Abstract: TH-OR71
Live Imaging Coupled with Image-Based Machine Learning Uncovers Potential Drivers and Therapeutic Targets in a Human Model of Ischaemic Reperfusion Injury
Session Information
- Mechanisms and Single-Cell Transcriptional Profiles in Transplant Rejection and Ischemia Reperfusion Injury
November 02, 2023 | Location: Room 115, Pennsylvania Convention Center
Abstract Time: 04:30 PM - 04:39 PM
Category: Transplantation
- 2101 Transplantation: Basic
Authors
- Cusack, Carmen M., University College London, London, United Kingdom
- Horsley, Harry, University College London, London, United Kingdom
- Salama, Alan D., University College London, London, United Kingdom
- Klootwijk, Enriko, University College London, London, United Kingdom
Background
During renal transplantation, kidneys are subjected to periods of hypoxia accompanied with nutrient deprivation, followed by reperfusion, stimulating the infiltration of immune cells into the kidney. There is growing interest in novel preservation methods to improve graft outcomes.
Methods
Primary proximal tubular epithelial cells (PTCs) were exposed to hypoxia (1% O2) for 72 hours in Hanks Balanced Salt Solution (HBSS) to mimic nutrient deprivation, with or without foetal bovine serum (FBS). PTCs were re-oxygenated (21% O2) in complete Dulbecco’s Modified Eagle Medium (DMEM). PTCs were imaged live using deconvolution laser-scanning confocal microscopy. A supervised machine learning (ML) random forest pixel classifier was trained to extract cellular morphometrics and injury data.
Results
Nutrient deprivation was associated with greater cell death and injury independent of hypoxia and is partially ameliorated by FBS supplementation (P≤.001). Furthermore, distinct cellular morphological differences were observed between conditions, indicating specific patterns of cellular injury and death.
Conclusion
Nutrient Deprivation is associated with the greatest degree of renal tubular injury, regardless of oxygen concentration. FBS may help to reduce tubular injury in transplanted organs. Further work is required to define the protective properties exhibited by FBS. Live imaging and ML are promising modalities for exploring cell death and potential therapeutics in ischemic reperfusion injury.
Figure 1: A) Nutrient deprived PTCs exposed to normoxia with no FBS (72h) and reoxygenated with complete DMEM (24h) B) ML mask generated from (A), C) Cell death ratios of PTCs +/- FBS generated from ML pipeline.