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

凋亡细胞清除在常染色体显性多囊肾病发病机制和治疗中的作用。

• 批准号: 2106178
• 金额: --
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2106178
• 关键词: role; apoptotic; cell; clearance; pathogenesis

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.

常染色体显性遗传性多囊肾病（ADPKD）是人类最常见的肾功能衰竭的遗传原因，其病理复杂，发病机制尚不清楚。第一个批准的治疗这种疾病的药物（托伐普坦）只有中等有效性，并伴有显著的副作用。ADPKD的两个突变基因之一PKD 2/TRPP 2编码一种非选择性阳离子通道。PKD 2在进化上是保守的，其同源物amo存在于果蝇（Drosophila melanogaster）中。功能丧失研究表明，在果蝇中，amo是生育、肌肉收缩和清除凋亡细胞（红细胞增多症）所必需的。在人类中，巨噬细胞增多症的失败与慢性炎症有关。同样，多囊肾的环境中含有许多凋亡和炎症细胞，巨噬细胞的流入与疾病进展的速度密切相关。在这个项目中，我们将使用患者来源的突变细胞和遗传学上易处理的模型（果蝇）的组合来测试假设，即红细胞增多症的缺陷有助于ADPKD的疾病进展。将PKD 2突变肾细胞和巨噬细胞的胞饮作用与正常细胞的胞饮作用进行比较，以确定PKD 2在每种细胞类型中在该过程中的相对贡献。将使用CRISPR/Cas9产生特定的人类PKD 2突变，例如导致通道活性丧失/获得的突变，以检查钙的作用。其次，我们将使用果蝇巨噬细胞的细胞凋亡的实时成像来确定导致amo突变胚胎中异常凋亡细胞清除的精确缺陷，确定是否有缺陷的识别，吞噬或巨噬细胞编程是使用RNAi和遗传相互作用方法的这些缺陷的基础。amo与钙稳态和钙库操作的钙进入有关，而钙内流是果蝇巨噬细胞JNK依赖性引发其随后行为的上游机制（Weavers等，第三，我们将在amo突变体背景中使用PKD 2的野生型或人类疾病变体进行拯救实验，以确定红细胞增多症是否是有缺陷的并且可以有助于疾病病理学。为此，我们将产生表达PKD 2的人类变体或突变等同于ADPKD患者中发现的突变的amo变体的转基因果蝇。精子活动力和肌肉收缩力提供了另一种检测抗精子抗体功能的方法。然后，我们将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。研究凋亡细胞清除缺陷是否是人类ADPKD肾细胞的特征2。使用果蝇3了解amo/PKD 2如何影响凋亡细胞清除的机制。在果蝇中进行拯救实验，以了解与人类PKD 2疾病变体相关的发病机制和钙信号传导的作用PKD 2依赖性过程的破坏如何促进多囊肾病理学的发展仍不清楚。然而，更好地了解PKD 2突变如何改变简单生物体中的这些功能，可以为疾病如何发生提供新的见解，并刺激新的方法来开发人类的新治疗方法。