Abstract: FR-PO767
Development of Novel Real-Time Biosensor Kidney Organoids to Study AKI
Session Information
- Development and Organoid Models
November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Development, Stem Cells, and Regenerative Medicine
- 500 Development, Stem Cells, and Regenerative Medicine
Authors
- Przepiorski, Aneta J., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
- Espiritu, Eugenel Bermudez, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
- Davidson, Alan J., University of Auckland, Auckland, New Zealand
- Hukriede, Neil A., University of Pittsburgh, Pittsburgh, Pennsylvania, United States
Background
Kidney disease is on the rise worldwide. Persistent acute kidney injury and chronic kidney disease inevitably both lead to tubular atrophy, kidney fibrosis and eventually end-stage renal disease. The exact mechanisms are poorly understood and there are currently no treatments. Recent advances in generating human kidney organoids in vitro, have provided an invaluable tool to study kidney disease and injury, and a new tool for small molecule screening. We utilize a recently developed human kidney organoid protocol from induced pluripotent stem cells (iPSCs) to generate a new kidney biosensor to deepen our understanding of acute kidney injury and fibrotic tissue development in this model.
Methods
As a real-time readout of acute injury and to test the efficacy of new compounds we developed an early apoptosis reporter, CytochromeC-GFP iPSC line. Healthy cells in the organoids express green GFP in the mitochondria, upon injury the GFP signal loses mitochondrial localization and becomes cytoplasmic before activating the Caspase 3 apoptotic pathway. We establish optimal dosage with known nephrotoxins and a real-time response using CytochromeC-GFP organoids to validate our biosensor.
Results
Using the CyochromeC-GFP biosensor iPSC line we show that the healthy iPSCs and organoids exhibit mitochondrial CytochromeC-GFP expression as shown by co-labelling with MitoTracker Red CMXRos. Upon injury with nephrotoxins the GFP signal in the injured cells loses localization to the mitochondria and becomes cytoplasmic. Co-labelling with apoptotic Caspase 3/7 stains showed that it co-localizes with the injured cells (cytoplasmic CytochromeC-GFP expression) in the tubules.
Conclusion
Using the CyochromeC-GFP biosensor iPSC line we show that the health of organoids can be monitored in real-time, and can be used to study cytotoxic response. It provides a new way to examine kidney health in a human based model that can be utilized in examining nephrotoxic effects during drug and small molecule compound screening.
Funding
- NIDDK Support