Molecular pathogenesis of autosomal recessive polycystic kidney disease (ARPKD): Fibrocystin as a regulator of cell metabolism and mitochondrial function

常染色体隐性多囊肾病（ARPKD）是一种儿童早期严重的遗传性疾病，其典型特征是子宫内纤维囊性肾脏增大，导致早发终末期肾病和强制性肝脏受累，表现为先天性肝纤维化。ARPKD的分子发病机制和受影响的纤维囊蛋白（FC）的细胞功能尚不完全清楚。有研究表明，FC可能影响相同的细胞内信号级联反应，这两种蛋白主要影响更常见的常染色体显性多囊肾病（ADPKD）。ARPKD和ADPKD之间重叠的临床和遗传发现已经被描述。为了深入了解FC的分子功能，我们对各种细胞模型进行了表征，包括患者来源的细胞系和转基因细胞系，例如通过RNASeq和蛋白质组学分析以及基于质谱的FC相互作用组研究获得了细胞特征。这些实验结果表明，FC除了在细胞纤毛上的功能外，还可能参与细胞代谢的调节。细胞代谢重编程已经被各种独立的小组描述为ADPKD。在建议的项目中，我们详细研究了FC与ARPKD中肾小管细胞代谢和线粒体变化之间的潜在联系，从而分析细胞代谢过程是否可以作为ARPKD的潜在治疗靶点。通过使用基于CRISPR/ cas9的pkhd1缺陷人类细胞模型，我们将表征fc依赖性对一般细胞方面的影响。然后，我们将定义特定的代谢信号谱，并研究线粒体功能、形态和蛋白质组成。在体内方法中，我们将通过小鼠ARPKD模型和产生双敲除小鼠来描述Caveolin-1在肾脏和肝脏表型中的作用，Caveolin-1是我们之前工作中鉴定的fc相互作用蛋白的候选蛋白。总之，该项目的结果将深入了解ARPKD的发病机制，并可能作为针对最具破坏性的儿童肾脏疾病之一的首次靶向治疗策略的起点。

Autosomal recessive polycysitc kidney disease (ARPKD) is a severe genetic disorder of early childhood that is typically characterized by intrauterine enlargement of fibrocystic kidneys resulting in early-onset end stage renal disease and obligatory hepatic involvement presenting as congenital hepatic fibrosis. The molecular pathogenesis of ARPKD and the cellular function of the affected protein fibrocystin (FC) remain incompletely understood. It has been suggested that FC may influence the same intracellular signaling cascades as the two proteins mainly affected in the more common autosomal dominant polycystic kidney disease (ADPKD). Overlapping clinical and genetic findings between ARPKD and ADPKD have been described.To gain insight into the molecular function of FC, we characterized various cellular models, including patient-derived cell lines and transgenic cell lines, and e.g. obtained cellular signatures by RNASeq and proteome analyses as well as mass spectromtry-based studies of the FC interactome. Findings from these experiments suggest that FC may, in addition to its function at the cellular cilium, contribute to the regulation of cellular metabolism. Celllular metabolic reprogramming has been described for ADPKD by various independent groups.In the suggested project we examine a potential link between FC and changes of the renal-tubular cell metabolism and mitochondria in ARPKD in detail and thus analyze whether cell metabolic processes could serve as a potential therapeutic target in ARPKD. By using CRISPR/Cas9-based PKHD1-deficient human cellular models we will characterize FC-dependent effects on general cellular aspects. We will then define specific metabolic signaling profiles and study mitochondrial function, morphology and protein composition. In an in vivo approach the role of Caveolin-1, a candidate for a FC-interacting protein identified in our previous work, for the renal and hepatic phenotype will be described by using a murine ARPKD modell and generating a double knockout mouse.In summary, the results of this project will give insights into the pathogenesis of ARPKD and may serve as a starting point for first targeted therapeutic strategies for one of the most devastating pediatric renal diseases.