The growing disparity between the demand for transplants and the available donor supply, coupled with an aging donor population and increasing prevalence of chronic diseases, highlights the urgent need for the development of platforms enabling reconditioning, repair, and regeneration of deceased donor organs. This necessitates the ability to preserve metabolically active kidneys ex vivo for days. However, current kidney normothermic machine perfusion (NMP) approaches allow metabolic preservation only for hours. Here we show that human kidneys discarded for transplantation can be preserved in a metabolically active state up to 4 days when perfused with a cell-free perfusate supplemented with TCA cycle intermediates at subnormothermia (25 °C). Using spatially resolved isotope tracing we demonstrate preserved metabolic fluxes in the kidney microenvironment up to Day 4 of perfusion. Beyond Day 4, significant changes were observed in renal cell populations through spatial lipidomics, and increases in injury markers such as LDH, NGAL and oxidized lipids. Finally, we demonstrate that perfused kidneys maintain functional parameters up to Day 4. Collectively, these findings provide evidence that this approach enables metabolic and functional preservation of human kidneys over multiple days, establishing a solid foundation for future clinical investigations.

Capillary electrophoresis-mass spectrometry (CE-MS) using a sheathless porous tip interface emerged as an attractive tool in metabolomics thanks to its numerous advantages. One of the main advantages compared to the classical co-axial sheath liquid interface is the increased sensitivity, while maintaining the inherent properties of CE, such as a high separation efficiency and low sample consumption. Specially, the ability to perform nanoliter-based injections from only a few microliters of material in the sample vial makes sheathless CE-MS a well-suited and unique approach for highly sensitive metabolic profiling of limited sample amounts. Therefore, in this work, we demonstrate the utility of sheathless CE-MS for metabolic profiling of biomass-restricted samples, namely for 20 µm-thick tissue sections of kidney from a mouse model of polycystic kidney disease (PKD). The extraction method was designed in such a way to keep a minimum sample-volume in the injection vial, thereby still allowing multiple nanoliter injections for repeatability studies. The developed strategy enabled to differentiate between different stages of PKD and as well changes in a variety of different metabolites could be annotated over experimental groups. These metabolites include carnitine, glutamine, creatine, betaine and creatinine. Overall, this study shows the utility of sheathless CE-MS for biomass-limited metabolomics studies.