RESUMEN
BACKGROUND: Chronic liver scarring from any cause leads to cirrhosis, portal hypertension, and a progressive decline in renal blood flow and renal function. Extreme renal vasoconstriction characterizes hepatorenal syndrome, a functional and potentially reversible form of acute kidney injury in patients with advanced cirrhosis, but current therapy with systemic vasoconstrictors is ineffective in a substantial proportion of patients and is limited by ischemic adverse events. Serelaxin (recombinant human relaxin-2) is a peptide molecule with anti-fibrotic and vasoprotective properties that binds to relaxin family peptide receptor-1 (RXFP1) and has been shown to increase renal perfusion in healthy human volunteers. We hypothesized that serelaxin could ameliorate renal vasoconstriction and renal dysfunction in patients with cirrhosis and portal hypertension. METHODS AND FINDINGS: To establish preclinical proof of concept, we developed two independent rat models of cirrhosis that were characterized by progressive reduction in renal blood flow and glomerular filtration rate and showed evidence of renal endothelial dysfunction. We then set out to further explore and validate our hypothesis in a phase 2 randomized open-label parallel-group study in male and female patients with alcohol-related cirrhosis and portal hypertension. Forty patients were randomized 1:1 to treatment with serelaxin intravenous (i.v.) infusion (for 60 min at 80 µg/kg/d and then 60 min at 30 µg/kg/d) or terlipressin (single 2-mg i.v. bolus), and the regional hemodynamic effects were quantified by phase contrast magnetic resonance angiography at baseline and after 120 min. The primary endpoint was the change from baseline in total renal artery blood flow. Therapeutic targeting of renal vasoconstriction with serelaxin in the rat models increased kidney perfusion, oxygenation, and function through reduction in renal vascular resistance, reversal of endothelial dysfunction, and increased activation of the AKT/eNOS/NO signaling pathway in the kidney. In the randomized clinical study, infusion of serelaxin for 120 min increased total renal arterial blood flow by 65% (95% CI 40%, 95%; p < 0.001) from baseline. Administration of serelaxin was safe and well tolerated, with no detrimental effect on systemic blood pressure or hepatic perfusion. The clinical study's main limitations were the relatively small sample size and stable, well-compensated population. CONCLUSIONS: Our mechanistic findings in rat models and exploratory study in human cirrhosis suggest the therapeutic potential of selective renal vasodilation using serelaxin as a new treatment for renal dysfunction in cirrhosis, although further validation in patients with more advanced cirrhosis and renal dysfunction is required. TRIAL REGISTRATION: ClinicalTrials.gov NCT01640964.
Asunto(s)
Enfermedades Renales/tratamiento farmacológico , Riñón/efectos de los fármacos , Cirrosis Hepática/tratamiento farmacológico , Relaxina/farmacología , Relaxina/uso terapéutico , Adolescente , Adulto , Anciano , Animales , Femenino , Tasa de Filtración Glomerular/efectos de los fármacos , Humanos , Riñón/irrigación sanguínea , Masculino , Persona de Mediana Edad , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Flujo Sanguíneo Regional/efectos de los fármacos , Escocia , Adulto JovenRESUMEN
UNLABELLED: Active myofibroblast (MF) contraction contributes significantly to the increased intrahepatic vascular resistance that is the primary cause of portal hypertension (PHT) in cirrhosis. We sought proof of concept for direct therapeutic targeting of the dynamic component of PHT and markers of MF activation using short-term administration of the peptide hormone relaxin (RLN). We defined the portal hypotensive effect in rat models of sinusoidal PHT and the expression, activity, and function of the RLN-receptor signaling axis in human liver MFs. The effects of RLN were studied after 8 and 16 weeks carbon tetrachloride intoxication, following bile duct ligation, and in tissue culture models. Hemodynamic changes were analyzed by direct cannulation, perivascular flowprobe, indocyanine green imaging, and functional magnetic resonance imaging. Serum and hepatic nitric oxide (NO) levels were determined by immunoassay. Hepatic inflammation was assessed by histology and serum markers and fibrosis by collagen proportionate area. Gene expression was analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting and hepatic stellate cell (HSC)-MF contractility by gel contraction assay. Increased expression of RLN receptor (RXFP1) was shown in HSC-MFs and fibrotic liver diseases in both rats and humans. RLN induced a selective and significant reduction in portal pressure in pathologically distinct PHT models, through augmentation of intrahepatic NO signaling and a dramatic reduction in contractile filament expression in HSC-MFs. Critical for translation, RLN did not induce systemic hypotension even in advanced cirrhosis models. Portal blood flow and hepatic oxygenation were increased by RLN in early cirrhosis. Treatment of human HSC-MFs with RLN inhibited contractility and induced an antifibrogenic phenotype in an RXFP1-dependent manner. CONCLUSION: We identified RXFP1 as a potential new therapeutic target for PHT and MF activation status.
