Denk Gerald U, Soroka Carol J, Takeyama Yasuaki, Chen Wen-Sheng, Schuetz John D, Boyer James L
Liver Center, Yale University School of Medicine, New Haven, CT 06520, USA.
J Hepatol. 2004 Apr;40(4):585-91. doi: 10.1016/j.jhep.2003.12.001.
BACKGROUND/AIMS: Multidrug resistance-associated protein 4 (Mrp4, ABCC4) transports cyclic nucleotides, anti-retroviral compounds, and sulfated bile acids. Mrp4 expression is increased in farnesyl/bile acid receptor knockout mice. Our aim was to investigate Mrp4 expression and function in rat liver and kidney in obstructive cholestasis.
Male Sprague-Dawley rats were subjected to bile duct ligation (BDL) or sham-surgery. Animals were sacrificed after 3, 7, and 14 days and tissues were harvested for Western blot analysis, real-time reverse transcriptase-mediated polymerase chain reaction (RT-PCR), and immunohistochemistry.
Western blot analysis revealed a progressive, more than seven-fold increase (P < 0.05) of Mrp4 expression in cholestatic livers, 14 days after BDL. In contrast, Mrp4 in 14-day BDL kidneys decreased to 26+/-4% of controls (P < 0.005). Immunohistochemistry localized Mrp4 to the basolateral hepatocyte membrane and corroborated its hepatic up-regulation after BDL. Real-time RT-PCR demonstrated no major changes of Mrp4 mRNA levels in liver and kidney after BDL. Cyclic adenosine monophosphate, an MRP4 substrate, was increased in plasma and urine, consistent with these findings.
Obstructive cholestasis in rats results in progressive up-regulation of Mrp4 protein in liver but down-regulation in kidney. The absence of corresponding changes in Mrp4 mRNA suggests posttranscriptional mechanisms as predominant regulators of Mrp4 expression in BDL rats.
背景/目的：多药耐药相关蛋白4（Mrp4，ABCC4）可转运环核苷酸、抗逆转录病毒化合物及硫酸化胆汁酸。在法尼基/胆汁酸受体敲除小鼠中，Mrp4表达增加。我们的目的是研究梗阻性胆汁淤积时大鼠肝脏和肾脏中Mrp4的表达及功能。
对雄性Sprague-Dawley大鼠进行胆管结扎（BDL）或假手术。在术后3、7和14天处死动物，采集组织用于蛋白质免疫印迹分析、实时逆转录介导的聚合酶链反应（RT-PCR）及免疫组织化学检测。
蛋白质免疫印迹分析显示，胆管结扎术后14天，胆汁淤积肝脏中Mrp4表达呈进行性增加，增幅超过7倍（P<0.05）。相比之下，胆管结扎术后14天，肾脏中Mrp4降至对照组的26±4%（P<0.005）。免疫组织化学将Mrp4定位于肝细胞基底外侧膜，并证实胆管结扎后其在肝脏中上调。实时RT-PCR显示胆管结扎后肝脏和肾脏中Mrp4 mRNA水平无明显变化。与这些发现一致，MRP4底物环磷酸腺苷在血浆和尿液中增加。
大鼠梗阻性胆汁淤积导致肝脏中Mrp4蛋白进行性上调，但在肾脏中下调。Mrp4 mRNA无相应变化表明转录后机制是胆管结扎大鼠中Mrp4表达的主要调节因素。
