Neumonía/patología , Neumonía/fisiopatología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Punciones , Sepsis/fisiopatología , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/metabolismoRESUMEN
The ß-common receptor (ßcR) plays a pivotal role in the nonhematopoietic tissue-protective effects of erythropoietin (EPO). Here we determined whether EPO reduces the acute kidney injury (AKI) caused by sepsis and whether this effect is mediated by the ßcR. In young (2 months old) C57BL/6 wild-type and ßcR knockout mice, lipopolysaccharide caused a significant increase in serum urea and creatinine, hence AKI. This AKI was not associated with any overt morphological alterations in the kidney and was attenuated by EPO given 1 h after lipopolysaccharide in wild-type but not in ßcR knockout mice. In the kidneys of endotoxemic wild-type mice, EPO enhanced the phosphorylation of Akt, glycogen synthase kinase-3ß, and endothelial nitric oxide synthase, and inhibited the activation of nuclear factor-κB. All these effects of EPO were lost in ßcR knockout mice. Since sepsis is more severe in older animals or patients, we tested whether EPO was renoprotective in 8-month-old wild-type and ßcR knockout mice that underwent cecal ligation and puncture. These older mice developed AKI at 24 h, which was attenuated by EPO treatment 1 h post cecal ligation and puncture in wild-type mice but not in ßcR knockout mice. Thus, activation of the ßcR by EPO is essential for the observed reduction in AKI in either endotoxemic young mice or older mice with polymicrobial sepsis, and for the activation of well-known signaling pathways by EPO.
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Lesión Renal Aguda/prevención & control , Lesión Renal Aguda/fisiopatología , Subunidad beta Común de los Receptores de Citocinas/metabolismo , Eritropoyetina/uso terapéutico , Riñón/metabolismo , Sepsis/complicaciones , Lesión Renal Aguda/metabolismo , Animales , Caspasa 3/metabolismo , Ciego/fisiopatología , Subunidad beta Común de los Receptores de Citocinas/deficiencia , Subunidad beta Común de los Receptores de Citocinas/genética , Modelos Animales de Enfermedad , Eritropoyetina/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Receptor Celular 1 del Virus de la Hepatitis A , Riñón/efectos de los fármacos , Ligadura , Lipopolisacáridos/efectos adversos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sepsis/inducido químicamente , Sepsis/etiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
There is limited evidence that the tissue-protective effects of erythropoietin are mediated by a heterocomplex of the erythropoietin receptor and the ß-common receptor ('tissue-protective receptor'), which is pharmacologically distinct from the 'classical' erythropoietin receptor homodimer that is responsible for erythropoiesis. However, the role of the ß-common receptor and/or erythropoietin in sepsis-induced cardiac dysfunction (a well known, serious complication of sepsis) is unknown. Here we report for the first time that the ß-common receptor is essential for the improvements in the impaired systolic contractility afforded by erythropoietin in experimental sepsis. Cardiac function was assessed in vivo (echocardiography) and ex vivo (Langendorff-perfused heart) in wild-type and ß-common receptor knockout mice, that were subjected to lipopolysaccharide (9 mg/kg body weight; young mice) for 16-18 hours or cecal ligation and puncture (aged mice) for 24 hours. Mice received erythropoietin (1000 IU/kg body weight) 1 hour after lipopolysaccharide or cecal ligation and puncture. Erythropoietin reduced the impaired systolic contractility (in vivo and ex vivo) caused by endotoxemia or sepsis in young as well as old wild-type mice in a ß-common-receptor-dependent fashion. Activation by erythropoietin of the ß-common receptor also resulted in the activation of well-known survival pathways (Akt and endothelial nitric oxide synthase) and inhibition of pro-inflammatory pathways (glycogen synthase kinase-3ß, nuclear factor-κB and interleukin-1ß). All the above pleiotropic effects of erythropoietin were lost in ß-common receptor knockout mice. Erythropoietin attenuates the impaired systolic contractility associated with sepsis by activation of the ß-common receptor, which, in turn, results in activation of survival pathways and inhibition of inflammation.