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Hipertensión Portal/prevención & control , Cirrosis Hepática/prevención & control , Hígado/efectos de los fármacos , Miofibroblastos/efectos de los fármacos , Relaxina/farmacología , Relaxina/uso terapéutico , Actinas/metabolismo , Animales , Tetracloruro de Carbono/efectos adversos , Células Cultivadas , Desmina/metabolismo , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/metabolismo , Hemodinámica/efectos de los fármacos , Hemodinámica/fisiología , Humanos , Hipertensión Portal/inducido químicamente , Hipertensión Portal/fisiopatología , Hígado/metabolismo , Hígado/fisiopatología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/fisiopatología , Masculino , Miofibroblastos/patología , Miofibroblastos/fisiología , Óxido Nítrico/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Relaxina/metabolismoRESUMEN
Although macrophages are widely recognized to have a profibrotic role in inflammation, we have used a highly tractable CCl(4)-induced model of reversible hepatic fibrosis to identify and characterize the macrophage phenotype responsible for tissue remodeling: the hitherto elusive restorative macrophage. This CD11B(hi) F4/80(int) Ly-6C(lo) macrophage subset was most abundant in livers during maximal fibrosis resolution and represented the principle matrix metalloproteinase (MMP) -expressing subset. Depletion of this population in CD11B promoter-diphtheria toxin receptor (CD11B-DTR) transgenic mice caused a failure of scar remodeling. Adoptive transfer and in situ labeling experiments showed that these restorative macrophages derive from recruited Ly-6C(hi) monocytes, a common origin with profibrotic Ly-6C(hi) macrophages, indicative of a phenotypic switch in vivo conferring proresolution properties. Microarray profiling of the Ly-6C(lo) subset, compared with Ly-6C(hi) macrophages, showed a phenotype outside the M1/M2 classification, with increased expression of MMPs, growth factors, and phagocytosis-related genes, including Mmp9, Mmp12, insulin-like growth factor 1 (Igf1), and Glycoprotein (transmembrane) nmb (Gpnmb). Confocal microscopy confirmed the postphagocytic nature of restorative macrophages. Furthermore, the restorative macrophage phenotype was recapitulated in vitro by the phagocytosis of cellular debris with associated activation of the ERK signaling cascade. Critically, induced phagocytic behavior in vivo, through administration of liposomes, increased restorative macrophage number and accelerated fibrosis resolution, offering a therapeutic strategy to this orphan pathological process.
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Antígenos Ly/inmunología , Intoxicación por Tetracloruro de Carbono/inmunología , Regulación de la Expresión Génica/inmunología , Cirrosis Hepática/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Animales , Antígenos Ly/genética , Antígeno CD11b/genética , Antígeno CD11b/inmunología , Tetracloruro de Carbono/toxicidad , Intoxicación por Tetracloruro de Carbono/genética , Intoxicación por Tetracloruro de Carbono/patología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/inmunología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/inmunología , Macrófagos/patología , Metaloproteinasa 12 de la Matriz/genética , Metaloproteinasa 12 de la Matriz/inmunología , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/inmunología , Ratones , Ratones Transgénicos , Monocitos/patologíaRESUMEN
UNLABELLED: Elastin has been linked to maturity of liver fibrosis. To date, the regulation of elastin secretion and its degradation in liver fibrosis has not been characterized. The aim of this work was to define elastin accumulation and the role of the paradigm elastase macrophage metalloelastase (MMP-12) in its turnover during fibrosis. Liver fibrosis was induced by either intraperitoneal injections of carbon tetrachloride (CCl(4) ) for up to 12 weeks (rat and mouse) or oral administration of thioacetamide (TAA) for 1 year (mouse). Elastin synthesis, deposition, and degradation were investigated by immunohistochemistry, quantitative polymerase chain reaction (qPCR), western blotting, and casein zymography. The regulation of MMP-12 elastin degradation was defined mechanistically using CD11b-DTR and MMP-12 knockout mice. In a CCl(4) model of fibrosis in rat, elastin deposition was significantly increased only in advanced fibrosis. Tropoelastin expression increased with duration of injury. MMP-12 protein levels were only modestly changed and in coimmunoprecipitation experiments MMP-12 was bound in greater quantities to its inhibitor TIMP-1 in advanced versus early fibrosis. Immunohistochemistry and macrophage depletion experiments indicated that macrophages were the sole source of MMP-12. Exposure of CCl(4) in MMP-12(-/-) mice led to a similar degree of overall fibrosis compared to wildtype (WT) but increased perisinusoidal elastin. Conversely, oral administration of TAA caused both higher elastin accumulation and higher fibrosis in MMP-12(-/-) mice compared with WT. CONCLUSION: Elastin is regulated at the level of degradation during liver fibrosis. Macrophage-derived MMP-12 regulates elastin degradation even in progressive experimental liver fibrosis. These observations have important implications for the design of antifibrotic therapies.
