ANNÉE 2015 THÈSE / UNIVERSITÉ DE RENNES 1 sous le sceau de l’Université Européenne de Bretagne pour le grade de DOCTEUR DE L’UNIVERSITÉ DE RENNES 1 Mention : Biologie et Sciences de la Santé École doctorale Vie-Agro-Santé présentée par Ahmad CHARANEK Préparée dans l’Unité de Recherche INSERM UMR 991 «Foie, Métabolismes et Cancer» (Pharmacie) Thèse soutenue à Rennes The Bile Canaliculus le 10 Juin 2015 devant le jury composé de : Revisited: Pr Chantal HOUSSET Professeur des Universités Morphological And Université de Paris 6 / rapporteur Pr Marc PALLARDY Functional Professeur des Universités Université de Paris 11 / rapporteur Alterations Induced Pr Pierre BRISSOT Professeur des Universités By Cholestatic Université de Rennes 1 / examinateur Dr Richard WEAVER Drugs In HepaRG Scientific Director INSTITUT DE RECHERCHES INTERNATIONALES SERVIER / examinateur Cells Dr Christiane GUGUEN-GUILLOUZO Directeur de Recherches Emérite Inserm Université de Rennes 1 / examinateur Pr André GUILLOUZO Professeur des Universités Université de Rennes 1 / directeur de thèse 1 The test of our progress is not whether we add more to the abundance of those who have much; it is whether we provide enough for those who have little. Franklin Delano Roosevelt 2 Acknowledgment Foremost, I would like to express my sincere gratitude to my supervisor Pr. André Guillouzo, for his constant guidance, continuous encouragement, and patience along the thesis preparation. I am grateful to the trust he gave me, and the moral support he provided. I highly appreciate his confidence in my ambitious goals, and his enormous efforts to provide me with all the required facilities. I am so thankful for his priceless advices that will pave the road for my future career. I would like to thank to all jury members: Mme Chantal Housset, Mr Marc Pallardy, Mr Pierre Brissot, and Mr Richard Weaver. Thank you for giving me the honor of judging my thesis work. I won’t be able to complete this journey without the support and guidance of Mme Guillouzo, I highly acknowledge her massive assistance, fruitful discussions, directions, patience, great scientific ideas, and attention to tiny details that taught me to strive towards my goal with steady steps. Special thanks and appreciation are expressed to Pr. Ziad Abdel-Razzak. Words cannot express my gratitude and respect for you. Your help and support are unforgettable. I can’t forget your patience, insistence, and massive effort you did to help me finding a way to d thios thesis “I won’t be able to do this thesis without your favor”. You were always supportive in need. May Allah reward you for all what you did. I would to express my sincere gratitude and deep thanks for the Lebanese Association for Scientific Research (LASeR) for supporting financially a part of my thesis work. 3 I would like also to express my gratitude for all the members of INSERM U991 especially, Mme Marie-Anne Robin, Mr Bernard Fromenty for all your support and help in need. Special and deep gratitude for my lab-mates, no words can express my appreciation and love for you all: Eva, Dounia, Anais, Camille, Nicolas, Sacha, Simon, Karima, Pégah, Yasmine, Sofie, Thomas O., Thomas G., Karim, Rozenn and Sebastien. I would like to thank Mr Dominique Rainteau and his team for their generous help and measurement of bile acids in his laboratory. I greatly appreciate Rémy Le Guével for his availability, effort, and enormous time he passed in image analysis. I won’t be able to complete this journey without the support of my Lebanese friends; Fida and Elise, since I arrived to Rennes you were very supportive and encouraging, thank you for the nice moments we passed together. Houssein you are special, you are a real brother, we shared all the good and bad moments of the thesis together, I don’t know how it could be without you, and how hard to be lonely. I will never forget your spontaneous support, kindness and generous help. Thank you because you don’t know how to say “NO”. Thank you for all the time you made me laugh when I was stressed. You compensated the feelings of loneliness since you arrived. May Allah bless you and compensate you by the best. Pamela, I can’t find the words to express my gratitude for you and your family; Ellie and Marcosa. Thank you for all what you did, for the amazing days we passed together I hope that we will have again the opportunity to work together, I greatly appreciate your assistance, support and advices. I can tell you there is only 4 cyclosporine in the life “hahahha”. I can’t compensate you for all what you did but Allah will do. My special appreciation and deep gratitude are expressed to you Audrey. You are exceptional. This work would not have been possible unless you were beside me, not only for the enormous experimental work you did and enormous effort in the success of this thesis, but also for your great will of assistance, support and encouragement. Your