Early Alterations of Bile Canaliculi Dynamics and the Rho Kinase/Myosin Light Chain Kinase Pathway Are Characteristics of Drug-Induced Intrahepatic Cholestasis
Early Alterations of Bile Canaliculi Dynamics and the Rho Kinase/Myosin Light Chain Kinase Pathway Are Characteristics of Drug-Induced Intrahepatic Cholestasis
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DOI：
10.1124/dmd.116.071373
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发表时间：
2016-11-01
影响因子：
3.9
通讯作者：
Guillouzo, Andre
中科院分区：
文献类型：
--
作者：
Burbank, Matthew G.;Burban, Audrey;Guillouzo, Andre
肝内胆汁淤积占药物性损伤的20%-40%，其中很大一部分仍然无法预测。我们的目的是研究药物诱导的胆汁淤积的机制，并提高其早期检测使用人类HepaRG细胞和一组12胆汁淤积药物和6非胆汁淤积药物。在这项研究中，我们分析了胆小管动力学，Rho激酶（ROCK）/肌球蛋白轻链激酶（MLCK）途径的影响，牛磺胆酸盐[主要的胆盐输出泵（BSEP）底物]的外排抑制，以及主要小管和基底外侧胆汁酸转运蛋白的表达。我们证明，12胆汁淤积药物分类的基础上报告的临床结果引起的胆管动力学紊乱，其特征在于无论是扩张或收缩，和改变的ROCK/MLCK信号通路，而非胆汁淤积化合物，相比之下，没有影响。与ROCK抑制剂Y-27632 [4-（1-氨乙基）-N-（4-吡啶基）环己烷甲酰胺二盐酸盐]和MLCK激活剂钙调蛋白共治疗分别减少了胆小管收缩和扩张，证实了这些途径在药物诱导的肝内胆汁淤积中的作用。相比之下，在所有胆汁淤积药物中均未观察到牛磺胆酸盐外排和/或膜囊中过表达的人BSEP的抑制;此外，据报道发现非胆汁淤积化合物的实例抑制BSEP。处理24小时后测定主要胆汁酸转运蛋白的转录水平。BSEP、Na+-牛磺胆酸盐共转运多肽和有机阴离子转运多肽B在大多数胆汁淤积性和一些非胆汁淤积性药物中下调，而多药耐药相关蛋白的失调变化更大，可能主要反映了继发性效应。总之，我们的研究结果表明，胆汁淤积药物一致导致与ROCK/MLCK调节相关的胆小管动力学的早期改变，这些变化比单独作为药物诱导胆汁淤积的预测性非临床标志物的外排抑制测量更特异。
Intrahepatic cholestasis represents 20%-40% of drug-induced injuries from which a large proportion remains unpredictable. We aimed to investigate mechanisms underlying drug-induced cholestasis and improve its early detection using human HepaRG cells and a set of 12 cholestatic drugs and six noncholestatic drugs. In this study, we analyzed bile canaliculi dynamics, Rho kinase (ROCK)/myosin light chain kinase (MLCK) pathway implication, efflux inhibition of taurocholate [a predominant bile salt export pump (BSEP) substrate], and expression of the major canalicular and basolateral bile acid transporters. We demonstrated that 12 cholestatic drugs classified on the basis of reported clinical findings caused disturbances of both bile canaliculi dynamics, characterized by either dilatation or constriction, and alteration of the ROCK/MLCK signaling pathway, whereas noncholestatic compounds, by contrast, had no effect. Cotreatment with ROCK inhibitor Y-27632 [4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] and MLCK activator calmodulin reduced bile canaliculi constriction and dilatation, respectively, confirming the role of these pathways in drug-induced intrahepatic cholestasis. By contrast, inhibition of taurocholate efflux and/or human BSEP overexpressed in membrane vesicles was not observed with all cholestatic drugs; moreover, examples of noncholestatic compounds were reportedly found to inhibit BSEP. Transcripts levels of major bile acid transporters were determined after 24-hour treatment. BSEP, Na+-taurocholate cotransporting polypeptide, and organic anion transporting polypeptide B were downregulated with most cholestatic and some noncholestatic drugs, whereas deregulation of multidrug resistance-associated proteins was more variable, probably mainly reflecting secondary effects. Together, our results show that cholestatic drugs consistently cause an early alteration of bile canaliculi dynamics associated with modulation of ROCK/MLCK and these changes are more specific than efflux inhibition measurements alone as predictive nonclinical markers of drug-induced cholestasis.