ABSTRACT
Non-alcoholic fatty liver (NAFLD) is a clinical entity whose importance has been increasing, because of its potential progression to chronic liver disease. The alteration of bile secretory function may be a relevant factor of hepatic injury in NAFLD. Objectives: To assess basal bile secretory function and protein mass of three major hepatobiliary transporters in an experimental NAFLD model. Materials and Methods: The bile secretory function was determined by conventional techniques in Sprague-Dawley control rats fed with a choline-deficient diet (CDD) for 8 weeks. Protein mass of Ntcp, Bsep and Mrp2 was measured by western blot. Results: An impaired bile secretory function was observed in rats fed with DDC (reduction of bile flow and secretion of bile acids and organic anions). In addition, DDC fed rats showed higher levels of serum aminotransferases. Ntcp protein mass decreased in rats with DDC, while Bsep and Mrp2 did not show quantitative variations in this experimental model. Conclusions: In this experimental model of NAFLD an impaired bile secretory function was observed, determining a cholestatic pattern. The decrease in Ntcp protein mass with unaltered Bsep and Mrp2 protein mass, associated with a significant decrease in bile secretion suggests a functional impairment of these transporters in rats fed with DDC diet.
El hígado graso no alcohólico (HGNA) es una entidad clínica de importancia creciente por su potencial progresión a daño hepático crónico. La alteración de la función secretora biliar puede ser un factor relevante en el daño o lesión hepática asociada al HGNA. Objetivos: Evaluar la función secretora biliar basal y los niveles de expresión proteica de tres de los principales transportadores hepatobiliares en un modelo de HGNA experimental. Materiales y Métodos: La función secretora biliar fue determinada por técnicas convencionales en ratas Sprague-Dawley control y alimentadas con una dieta deficiente en colina (DDC) durante 8 semanas. Los niveles de expresión proteica de Ntcp, Bsep y Mrp2 fueron cuantificados por western blot. Resultados: Se observó un deterioro de la función secretora biliar en las ratas alimentadas con DDC (reducción del flujo biliar y de secreción de ácidos biliares y aniones orgánicos). Además, las ratas con DDC presentaron niveles más altos de transaminasas séricas. Los niveles de expresión proteica de Ntcp disminuyeron en las ratas con DDC, mientras que Bsep y Mrp2 no presentaron variaciones cuantitativas en este modelo experimental. Conclusiones: En este modelo de HGNA experimental se observó una función secretora biliar alterada, determinando un patrón colestásico. La disminución de los niveles de expresión proteica de Ntcp junto con la mantención de Bsep y Mrp2, asociados a una disminución significativa de la secreción biliar, sugiere un deterioro funcional de estos transportadores en ratas alimentadas con dieta DDC.
Subject(s)
Animals , Rats , Bile , Fatty Liver/physiopathology , Fatty Liver/metabolism , Cholestasis/metabolism , Choline Deficiency , Liver/pathology , Multidrug Resistance-Associated Proteins/metabolism , Rats, Sprague-Dawley , Organ Size , Organic Anion Transporters, Sodium-Dependent/metabolism , Blotting, Western , Bile Acids and Salts/metabolismABSTRACT
The recent upsurge of antimony (Sb) resistance is a major impediment to successful chemotherapy of visceral leishmaniasis (VL). Mechanisms involved in antimony resistance have demonstrated an upregulation of drug efflux pumps; however, the biological role drug efflux pumps in clinical isolates remains to be substantiated. Thus, in this study, the functionality of drug efflux pumps was measured in promastigotes and axenic amastigotes isolated from VL patients, who were either Sb-sensitive (AG83, 2001 and MC9) or resistant (NS2, 41 and GE1) using rhodamine123 as a substrate for multidrug resistant (MDR) pumps and calcein as a substrate for multidrug resistance-associated proteins (MRP) respectively; their specificity was confirmed using established blockers. Sb-resistant (Sb-R) isolates accumulated higher amounts of R123, as compared to Sb-sensitive (Sb-S) isolates. Verapamil, a MDR inhibitor failed to alter R123 accumulation, suggesting absence of classical MDR activity. In Sb-R isolates, both promastigotes and axenic amastigotes accumulated significantly lower amounts of calcein than Sb-S isolates and probenecid, an established pan MRP blocker, marginally increased calcein accumulation. Depletion of ATP dramatically increased calcein accumulation primarily in Sb-R isolates, indicating existence of a MRP-like pump, which was more active in Sb-R isolates. In conclusion, our data suggested that overfunctioning of a MRP-like pump contributed towards generation of Sb-R phenotype in L. donovani field isolates.