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Nanoparticle Release by Extended Criteria Donor Kidneys During Normothermic Machine Perfusion

Woud, Wouter W., MSc1; Merino, Ana, PhD1; Hoogduijn, Martin J., PhD1; Boer, Karin, PhD1; van den Hoogen, Martijn W. F., PhD, MD1; Baan, Carla C., PhD1; Minnee, Robert C., PhD, MD2

doi: 10.1097/TP.0000000000002642
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1 Department of Internal Medicine, Section of Nephrology and Transplantation, Erasmus MC, University Medical Center Rotterdam, The Netherlands.

2 Department of Surgery, Division of Hepato-Pancreato-Biliary and Transplant Surgery, Erasmus MC, University Medical Center Rotterdam, The Netherlands.

Received 19 December 2018. Revision received 17 January 2019.

Accepted 17 January 2019.

The authors declare no funding or conflicts of interest.

W.W.W. participated in the research design, performance of the research, article drafting and is the corresponding author. A.M. participated in data acquisition and article revision. M.J.H. and K.B. participated in the article revision and research design. M.W.F.H. and C.C.B. participated in the article revision. R.C.M. performed surgery, supplied machine perfusion samples, and participated in the article revision.

Correspondence: Wouter W. Woud, MSc, Department of Internal Medicine, Section of Nephrology and Transplantation, Erasmus MC, University Medical Center Rotterdam, Room Na-524, Doctor Molewaterplein 40, 3015GD Rotterdam, the Netherlands. (w.woud@erasmusmc.nl).

The poor outcomes of transplantations with kidneys from extended criteria donors require new methods of organ preservation and assessment given the more severe ischemia/reperfusion injuries compared with standard criteria donors.1 Machine perfusion (MP), aimed at reducing ischemia/reperfusion injuries and increasing graft function, is extensively being researched and allows for the examination of the isolated kidneys ex vivo through the analysis of perfusion fluids.1,2 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 assess organ function posttransplantation.3 We postulate that the analysis of nanoparticles, including EVs, in perfusion fluid during normothermic MP may allow for the assessment of kidney quality before transplantation.

In this pilot trial, three extended criteria donor kidneys, (2 donors after circulatory death, 1 donor after brain death, comparable warm ischemia times of 15 minutes followed by 12 hours of cold ischemia, age 66/73/65, all male) were perfused at 37°C for 2 hours during which perfusate samples were taken at 30-minute intervals. Samples were centrifuged at 16.000g for 10 minutes to discard platelets and supernatant was diluted 10× in 0.22-µm filtered PBS before the 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 to 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.

FIGURE 1

FIGURE 1

In the perfusate samples, the average particle size remained unchanged (~155 ± 7.6 nm, data not shown), while ~7.75-fold increase in cumulative nanoparticle concentration was observed over time: 9.03E9 particles/mL after 120 minutes compared with 1.17E9 particles/mL after 0 minutes of perfusion (Figure 1C). Particle excretion was observed to be the highest from the donor after brain death 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 the analysis of perfusion fluid may be utilized to assess renal quality before transplantation.

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REFERENCES

1. Brat A, Pol RA, Leuvenink HGNovel preservation methods to increase the quality of older kidneys. Curr Opin Organ Transplant. 2015;20:438–443.
2. Hosgood SA, Saeb-Parsy K, Wilson C, et alProtocol of a randomised controlled, open-label trial of ex vivo normothermic perfusion versus static cold storage in donation after circulatory death renal transplantation. BMJ Open. 2017;7:e012237.
3. Morelli AEExosomes: from cell debris to potential biomarkers in transplantation. Transplantation. 2017;101:2275–2276.
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