In vivo Pathway Discovery in Autosomal Dominant Polycystic Kidney Disease

摘要常染色体显性遗传性多囊肾病(ADPKD)是最常见的单基因遗传病之一 疾病，影响全球1：1000人。它的特点是巨大的充满液体的肾囊重塑， 压迫和破坏周围的正常组织，并逐渐降低肾功能，导致 大约50%的患者到了60岁时就会出现肾脏疾病。大多数ADPKD是由基因突变引起的 两个基因，即编码多囊蛋白-1蛋白(PC1)的PKD1和编码多囊蛋白-2的PKD2 蛋白质(PC2)。PC1和PC2相互作用，并被认为在纤毛信号中发挥作用。它是 人们普遍认为纤毛是推动ADPKD发病的中心成分。 尽管它们与纤毛中功能性PC复合体的机制联系尚不清楚，但许多信号 囊中的通路被扰乱了。在过去的几年里，与疾病相关的途径的清单已经增长到 新证据表明，代谢是一种新的途径，在ADPKD中受到深刻影响，可能 两者都参与了疾病的发病机制，并成为治疗发展的靶点。在它还在继续的时候 确定新发现的以ADPKD为特征的代谢紊乱是否是直接驱动因素 对于包囊的形成，很明显，细胞的许多和不同的相互交织的代谢回路的性质和活动 在确定其投资所需的能源以参与 包囊生长所需的增殖和活跃的溶质和液体运输。这项提案的主要目标是 是向研究界提供新的工具和数据集，这些工具和数据集将大大加强以下努力 探索和利用ADPKD的代谢变化特征。我们将制作一款独特的设计和 基于新的细胞特异性转录和线粒体体内模型的严格精选资源 PC蛋白丢失后最早阶段产生的蛋白质组和线粒体代谢效应 而伴随的纤毛丧失和PC蛋白重新激活的影响进一步告知这一点。本节目 将使用成人可诱导的条件性PC基因敲除小鼠模型，并将采用允许 有条件分离核糖体(TRAP)和有条件分离线粒体，从而使细胞类型- 特定的转录组和线粒体蛋白质组和代谢组学研究。体内代谢的最新进展 通量研究将应用于这些新的遗传小鼠模型的肾皮质。这些研究的结果 分析将结合在一起，以产生健壮的生物数据集，这些数据集将通过应用 必要的信息学机制，以便将这些数据传播给更广泛的研究界 近乎实时。关键的是，我们的活体研究旨在发现肾脏之后发生的最早的变化 肾小管失去多囊蛋白的表达--在囊形成之前的时间点。研究小组带来了 在ADPKD动物模型、PC信号和生物学以及体内的广泛和互补的专业知识 代谢研究加上强大的生物统计学和生物信息学支持，以产生拟议的资源。

Abstract Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common monogenic diseases, affecting >1:1000 individuals worldwide. It is characterized by large fluid-filled renal cysts that remodel, compress and destroy surrounding normal tissue, and that progressively reduce kidney function, leading to end stage renal disease in about 50% of patients by the sixth decade of life. Most ADPKD results from mutations in two genes, PKD1, which encodes the polycystin-1 protein (PC1), and PKD2, which encodes the polycystin-2 protein (PC2). PC1 and PC2 interact with one another and are thought to play a role in cilia signaling. It is generally accepted that the cilium is a central component in the pathways that drive ADPKD pathogenesis. Although their mechanistic connection to the functional PC complex in cilia is unclear, numerous signaling pathways are perturbed in cysts. In the past few years the list of disease-related pathways has grown through new evidence that implicates metabolism as a novel pathway that is profoundly affected in ADPKD and that may both participate in disease pathogenesis and serve as a target for therapeutic development. While it remains to be established whether the newly-identified metabolic derangements that characterize ADPKD are direct drivers of cyst formation, it is clear that the nature and activities of a cell's many and varied intertwined metabolic circuits plays a central role in determining its capacity to invest the energy required in order to participate in the proliferation and active solute and fluid transport that are required for cyst growth. The main goal of this proposal is to provide the research community with novel tools and data sets that will substantially enhance efforts to explore and exploit the metabolic changes that characterize ADPKD. We will produce a uniquely designed and rigorously curated resource based upon novel in vivo models of the cell specific transcriptomic, mitochondrial proteomic and mitochondrial metabolic effects that result from the earliest stages after loss of the PC proteins and that are further informed by the effects of concomitant cilia loss and PC protein reactivation. This program will make use of adult inducible conditional PC knockout mouse models and will employ strategies that will permit conditional isolation of ribosomes (TRAP) and conditional isolation of mitochondria, thus enabling cell-type- specific transcriptomic and mitochondrial proteomic and metabolomic studies. State of the art in vivo metabolic flux studies will be applied to the kidney cortices of these novel genetic mouse models. The results of these analyses will be combined to produce robust biological data sets that will be assembled through application of the requisite informatics mechanisms in order to disseminate these data to the broader research community in near real time. Critically, our in vivo studies are designed to discover the earliest changes that occur after kidney tubules lose polycystin protein expression—at time points well before cysts form. The research team brings together extensive and complementary expertise in ADPKD animal models, PC signaling and biology and in vivo metabolic studies coupled with strong biostatistical and bioinformatics support to produce the proposed resource.