Abstract: TH-PO1084
Automated High Content Imaging to Identify New Therapeutics for Podocytopathies
Session Information
- Glomerular Diseases: Podocyte Biology - I
November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1204 Podocyte Biology
Authors
- Romoli, Simone, AstraZeneca, Cambridge, United Kingdom
- Zabell, Thea S., AstraZeneca, Cambridge, United Kingdom
- Pijacka, Wioletta, AstraZeneca, Cambridge, United Kingdom
- Matthews, Carl, AstraZeneca, Cambridge, United Kingdom
- Rust, Steven, AstraZeneca, Cambridge, United Kingdom
- Woollard, Kevin, AstraZeneca, Cambridge, United Kingdom
- Irving, Lorraine M., AstraZeneca, Cambridge, United Kingdom
Group or Team Name
- Bioscience Renal In-vitro Team UK
Background
Damage and eventual loss of podocytes is a hallmark of CKD. A key hurdle in the investigation of podocyte loss/damage is the lack of automated relevant cell-based screens. Existing techniques are time consuming and low throughput and cannot be used effectively in drug discovery. Here we describe a novel automated high throughput assay to evaluate and quantify podocytes in animal models of kidney disease.
Methods
Sprague Dawley rat glomeruli were isolated by differential sieving without enzymatic digestion, and plated in 384 well plates. Podocyte quantification was performed using a triple immunofluorescence detection of WT1, Nephrin and DAPI using confocal microscopy Opera QEHS. 3D glomeruli images created by 60X acquisition of 42 planes with 2 μm spacing were quantified using a customised algorithm using Columbus Analysis System that was developed to exclude artefacts, allow normalisation and ensure unbiased analysis. Tailored readouts include glomeruli number and morphology, podocyte number and nephrin expression.
Results
Podocyte identification was based on co-localization of WT1 and nephrin in a specific pattern in a 3D tilt glomerular reconstruction. Capsule cell positive for WT1 were excluded. Analysis was normalized for glomerular area by DAPI staining. We validated the assay using various doses of adriamycin (ADR) to induce podocyte loss. At low dose (50 nM), ADR decreases nephrin expression and changed its localisation, consistent with podocyte damage. At high ADR doses 500nM and 1µM, podocyte loss was evident (31.5-50%), with extensive glomerular damage at 1 μM. We then studied the effect of Ang II on podocyte damage using the same technique: preliminary data indicates that Ang II (100pM-1µM) caused significant podocyte damage (nephrin membrane reorganization) but no podocyte loss.
Conclusion
We have successfully developed a tool to allow high throughput automated assessment of podocyte health in isolated glomeruli. This automated 3D-High Content Imaging platform enables candidate target validation and drug identification.
Funding
- Commercial Support –