Extracellular Vesicles Released During Normothermic Machine Perfusion Are Associated With Human Donor Kidney Characteristics

Abstract

Background. Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. The characterization of EVs released by kidney grafts during normothermic machine perfusion (NMP) may present a promising avenue to assess graft status before transplantation. Methods. We phenotyped and determined the concentrations of EVs in the perfusate of 8 discarded expanded-criteria donor human kidneys during 6 h of NMP. Perfusate samples were taken at 0/60/180/360 min and examined with nanoparticle tracking analysis and imaging flow cytometry (IFCM). Using IFCM, EVs were identified by their expression of common EV markers CD9, CD63, and CD81 (tetraspanins) in combination with either platelet endothelial cell adhesion molecule (CD31), pan-leukocyte protein (CD45), or carboxyfluorescein succiminidyl ester (CFSE) fluorescence. Results. Nanoparticle tracking analysis measurements revealed the release of nanoparticles <400 nm into the perfusate during NMP. With IFCM, tetraspanin protein signatures of the released nanoparticles were characterized, and the majority (~75%) of CFSE+ EVs were found to be CD81+, whereas ~16% were CD9+ and ~8% CD63+. Correlation analysis of concentrations of identified EV subsets with crude donor characteristics and NMP viability characteristics revealed significant correlations with cold ischemia time, donor age, and renal flow. Conclusions. Our findings demonstrate that discarded expanded-criteria donor kidney grafts release distinct EV subsets during NMP. Because these subsets correlate with well-established indicators of transplant outcome, EVs might represent new potential candidates for assessment of kidney graft quality.

Proteomics reveals specific biological changes induced by the normothermic machine perfusion of donor kidneys with a significant up-regulation of Latexin

“…For example, mass spectrometry was used in one recent study to show that NMP with urine recirculation preserves and revitalizes kidney metabolism, primarily by reducing the abundance of damage-associated molecular patterns (DAMPs), improving mitochondrial glucose metabolism, and minimizing biochemical alterations induced by I/R injury 9 . Furthermore, nanoparticle tracking analysis demonstrated that discarded ECD kidney grafts release distinct extracellular vesicles (EVs) during NMP that may be suitable for the assessment of kidney graft quality 10 .…”

Genetic Modulation: Future Trends Toward Graft Optimization During Machine Perfusion

“…This suggests that EVs not only hold the potential to deliver therapeutics but also serve as pretransplant biomarkers. 56 …”

Machine perfusion organ preservation: Highlights from the American Transplant Congress 2021

“…This conjugation can both simplify and enhance the use of nanomaterials to target graft endothelial cells, opening opportunities to deliver therapeutics with efficacy prior to transplant. Woud 10 and the team from Rotterdam, Netherlands, analyzed the excretion of small Extracellular Vesicles (EV) during normothermic machine perfusion of kidneys as a potential biomarker both before and after transplantation. Eight discarded human kidneys were perfused in a closed system at 37°C for 6 h. Perfusate samples were taken before and at 1, 3, 6 h. They observed a positive correlation of EV levels with donor age and kidney weight, while negative correlations were found for ischemia time.…”