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Elastina/metabolismo , Cirrosis Hepática Experimental/metabolismo , Macrófagos/enzimología , Metaloproteinasa 12 de la Matriz/fisiología , Animales , Tetracloruro de Carbono/toxicidad , Hígado/metabolismo , Metaloproteinasa 12 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Ratas , Inhibidor Tisular de Metaloproteinasa-1/genética , Tropoelastina/biosíntesisRESUMEN
Background and Aims: The prevalence of chronic liver disease in adults exceeds 30% in some countries and there is significant interest in developing tests and treatments to help control disease progression and reduce healthcare burden. Breath is a rich sampling matrix that offers non-invasive solutions suitable for early-stage detection and disease monitoring. Having previously investigated targeted analysis of a single biomarker, here we investigated a multiparametric approach to breath testing that would provide more robust and reliable results for clinical use. Methods: To identify candidate biomarkers we compared 46 breath samples from cirrhosis patients and 42 from controls. Collection and analysis used Breath Biopsy OMNI™, maximizing signal and contrast to background to provide high confidence biomarker detection based upon gas chromatography mass spectrometry (GC-MS). Blank samples were also analyzed to provide detailed information on background volatile organic compounds (VOCs) levels. Results: A set of 29 breath VOCs differed significantly between cirrhosis and controls. A classification model based on these VOCs had an area under the curve (AUC) of 0.95±0.04 in cross-validated test sets. The seven best performing VOCs were sufficient to maximize classification performance. A subset of 11 VOCs was correlated with blood metrics of liver function (bilirubin, albumin, prothrombin time) and separated patients by cirrhosis severity using principal component analysis. Conclusions: A set of seven VOCs consisting of previously reported and novel candidates show promise as a panel for liver disease detection and monitoring, showing correlation to disease severity and serum biomarkers at late stage.
RESUMEN
Liver fibrosis and its end stage, cirrhosis, represent the final common pathway of virtually all chronic liver diseases. As our understanding of the pathogenesis of liver fibrosis has progressed, it has become evident that the liver provides a useful generic model of inflammation and repair, demonstrating interplay between the epithelial, inflammatory, myofibroblast and extracellular matrix components of the mammalian wound healing response. In this review, the paradigm that liver fibrosis is a potentially reversible process-demonstrating both fibrosis (scarring) and resolution with remodeling and restitution of normal or near-normal tissue architecture-will be explored. The remarkable progress in unraveling the complexities of liver fibrosis has been due to developments in technologies including the isolation of discrete liver cell populations which have facilitated studies of their behavior in tissue culture and in vivo. More recently, animal models that mimic chronic liver diseases have been established. These models are tractable and can be applied in gene knockout and transgenic mice. This article will highlight recent studies that reveal key mechanisms mediating the regression of liver fibrosis which have derived from the use of such complementary animal and human model systems and describe how our greater understanding of this dynamic process is likely to inform the development of directed and effective anti-fibrotic approaches.
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Modelos Animales de Enfermedad , Cirrosis Hepática/fisiopatología , Regeneración Hepática/fisiología , Animales , Apoptosis/fisiología , Células Cultivadas , Progresión de la Enfermedad , Humanos , Cirrosis Hepática/patologíaRESUMEN
INTRODUCTION: Liver cirrhosis and its complication - hepatocellular carcinoma (HCC) - have been associated with increased exhaled limonene. It is currently unclear whether this increase is more strongly associated with the presence of HCC or with the severity of liver dysfunction. METHODS: We compared the exhaled breath of 40 controls, 32 cirrhotic patients, and 12 cirrhotic patients with HCC using the Breath Biopsy platform. Breath samples were analyzed by thermal desorption-gas chromatography-mass spectrometry. Limonene levels were compared between the groups and correlated to bilirubin, albumin, prothrombin time international normalized ratio, and alanine aminotransferase. RESULTS: Breath limonene concentration was significantly elevated in subjects with cirrhosis-induced HCC (M: 82.1 ng/L, interquartile range [IQR]: 16.33-199.32 ng/L) and cirrhosis (M: 32.6 ng/L, IQR: 6.55-123.07 ng/L) compared with controls (M: 6.2 ng/L, IQR: 2.62-9.57 ng/L) (P value = 0.0005 and 0.0001, respectively) with no significant difference between 2 diseased groups (P value = 0.37). Levels of exhaled limonene correlated with serum bilirubin (R = 0.25, P value = 0.0016, r = 0.51), albumin (R = 0.58, P value = 5.3e-8, r = -0.76), and international normalized ratio (R = 0.29, P value = 0.0003, r = 0.51), but not with alanine aminotransferase (R = 0.01, P value = 0.36, r = 0.19). DISCUSSION: Exhaled limonene levels are primarily affected by the presence of cirrhosis through reduced liver functional capacity, as indicated by limonene correlation with blood metrics of impaired hepatic clearance and protein synthesis capacity, without further alterations observed in subjects with HCC. This suggests that exhaled limonene is a potential non-invasive marker of liver metabolic capacity (see Visual abstract, Supplementary Digital Content 1, http://links.lww.com/CTG/A388).
