The impact of normothermic and hypothermic preservation methods on kidney lipidome – comparative study using chemical biopsy with microextraction probes Provisionally Accepted

Natalia Warmuzińska¹ Kamil Łuczykowski¹ Iga Stryjak¹ Hernando Rosales-Solano² Peter Urbanellis³ Janusz Pawliszyn² Markus Selzner³ Barbara Bojko^1*

• ¹Nicolaus Copernicus University in Toruń, Poland
• ²University of Waterloo, Canada
• ³Ajmera Transplant Centre, Toronto General Hospital, Canada

Introduction: Normothermic ex vivo kidney perfusion (NEVKP) is designed to replicate physiological conditions to improve graft outcomes. A comparison of the impact of hypothermic and normothermic preservation techniques on graft quality was performed by lipidomic profiling using solid-phase microextraction (SPME) chemical biopsy as a minimally invasive sampling approach.
Methods: Direct kidney sampling was conducted using SPME probes coated with a mixed-mode extraction phase in a porcine autotransplantation model of the renal donor after cardiac death, comparing three preservation methods: static cold storage (SCS), NEVKP, and hypothermic machine perfusion (HMP). The lipidomic analysis was done using ultra-high-performance liquid chromatography coupled with a Q-Exactive Focus Orbitrap mass spectrometer.
Results: Chemometric analysis showed that the NEVLP group was separated from SCS and HMP groups. Further in-depth analyses indicated significantly (p<0.05, VIP>1) higher levels of acylcarnitines, phosphocholines, ether-linked and longer-chain phosphoethanolamines, triacylglycerols and most lysophosphocholines and lysophosphoethanolamines in the hypothermic preservation group. The results showed that the preservation temperature has a more significant impact on the lipidomic profile of the kidney than the preservation method's mechanical characteristics.
Conclusions: Higher levels of lipids detected in the hypothermic preservation group may be related to ischemia-reperfusion injury, mitochondrial dysfunction, pro-inflammatory effect, and oxidative stress. Obtained results suggest the NEVKP method's beneficial effect on graft function and confirm that SPME chemical biopsy enables low-invasive and repeated sampling of the same tissue, allowing tracking alterations in the graft throughout the entire transplantation procedure.