The role of apoptotic cell clearance in the pathogenesis and treatment of autosomal dominant polycystic kidney disease.
Lead Research Organisation:
University of Sheffield
Department Name: Infection Immunity & Cardiovasc Disease
Abstract
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 cells
2. Understand mechanisms of how amo/PKD2 impacts apoptotic cell clearance using Drosophila
3. 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.
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 cells
2. Understand mechanisms of how amo/PKD2 impacts apoptotic cell clearance using Drosophila
3. 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.
Organisations
People |
ORCID iD |
| Albert Ong (Primary Supervisor) | |
| Elliot Brooks (Student) |