Abstract: PO2504
Leveraging High-Content Imaging Platforms for Drug Discovery
Session Information
- CKD: Metabolism, Epigenetics, and Signaling
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 2103 CKD (Non-Dialysis): Mechanisms
Authors
- Pan, Xiang, Goldfinch Bio Inc, Cambridge, Massachusetts, United States
- Norman, Timothy A., Goldfinch Bio Inc, Cambridge, Massachusetts, United States
- Fanelli, Alyssa, Goldfinch Bio Inc, Cambridge, Massachusetts, United States
- Gallagher, Justin Anthony, Goldfinch Bio Inc, Cambridge, Massachusetts, United States
- Kyrychenko, Sergii, Goldfinch Bio Inc, Cambridge, Massachusetts, United States
- Gallagher, Rachel, Goldfinch Bio Inc, Cambridge, Massachusetts, United States
- Gustafson, Thomas A., Goldfinch Bio Inc, Cambridge, Massachusetts, United States
- Lalioti, Maria, Goldfinch Bio Inc, Cambridge, Massachusetts, United States
Background
Disruption of the structure and function of the glomerular filtration barrier leading to proteinuria is a hallmark of several podocytopathies. Efficacy in translatable in vitro models is a critical first step to develop new therapies. For example, free fatty acids such as palmitic acid and protamine sulfate are well characterized in-vitro models to model DN and FSGS. However, these models are low throughput, making them unsuitable for target and compound screening.
Methods
We used immortalized murine podocytes and adapted the readouts of in vitro assays to a high content imaging /screening (HCS) platform. Readouts in response to Palmitic Acid (PA) included: apoptosis and cell viability by annexinV and propidium iodide staining or MTT; mitochondrial membrane potential by JC-1 and Mitotracker Deep Red; and mitochondrial and cytosolic reactive oxygen species by MitoSOX and DCF. Actin cytoskeleton dynamics were assessed by quantification of actin aggregation (Phalloidin) and synaptopodin reduction in response to Protamine Sulfate (PS). Fluorescent staining followed by high content imaging and custom image analysis were performed on the BioTek-Cytation 5 cell imaging reader.
Results
Podocyte apoptosis and cell death was comparable to the readout by FACS, but capacity was increased by at least 4x. We observed a dose-dependent, incremental change in mitochondrial ROS and membrane depolarization. Mitotracker Deep Red was less sensitive than other assays for PA injury.
PS treatment resulted in loss of Synaptopodin and increase of Phalloidin aggregation, and is usually assessed by confocal microscopy, which is difficult to quantify. We developed an automated imaging pipeline to quantify 9 fields per well of a 96-well plate. We showed that there was a dose- and time-dependent actin aggregation that was partially rescued by cyclosporin A, a known positive control.
Conclusion
We established a reliable and semi-automated high-content imaging platform, which will facilitate a better mechanistic understanding of podocyte injury, as well as drug discovery, including target validation and compound screening in podocytes.
Funding
- Commercial Support – Goldfinch Bio