Roles for Pkd1l1 in bile duct development

项目摘要 胆道闭锁（BA）是1岁以下婴儿移植任何实体器官的主要指征， 几十年来一直未能找到病因学和病理生理学的重大发现。BA是一种新生儿肝脏疾病， 以肝内和肝外胆管的纤维炎性阻塞为特征。通过外显子组 对具有异位（偏侧性）特征的BA个体的子集（具有BA脾脏的那些）进行测序， 畸形综合征），几名参与者被确定与双等位基因损伤突变的纤毛基因 PKD 1 L1（多囊肾1型）。探索PKD 1 L1受损的机制后果 在人类的信号转导中，我们开发了肝内胆管细胞限制性Pkd 1 l1 Fl/Fl;Afp-Cre（LKO）小鼠。 最近发表的数据表明，发育中的小鼠肝脏中Pkd 1 l1的缺失导致早期胆管炎。 在成年期，胆道畸形和增强的胆管周围纤维炎症，在远端 胆管结扎后梗阻（BDL）。这些组织学特征与人类BA肝脏中观察到的特征非常相似， 提供了一个新的机会，发现特定的细胞和分子的见解BA的快速和深刻的 发病机制有两种范式必须揭示，以提供更透彻的理解， Pkd 1 l1，从而BA的分子发病机制。首先，在发育中缺乏Pkd 1 l1的后果 导致成年期胆道纤维炎症的胆道系统尚不清楚。二、Pkd 1 l1的作用机制 胆管细胞内的信号传导，当其不存在时会导致胆道病理学，仍有待阐明。 在这项提议中，首要的假设是，Pkd 1 l1是胆道正常发育所必需的 和信号。这一假设将通过以下两个目标进行检验。目标1探讨了 以及发育中的产前和出生后早期肝脏的早期胆管畸形的表征。这一目标 将利用最近开发的Pkd 111 kr/Fl（NullFl）和Pkd 111 kr/Fl;Afp-cre（NullLKO）小鼠系。目标二是 一组使用分离胆管细胞研究的体外实验，以确定Pkd 1 l1相互作用物和信号传导 结果，特别是对各种胆汁酸的反应，在分离的Pkd 1 l1 Fl/Fl和LKO胆管细胞。 综上所述，我们预期这两个目标将发现新的细胞和分子机制的胆汁， 束发育和信号传导。此外，来自这些基于Pkd 1 l1的小鼠模型的信息将 有助于提供支持性临床前证据，以解决目前缺乏有效的医学治疗， BA.

PROJECT SUMMARY Biliary atresia (BA) is the main indication to transplant any solid organ in infants less than 1 year of age and has eluded major discoveries of etiology and pathophysiology for decades. BA is a neonatal liver disease that is best characterized by fibroinflammatory obstruction of both intra- and extrahepatic bile ducts. Through exome sequencing of a subset of BA individuals with heterotaxic (laterality) features (those with the BA Splenic Malformation syndrome), several participants were identified with biallelic damaging mutations in the ciliary gene PKD1L1 (Polycystic kidney disease 1 like 1). To explore mechanistic consequences of impaired PKD1L1 signaling in humans, we developed an intrahepatic cholangiocyte-restricted Pkd1l1Fl/Fl;Afp-Cre (LKO) mouse. Recently published data indicates that the absence of Pkd1l1 in the developing mouse liver leads to early biliary dysmorphology and enhanced peribiliary fibroinflammation at adult ages, moreso in the setting of distal obstruction after bile duct ligation (BDL). These histologic features strongly mimic those seen in human BA livers, offering a novel opportunity to discover specific cellular and molecular insights into BA’s rapid and profound pathogenesis. There are two paradigms that must be uncovered to provide a more thorough understanding of Pkd1l1, and thus the molecular pathogenesis of BA. First, the consequences of absent Pkd1l1 in the developing biliary tree that leads to adulthood biliary fibroinflammation are unknown. Second, the mechanism of Pkd1l1 signaling within cholangiocytes, which when absent contributes to biliary pathology, remains to be elucidated. The overarching hypothesis in this proposal is that Pkd1l1 is required for proper biliary development and signaling. This hypothesis will be tested through the following two aims. Aim 1 explores the delineation and characterization of early bile duct dysmorphology in developing prenatal and early postnatal livers. This aim will utilize the recently developed Pkd1l1Null/Fl (NullFl) and Pkd1l1Null/Fl;Afp-cre (NullLKO) mouse lines. Aim 2 is an in vitro set of experiments with isolated cholangiocyte studies to define Pkd1l1-interactors and signaling consequences, specifically in response to various bile acids, in isolated Pkd1l1Fl/Fl and LKO cholangiocytes. Taken together, we anticipate that these two aims will discover new cellular and molecular mechanisms of biliary tract development and signaling. In addition, information stemming from these Pkd1l1-based mouse models will help provide supportive pre-clinical evidence to address the current paucity of effective medical therapeutics in BA.