Cellular responses during kidney normothermic machine perfusion reflect graft outcomes

There are currently no molecular tools that can reliably predict post-transplant outcome of kidney grafts during normothermic machine perfusion (NMP). In this study we investigated whether the cellular responses of donor kidneys during NMP can predict post-transplant outcome via transcriptomic analyses. Snap-frozen biopsies of 28 kidneys were taken before and after 2-hour NMP. 6 kidneys were included for single nucleus RNA sequencing (snRNA-seq) and 22 were for quantitative real-time PCR (qPCR). snRNA-seq analyses revealed a total of 38,451 cells distributed in 11 distinct cell types. NMP induced an upregulation of genes for ATP production-related proteins, heat shock proteins (HSP), transporter proteins and proteins that prevent protein misfolding. These findings were confirmed by the qPCR analyses. We observed no significant gene differences between the delayed graft function (DGF) and non-DGF kidneys. However, after NMP, the expression of HSP was above average in 3 kidneys with primary non-function (PNF) and kidneys that experienced acute rejection in the first month after transplantation compared to well-functioning kidneys. Overall, this study demonstrates that 2-hour NMP impacts gene expression profiles of the majority of cell types of the kidney. It suggests that NMP may serve to predict kidney function post-transplantation by triggering differential gene expression patterns. Snap-frozen biopsies from 6 kidneys taken before and after 2-hour NMP were subjected to snRNA-seq. Nuclei were isolated by nuclei isolation buffer (Nuclei EZ lysis buffer, N3408-200ML, Sigma-Aldrich). They were then loaded into the chromium controller (PN-120223, 10x Genomics) on a Chromium Next GEM Chip G (PN-2000177, 10x Genomics) and processed following the manufacturer’s original protocol. Data analysis was processed with Seurat (v4.2.0). Biopsies from another 22 kidneys taken at the same time points were included for qPCR to confirm the snRNA-seq data. Estimated glomerular filtration rates (eGFR) until 30 days post-transplant were used to determine post-transplant function. 2 discarded kidneys were collected as well for the control of "poor functional kidneys" .