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Carcinoma Hepatocelular/diagnóstico , Limoneno/análisis , Cirrosis Hepática/diagnóstico , Neoplasias Hepáticas/diagnóstico , Compuestos Orgánicos Volátiles/análisis , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/análisis , Pruebas Respiratorias , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/fisiopatología , Estudios de Casos y Controles , Estudios Transversales , Femenino , Humanos , Hígado/patología , Hígado/fisiopatología , Cirrosis Hepática/patología , Cirrosis Hepática/fisiopatología , Pruebas de Función Hepática/métodos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/fisiopatología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Índice de Severidad de la EnfermedadRESUMEN
A fatal concentration of pentobarbital found in a coroner's case where the history had not indicated use of this drug prompted a review of fatalities in Ontario from 2012 to 2015. Coroner's case files, including police and toxicology reports, were reviewed in twenty deaths, in which pentobarbital was identified as the primary cause of death. In all of the deaths (11 females, 9 males), the blood concentration of pentobarbital was greater than 10 mg/L. There were three to eight deaths per year and each was classified as suicide. In 11 cases, there was clear evidence that the drug was purchased over the internet from Mexico or China and imported into Canada. In four cases, it appears that the pentobarbital was labeled as a different, innocuous chemical to facilitate crossing the border without scrutiny. The findings underscore the value of a thorough scene investigation, including details of evidence that may be considered unrelated.
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Hipnóticos y Sedantes/envenenamiento , Pentobarbital/envenenamiento , Suicidio , Adulto , Anciano de 80 o más Años , Enfermedad Crónica/psicología , Sobredosis de Droga , Femenino , Humanos , Hipnóticos y Sedantes/sangre , Masculino , Trastornos Mentales/psicología , Persona de Mediana Edad , Ontario , Pentobarbital/sangre , Adulto JovenRESUMEN
The source of acellular specimens is not infrequently challenged, especially for toxicology specimens, but such specimens may not be amenable to conventional genetic testing to confirm the source. Our evaluation of genomic and mitochondrial DNA (mtDNA) amplicons using polymerase chain reaction (PCR) from centrifuged, filtered, or whole urine specimens demonstrated higher sensitivity of detection of mtDNA than genomic DNA and a higher detection rate for the mtDNA markers than genomic markers in all sample sets. The mitochondrial amplicons were sequenced to identify specific single nucleotide polymorphisms (SNPs). Subsequently, a real-time PCR technique using fluorescence resonance energy transfer (FRET) probes designed to hybridize to the published mtDNA sequence over known SNP locations present within the mitochondrial control region was developed. Our results demonstrate the feasibility of using a FRET-based assay of mitochondrial genotypes with acellular laboratory specimens to screen for specimen mix-ups or to confirm sources of controversial toxicology specimens.
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ADN Mitocondrial/análisis , Secuencia de Bases , ADN Mitocondrial/orina , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: There is an increased risk of death due to drug toxicity after release from incarceration. The purpose of this study was to describe the timing, rate and circumstances of drug toxicity deaths following release from incarceration. This information can be used to help design potential preventive interventions. METHODS AND FINDINGS: We reviewed coroner's files to identify deaths in adults in Ontario between 2006 and 2013 caused by drug toxicity (n = 6,978) and these records were matched with provincial correctional records to identify individuals who died within one year of being released from incarceration (n = 702). Twenty percent (n = 137) of the 702 deaths occurred within one week of release. The majority (77%, n = 538) of deaths after release involved one or more opioids. Of the deaths involving opioids, intervention by another person may have been possible in 318 cases. CONCLUSIONS: Between 2006 and 2013 in Ontario, one in ten drug toxicity deaths in adults occurred within one year of release from provincial incarceration. These findings may help to inform the implemention and assessment of interventions aimed at reducing drug toxicity deaths following release from incarceration.