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphate/metabolism , Animals , Antimony/pharmacology , Antiprotozoal Agents/pharmacology , Drug Resistance, Multiple , Fluoresceins/metabolism , Humans , Leishmania donovani/drug effects , Leishmania donovani/metabolism , Leishmaniasis, Visceral/drug therapy , Leishmaniasis, Visceral/parasitology , Leishmaniasis, Visceral/physiopathology , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Multidrug Resistance-Associated Proteins/metabolism , Ofloxacin/pharmacology , Probenecid/pharmacology , Protozoan Proteins/metabolism , Rhodamine 123/metabolism , Verapamil/pharmacologyABSTRACT
CONTEXT AND OBJECTIVE: Overexpression of the multidrug resistance-associated protein 1 (MRP1) gene has been linked with resistance to chemotherapy in vitro, but little is known about its clinical impact on acute leukemia patients. Our aim was to investigate the possible association between MRP1 gene expression level and clinical outcomes among Iranian leukemia patients. DESIGN AND SETTING: This was an analytical cross-sectional study on patients referred to the Hematology, Oncology and Stem Cell Research Center, Sharyatee Public Hospital, whose diagnosis was acute myelogenous leukemia (AML) or acute lymphoblastic leukemia (ALL). All molecular work was performed at NIGEB (public institution). METHODS: To correlate with prognostic markers and the clinical outcome of acute leukemia, MRP1 gene expression was assessed in 35 AML cases and 17 ALL cases, using the quantitative real-time polymerase chain reaction and comparing this to the chemotherapy response type. RESULTS: Mean expression in AML patients in complete remission (0.032 ± 0.031) was significantly lower than in relapsed cases (0.422 ± 0.297). In contrast, no significant difference in MRP1 mRNA level was observed between complete remission and relapsed ALL patients. There was a difference in MRP1 expression between patients with unfavorable and favorable cytogenetic prognosis (0.670 ± 0.074 and 0.028 ± 0.013, respectively). MRP1 expression in M5 was significantly higher (p-value = 0.001) than in other subtypes. CONCLUSIONS: The findings suggest that high MRP1 expression was associated with poor clinical outcome and was correlated with the M5 subtype and poor cytogenetic subgroups among AML patients but not among ALL patients.
CONTEXTO E OBJETIVO: A superexpressão do gene de resistência a múltiplas drogas associado à proteína 1 (MRP1) tem sido ligada à resistência à quimioterapia in vitro, porém pouco é conhecido sobre seu impacto clínico nos pacientes com leucemia aguda. Nosso objetivo foi investigar a possível associação entre a expressão do gene MRP1 e os desfechos clínicos em pacientes iranianos com leucemia. DESENHO E LOCAL: Este foi um estudo analítico transversal em pacientes encaminhados ao Centro de Pesquisa em Hematologia, Oncologia e Células Tronco do Hospital Público de Sharyatee, com diagnóstico de leucemia mielóide aguda (LMA) ou leucemia linfoblástica aguda (LLA). Todo trabalho molecular foi realizado no NIGEB (instituição pública). MÉTODOS: Para correlação de marcadores prognósticos e desfechos clínicos da leucemia aguda, a expressão do MRP1 foi avaliada em 35 casos de LMA e 17 de LLA, usando a reação da cadeia de polimerase quantitativa em tempo real, e comparando este dado ao tipo de resposta à quimioterapia. RESULTADOS: A média da expressão em pacientes com LMA em remissão completa (0,032 ± 0,031) foi significativamente menor que aquela dos casos recidivantes (0,422 ± 0,297). Por outro lado, não foram observadas diferenças significativas nos níveis de mRNA para MRP1 entre os casos de LLA com remissão completa e os casos recidivantes. Houve uma diferença na expressão de MRP1 entre pacientes com prognóstico citogenético não-favorável e favorável (0,670 ± 0,074 e 0,028 ± 0,013, respectivamente). A expressão de MRP1 em M5 foi significativamente maior (valor de p = 0,001) do que em outros subtipos. CONCLUSÕES: Os achados sugerem que a alta expressão de MRP1 se associou com o pior desfecho clínico, estando correlacionada com o subtipo M5 e os subgrupos citogenéticos menos favoráveis para os pacientes com LMA, mas não para pacientes com LLA.