Chapter 2 24 The poor outcomes of transplantations with kidneys from extended criteria donors (ECD) requires new methods of organ preservation and assessment given the more severe ischemia/reperfusion injuries (IRI) compared to standard criteria donors1. Machine perfusion (MP), aimed at reducing IRI and increasing graft function, is extensively being researched and allows for the examination of the isolated kidneys ex vivo through analysis of perfusion fluids1,2. Donor-derived Extracellular Vesicles (EVs), which may reflect the conditional state of their tissue of origin, are known to be excreted in vivo in blood/urine and as such have been used to asses organ function post transplantation3. We postulate that analysis of nanoparticles, including EVs, in perfusion fluid during normothermic MP may allow for the assessment of kidney quality prior to transplantation. In this pilot trial, three ECD kidneys, (2 donors after cardiac death (DCD), 1 donor after brain death (DBD), comparable warm ischemia times of 15 minutes followed by 12 hours of cold ischemia, age 66/73/65, all male) were perfused at 37 oC for 2 hours during which perfusate samples were taken at 30 minutes intervals. Samples were centrifuged at 16.000x g for 10 minutes to discard platelets and supernatant was diluted 10x in 0.22 µm filtered PBS prior to analysis by Nanoparticle Tracking Analysis (NTA) (Figure 1A) to determine nanoparticle size and concentration (Figure 1B). Samples were measured by the Malvern Panalytical NanoSight NS300 and analyzed with NTA software version 3.2.16. In brief, NTA tracks the Brownian motion of individual nanoparticles in suspension on a frame-by-frame basis and correlates this movement with particle size through the Stokes-Einstein equation. Per sample, 10 videos of 15 seconds with 20-60 particles in the field of focus were recorded with camera level 11 and analyzed with detection threshold 5. This threshold was found to eliminate most of the protein background in our analysis and allowed us to focus on more complex particles such as EVs. In the perfusate samples the average particle size remained unchanged (~155 ± 7.6 nm, data not shown), while an ~7.75-fold increase in cumulative nanoparticle concentration was observed over time: 9.03E9 particles/mL after 120 minutes compared to 1.17E9 particles/mL after 0 minutes of perfusion (Figure 1C). Particle excretion was observed to be highest from the DBD kidney during the entire normothermic MP procedure. Whether this increased nanoparticle release reflects better kidney function requires further research; the released nanoparticles contain kidney-derived EVs which may be indicative for renal quality. These preliminary results indicate that analysis of perfusion fluid may be utilized to assess renal quality prior to transplantation.
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