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Subunidad beta Común de los Receptores de Citocinas/metabolismo , Eritropoyetina/farmacología , Corazón/efectos de los fármacos , Corazón/fisiopatología , Sepsis/fisiopatología , Animales , Ciego/patología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Endotoxemia/complicaciones , Endotoxemia/diagnóstico por imagen , Endotoxemia/patología , Endotoxemia/fisiopatología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Pruebas de Función Cardíaca , Técnicas In Vitro , Interleucina-1beta/metabolismo , Ligadura , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/metabolismo , Perfusión , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Punciones , Sepsis/complicaciones , Sepsis/diagnóstico por imagen , Sepsis/patología , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/metabolismo , UltrasonografíaRESUMEN
INTRODUCTION: The effects of dopexamine, a ß2-agonist, on perioperative and sepsis-related hemodynamic, microvascular, immune, and organ dysfunction are controversial and poorly understood. We investigated these effects in a rodent model of laparotomy and endotoxemia. METHODS: In two experiments, 80 male Wistar rats underwent laparotomy. In 64 rats, this was followed by administration of endotoxin; the remainder (16) underwent sham endotoxemia. Endotoxemic animals received either dopexamine at 0.5, 1, or 2 µg/kg/min or 0.9% saline vehicle (controls) as resuscitation fluid. The effects of dopexamine on global hemodynamics, mesenteric regional microvascular flow, renal and hepatic function and immune activation were evaluated. RESULTS: Endotoxin administration was associated with a systemic inflammatory response (increased plasma levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and IL-10, as well as cell-adhesion molecules CD11a and CD11b), and increased pulmonary myeloperoxidase (MPO) activity (indicating pulmonary leukocyte infiltration), whereas biochemical changes demonstrated lactic acidosis with significant renal and hepatic injury. Dopexamine administration was associated with less-severe lactic acidosis (pooled dopexamine versus controls, (lactate, 2.2 mM±0.2 mM versus 4.0 mM±0.5 mM; P<0.001) and reductions in the systemic inflammatory response (pooled dopexamine versus control, 4 hour (TNF-α): 324 pg/ml±93 pg/ml versus 97 pg/ml±14 pg/ml, p<0.01), pulmonary myeloperoxidase (MPO) activity, and hepatic and renal injury (pooled dopexamine versus control (ALT): 81 IU/L±4 IU/L versus 138 IU/L±25 IU/L; P<0.05; (creatinine): 49.4 µM±3.9 µM versus 76.2 µM±9.8 µM; P<0.005). However, in this study, clinically relevant doses of dopexamine were not associated with clinically significant changes in MAP, CI, or gut regional microvascular flow. CONCLUSIONS: In this model, dopexamine can attenuate the systemic inflammatory response, reduce tissue leukocyte infiltration, and protect against organ injury at doses that do not alter global hemodynamics or regional microvascular flow. These findings suggest that immunomodulatory effects of catecholamines may be clinically significant when used in critically ill surgical patients and are independent of their hemodynamic actions.
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Dopamina/análogos & derivados , Endotoxemia/prevención & control , Hemodinámica/efectos de los fármacos , Microcirculación/efectos de los fármacos , Flujo Sanguíneo Regional/efectos de los fármacos , Vasodilatadores/uso terapéutico , Animales , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Velocidad del Flujo Sanguíneo/fisiología , Dopamina/farmacología , Dopamina/uso terapéutico , Endotoxemia/fisiopatología , Hemodinámica/fisiología , Inflamación/fisiopatología , Inflamación/prevención & control , Masculino , Microcirculación/fisiología , Sustancias Protectoras/farmacología , Sustancias Protectoras/uso terapéutico , Distribución Aleatoria , Ratas , Ratas Wistar , Flujo Sanguíneo Regional/fisiología , Vasodilatadores/farmacologíaRESUMEN
Pre-treatment with erythropoietin (EPO) has been demonstrated to exert tissue-protective effects against 'ischemia-reperfusion'-type injuries. This protection might be mediated by mobilization of bone marrow endothelial progenitor cells (EPCs), which are thought to secrete paracrine factors. These effects could be exploited to protect against tissue injury induced in cases where hemorrhage is foreseeable, for example, prior to major surgery. Here, we investigate the effects of EPO pre-treatment on the organ injury and dysfunction induced by hemorrhagic shock (HS). Recombinant human EPO (1000 IU/kg/day i.p.) was administered to rats for 3 days. Rats were subjected to HS on day 4 (pre-treatment protocol). Mean arterial pressure was reduced to 35 ± 5 mmHg for 90 minutes, followed by resuscitation with 20 ml/kg Ringer's lactate for 10 minutes and 50% of the shed blood for 50 minutes. Rats were sacrificed 4 hours after the onset of resuscitation. EPC (CD34(+)/flk-1(+) cell) mobilization was measured following the 3-day pre-treatment with EPO and was significantly increased compared with rats pre-treated with phosphate-buffered saline. EPO pre-treatment significantly attenuated organ injury and dysfunction (renal, hepatic and neuromuscular) caused by HS. In livers from rats subjected to HS, EPO enhanced the phosphorylation of Akt (activation), glycogen synthase kinase-3ß (GSK-3ß; inhibition) and endothelial nitric oxide synthase (eNOS; activation). In the liver, HS also caused an increase in nuclear translocation of p65 (activation of NF-κB), which was attenuated by EPO. This data suggests that repetitive dosing with EPO prior to injury might protect against the organ injury and dysfunction induced by HS, by a mechanism that might involve mobilization of CD34(+)/flk-1(+) cells, resulting in the activation of the Akt-eNOS survival pathway and inhibition of activation of GSK-3ß and NF-κB.
