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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

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.