Abstract: PO2006
Novel Podocyte Protective Compounds Identified Using Ultra-Miniaturized High-Content Screening (HCS) Assays
Session Information
- Podocyte Biology
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1204 Podocyte Biology
Authors
- Noben, Manuel, Rush University Medical Center, Chicago, Illinois, United States
- Qavi, Danish, Rush University Medical Center, Chicago, Illinois, United States
- Khanna, Shreyaa, Rush University Medical Center, Chicago, Illinois, United States
- Lee, Ha Won, Rush University Medical Center, Chicago, Illinois, United States
- Altintas, Mehmet M., Rush University Medical Center, Chicago, Illinois, United States
- Reiser, Jochen, Rush University Medical Center, Chicago, Illinois, United States
- Heynen-Genel, Susanne, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, United States
- Gupta, Vineet, Rush University Medical Center, Chicago, Illinois, United States
Background
Podocytes are specialized epithelial cells which are part of the filtration barrier in the kidney. Podocyte dysfunction is part of kidney pathology hallmarked by proteinuria. Using a high-content imaging based assay, we have shown that podocytes can be used to identify novel therapeutic compounds.
Methods
Differentiated mouse podocytes were seeded on collagen-I coated multi-well plates. After 10-14 days of differentiation, cells were exposed to puromycin aminonucleoside (PAN, podocyte injury inducing agent), with compounds from the screening library or newly identified targets, or DMSO as control, for 48 hours. After, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were labeled with cytoplasmic stain HCS CellMask Green, and actin fibers were detected by using labelled phalloidin. Cell images were taken with using Opera High-Content Screening (HCS) System. Columbus software was used to quantify morphology properties such as roundness, as well as the overall F-actin signal. We utilized commercial libraries containing >50k unique compounds to identify podocyte protective hits.
Results
Using PAN as a podocyte damaging agent, we noticed marked reduction in F-actin fiber numbers and intensity, and increased roundness in podocytes. Screening of a library of chemical compounds identified >25 hits which had favorable profiles.
Conclusion
Using our optimized podocytes high-throughput screening assay in 1536-well plates, we have identified a number of highly novel compounds. Further validation on smaller well formats reproduced these findings. In vitro and in vivo mechanistic studies provide new insights about podocyte pathways that can be therapeutically targeted.
Funding
- NIDDK Support