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Eritropoyetina/uso terapéutico , Especificidad de Órganos , Choque Hemorrágico/tratamiento farmacológico , Choque Hemorrágico/fisiopatología , Animales , Antígenos CD34/metabolismo , Circulación Sanguínea/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Modelos Animales de Enfermedad , Eritropoyetina/sangre , Eritropoyetina/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Hematócrito , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Óxido Nítrico Sintasa de Tipo III/metabolismo , Especificidad de Órganos/efectos de los fármacos , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Choque Hemorrágico/sangre , Choque Hemorrágico/enzimología , Factor de Transcripción ReIA/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Erythropoietin (EPO) is known to have numerous biological functions. Its primary function in the body is to increase red blood cell numbers by way of preventing the apoptosis of erythroid progenitor cells via the homodimeric EPO receptor. The discovery that the local production of EPO within the brain in response to hypoxia or ischemia protects neurons against injury via an anti-apoptotic effect formed the basis of the hypothesis that the local generation of EPO limits the extent of injury. Although the hypothesis proved to be true in pre-clinical models of ischemia/reperfusion injury and inflammation, the randomized, controlled clinical trials that followed demonstrated serious adverse events of EPO due to activation of the hematopoietic system. Consequently, derivatives of EPO that lacked erythropoietic activity were discovered to reduce injury in many pre-clinical models associated with ischemia and inflammation. Unfortunately, there are no published clinical trials to determine the efficacy of non-erythropoietic derivatives of EPO in humans.
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Cuidados Críticos , Eritropoyetina/uso terapéutico , Eritropoyetina/análogos & derivados , Eritropoyetina/farmacología , HumanosRESUMEN
In preclinical studies, erythropoietin (EPO) reduces ischemia-reperfusion-associated tissue injury (for example, stroke, myocardial infarction, acute kidney injury, hemorrhagic shock and liver ischemia). It has been proposed that the erythropoietic effects of EPO are mediated by the classic EPO receptor homodimer, whereas the tissue-protective effects are mediated by a hetero-complex between the EPO receptor monomer and the ß-common receptor (termed "tissue-protective receptor"). Here, we investigate the effects of a novel, selective-ligand of the tissue-protective receptor (pyroglutamate helix B surface peptide [pHBSP]) in a rodent model of acute kidney injury/dysfunction. Administration of pHBSP (10 µg/kg intraperitoneally [i.p.] 6 h into reperfusion) or EPO (1,000 IU/kg i.p. 4 h into reperfusion) to rats subjected to 30 min ischemia and 48 h reperfusion resulted in significant attenuation of renal and tubular dysfunction. Both pHBSP and EPO enhanced the phosphorylation of Akt (activation) and glycogen synthase kinase 3ß (inhibition) in the rat kidney after ischemia-reperfusion, resulting in prevention of the activation of nuclear factor-κB (reduction in nuclear translocation of p65). Interestingly, the phosphorylation of endothelial nitric oxide synthase was enhanced by EPO and, to a much lesser extent, by pHBSP, suggesting that the signaling pathways activated by EPO and pHBSP may not be identical.
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Lesión Renal Aguda/tratamiento farmacológico , Oligopéptidos/administración & dosificación , Lesión Renal Aguda/metabolismo , Animales , Clusterina/sangre , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Glomérulos Renales/efectos de los fármacos , Glomérulos Renales/fisiopatología , Túbulos Renales/efectos de los fármacos , Túbulos Renales/fisiopatología , Masculino , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Osteopontina/sangre , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Recent evidence suggests that cell therapy such as the injection of bone marrow-derived mononuclear cells (BMMNCs) can exert protective effects in various conditions associated with ischemia-reperfusion injury. Here, we investigate the effects of BMMNCs on the organ injury/dysfunction induced by hemorrhagic shock (HS). Thirty-seven anesthetized male Wistar rats were subjected to hemorrhage by reducing mean arterial pressure to 35 ± 5 mmHg for 90 min, followed by resuscitation with 20 mL/kg Ringer's lactate administered over 10 min and 50% of the shed blood over 50 min. Rats were killed 4 h after the onset of resuscitation. Bone marrow-derived mononuclear cells were freshly isolated from rat tibias and femurs using Percoll density gradient centrifugation, and BMMNCs (1 × 10 cells per rat in 1 mL/kg phosphate-buffered saline, i.v.) were administered on resuscitation. Hemorrhagic shock resulted in significant organ injury/dysfunction (renal, hepatic, neuromuscular) and inflammation (hepatic, lung). In rats subjected to HS, administration of BMMNCs significantly attenuated (i) organ injury/dysfunction (renal, hepatic, neuromuscular) and inflammation (hepatic, lung), (ii) increased the phosphorylation of Akt and glycogen synthase kinase-3ß, (iii) attenuated the activation of nuclear factor-κB, (iv) attenuated the increase in extracellular signal-regulated kinase 1/2 phosphorylation, and (v) attenuated the increase in expression of intercellular adhesion molecule-1. Our findings suggest that administration of BMMNCs protects against the induction of early organ injury/dysfunction caused by severe HS by a mechanism that may involve activation of Akt and the inhibition of glycogen synthase kinase-3ß and nuclear factor-κB.