Background: Kidneys donated after circulatory death (DCD) and kidneys from older donors demonstrate increased susceptibility to ischemia reperfusion injury (IRI) and immune attack after transplantation. Consequently, these kidneys face an increased likelihood of delayed graft function (DGF), an elevated risk of acute rejection and enhanced propensity for graft loss. There are currently no assessment tools that can reliably predict post-transplant outcome of donor kidneys. In this study we investigated whether the cellular responses of kidneys during normothermic machine perfusion (NMP) can predict post-transplant outcome.
Methods: 12 Snap-frozen biopsies from 6 kidneys taken before and after 2-hour NMP were subjected to single nucleus RNA sequencing (snRNA-seq). Another 44 biopsies from 22 kidneys taken at the same time points were included for real-time PCR to confirm the snRNA-seq data. Additionally, biopsies from 2 kidneys that were not suitable for transplantation due to prolonged warm ischemia, but that underwent 2h NMP were collected. Estimated glomerular filtration rates (eGFR) until 30 days post-transplant were used to determine post-transplant function. 
Results: snRNA-seq included a total 38,451 cells distributed in 11 distinct cell types. Significant effects of NMP on overall gene expression were observed in 9 out of 11 cell types. The differences were characterized by the upregulation of genes for ATP production-related proteins, transporter proteins, heat shock proteins (HSP) and proteins that prevent protein misfolding after NMP. We observed no significant gene expression differences between the DGF and non-DGF kidneys in both snRNA seq and real time PCR cohorts. However, we observed that after NMP, the expression of HSP genes was significantly increased in kidneys with primary non-function (PNF) and kidneys that experienced an acute rejection in the first month after transplantation.
Conclusions: This study demonstrates that 2-hour NMP impacts gene expression profiles of the majority of cell types of the kidney. Furthermore, it suggests that NMP may serve to predict kidney function post-transplantation by triggering differential gene expression patterns in suboptimal kidneys.