The role of apoptotic cell clearance in the pathogenesis and treatment of autosomal dominant polycystic kidney disease.

常染色体显性多囊肾病（ADPKD）是男性肾衰竭最常见的遗传原因。其病理复杂，发病机制尚不清楚。第一种被批准用于治疗这种疾病的药物（托伐普坦）只有中等疗效，并且伴有明显的副作用。迫切需要开发更有效的治疗方法。在ADPKD中发生突变的两个基因之一PKD2/TRPP2编码一个非选择性阳离子通道。PKD2在进化上是保守的，其同源物amo存在于果蝇中。功能丧失研究表明，amo是果蝇生育、肌肉收缩和清除凋亡细胞（efferocytosis）所必需的。在人的慢性炎症条件下，effocytosis的失败是相关的。同样，多囊肾的环境中含有许多凋亡细胞和炎症细胞，巨噬细胞内流与疾病进展率密切相关。在这个项目中，我们将使用患者衍生的突变细胞和遗传可处理的模型（果蝇）的组合来验证efferocytosis缺陷有助于ADPKD疾病进展的假设。遗传定义的肾小管细胞（PKD2）已经从人类患者和小鼠突变肾脏中分离出来。将PKD2突变肾细胞和巨噬细胞与正常细胞的Efferocytosis进行比较，以确定PKD2在每种细胞类型中这一过程中的相对贡献。将使用CRISPR/Cas9产生特异性的人类PKD2突变，例如导致通道活性的丧失/获得，以检测钙的作用。其次，我们将使用果蝇巨噬细胞的efferocytosis实时成像来描述导致amo突变胚胎中异常凋亡细胞清除的精确缺陷，并使用RNAi和遗传相互作用方法确定识别、吞噬或巨噬细胞编程缺陷是否在这些缺陷的基础上。amo与钙稳态和储存操作的钙进入有关，而钙的流入是果蝇巨噬细胞jnk依赖性启动其后续行为的上游机制（Weavers et al., 2016 Cell）。第三，我们将在amo突变背景下使用PKD2的野生型或人类疾病变体进行救援实验，以确定effocytosis是否存在缺陷并可能导致疾病病理。为此，我们将产生转基因果蝇，表达PKD2的人类变体或与ADPKD患者中发现的突变相同的amo变体。精子活力和肌肉收缩力提供了amo功能的替代测定方法。然后，我们将寻求通过改变下游信号通路来改善果蝇的表型，例如钙储存的遗传操作。因此，主要目标是：1。研究凋亡细胞清除缺陷是否是人类ADPKD肾细胞的一个特征。利用果蝇a3了解amo/PKD2影响凋亡细胞清除的机制。在果蝇中进行救援实验，以了解与人类PKD2疾病变异和钙信号作用相关的发病机制，PKD2依赖性过程的破坏如何促进多囊肾病理的发展尚不清楚。然而，更好地了解PKD2突变如何改变简单生物体中的这些功能，可以为疾病如何产生提供新的见解，并刺激开发人类新疗法的新方法。

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of kidney failure in man. Its pathology is complex and its pathogenesis poorly understood. The first approved drug to treat this disease (tolvaptan) is only moderately effective and associated with significant side-effects. There is a pressing need to develop more effective treatments.One of the two genes mutated in ADPKD, PKD2/TRPP2, encodes a non-selective cation channel. PKD2 is evolutionarily conserved, with a homologue, amo, present in the fruit fly, Drosophila melanogaster. Loss-of-function studies show amo is required for fertility, muscle contraction and clearance of apoptotic cells (efferocytosis) in flies. Failures in efferocytosis are associated with chronic inflammatory conditions in man. Similarly, the milieu of polycystic kidneys contains many apoptotic and inflammatory cells, with macrophage influx closely correlated with rates of disease progression. In this project, we will test the hypothesis that defects in efferocytosis contribute to disease progression in ADPKD, using a combination of patient-derived mutant cells and a genetically-tractable model (Drosophila fruit flies).Genetically-defined kidney tubular cells (PKD2) have been isolated from human patients and mouse mutant kidneys. Efferocytosis by PKD2 mutant kidney cells and macrophages will be compared to that of normal cells to determine the relative contribution of PKD2 in this process in each cell type. Specific human PKD2 mutations, e.g. which lead to loss/gain of channel activity, will be generated using CRISPR/Cas9 to examine the role of calcium.Secondly, we will use live imaging of efferocytosis by Drosophila macrophages to characterise the precise defects that lead to aberrant apoptotic cell clearance in amo mutant embryos, determining whether defective recognition, engulfment or macrophage programming underlies these defects using RNAi and genetic interaction approaches. amo is implicated in calcium homeostasis and store-operated calcium entry, while influx of calcium is an upstream mechanism in JNK-dependent priming of Drosophila macrophages to their subsequent behaviours (Weavers et al., 2016 Cell).Thirdly, we will perform rescue experiments using wild-type or human disease variants of PKD2 in amo mutant backgrounds to establish whether efferocytosis is defective and can contribute to disease pathology. To do this we will generate transgenic flies expressing either human variants of PKD2 or amo variants with mutations equivalent to those found in ADPKD patients. Sperm motility and muscle contractility provide alternative assays of amo function. We will then seek to ameliorate Drosophila phenotypes by altering downstream signalling pathways, for example genetic manipulation of calcium storage.Therefore, the major aims are to:1. Investigate whether defective apoptotic cell clearance is a feature of human ADPKD kidney cells2. Understand mechanisms of how amo/PKD2 impacts apoptotic cell clearance using Drosophila3. Perform rescue experiments in flies to understand the pathogenesis associated with human PKD2 disease variants and role of calcium signalling How disruption of PKD2-dependent processes contributes to development of polycystic kidney pathology remains unclear. However, a better understanding of how mutations in PKD2 alter these functions in a simple organism could provide fresh insight into how disease arises and stimulate new approaches to develop new treatments in man.