generosity, love and care compensated the feelings of loneliness, foreignness, and weakness I have experienced during hard moments away from my family. Thank you for cheering me up and stood by me through all the times. Sure I won’t forget to thank you Matthew, you deserves special and best thanks not only for participating in the success of the thesis, but also for the amazing moments, jokes and the interesting ambience we experienced together. I wont forget how much you were attentive with me especialy when I was sick. At the end, I will not forget my family, I would like to express my profound gratitude and high appreciation to you Rawaa, Rima, Nijmi, Mohammad and Omar for your precious support, and your continuous encouragement and prays, for all the sacrifices that you have made on my behalf. This work is dedicated for you MOM and DAD, how can I compensate everything you did for me, I am indebted for you. May Allah guard and reward you. I feel so blessed with everything, thanks to almighty Allah for everything. 5 6 Abstract Cholestasis is one of the most common manifestations of drug-induced liver injury (DILI). Since up to now it is unpredictable in 40% of all cases its accurate prediction represents a major challenge. First, we validated that differentiated HepaRG human liver cells are a suitable in vitro model to study drug-induced cholestasis, by comparing localization of influx and efflux transporters and their functional activity in these cells and primary human hepatocytes. All tested influx and efflux transporters were correctly localized to canalicular (BSEP, MRP2, MDR1, and MDR3) or basolateral (NTCP, MRP3) membrane domains and were functional. In addition, the HepaRG cell line also exhibits bile acids (BAs) metabolizing enzymes and has the capacity to synthesize BAs and to further amidate these BAs with taurine and glycine as well as sulfate, at a rate similar to that of primary hepatocytes. Concentration- dependent changes were observed in total BAs disposition after treatment of HepaRG cells by the cholestatic drug cyclosporine A (CsA). Inhibition of efflux and uptake of taurocholate was evidenced as early as 15 min and 1 h respectively. These early effects were associated with deregulation of cPKC pathway and induction of endoplasmic reticulum stress that preceded generation of oxidative stress. We also showed for the first time intracellular accumulation of endogenous BAs by a cholestatic drug in vitro. In addition, our work brings evidences that motility of bile canaliculi (BC) is essential for BAs clearance where ROCK pathway and actomyosin complex are highly implicated. We provided the first demonstration that ROCK pathway and BC dynamics are major targets of cholestatic compounds. Our data should help in the development of screening methods for early prediction of drug- induced cholestatic side effects. 7 Résumé La cholestase est l’une des manifestations les plus courantes des lésions induites par des médicaments. Dans 40% des cas elle n’est pas prévisible; une meilleure prédictibilité représente donc un défi majeur. Tout d’abord, nous avons démontré que les cellules hépatiques humaines HepaRG différenciées sont un modèle approprié pour étudier la cholestase induite par les médicaments en comparant la localisation et l’activité des transporteurs d’influx et d’efflux avec les hépatocytes humains primaires. Tous les transporteurs d’efflux et d’influx testés ont été correctement localisés au niveau des membranes canaliculaire (BSEP, MRP2, MDR1 et MDR3) et basolatéral (NTCP, MRP3) et sont fonctionnels. En outre, ces cellules expriment également les enzymes qui métabolisent les acides biliaires (ABs) et ont la capacité de les synthétiser et de les conjuguer avec la taurine, la glycine et le sulfate, à un taux similaire à celui des hépatocytes primaires. Des changements ont été observés sur la répartition des ABs totaux après traitements de cellules HepaRG par un médicament cholestatique, la cyclosporine A (CsA), de manière concentration- dépendante. L’inhibition de l’efflux et de l’influx de taurocholate a été observée après 15 min et 1 h respectivement. Ces premiers effets ont été associés à la dérégulation de la voie des cPKC et l’induction d’un stress du réticulum endoplasmique puis d’un stress oxydant. Nous avons également montré pour la première fois une accumulation intracellulaire d’ABs endogènes avec un médicament cholestatique in vitro. En outre, notre travail apporte des preuves que la motilité des canalicules biliaires (BC) est indispensable à la clairance des ABs. La voie ROCK et le complexe actomyosine sont fortement impliqués. Nous avons fourni la première démonstration que la voie ROCK et les dynamiques des BC sont des cibles majeures des composés cholestatiques. Nos données devraient contribuer à l’élaboration de méthodes de screening pour la prédiction précoce des effets secondaires induits par les médicaments cholestatiques.