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BACKGROUND Machine cold perfusion is beneficial to the preservation of kidneys for transplantation. At the end of preservation, the perfusion solution contains many proteins. Using a proteomics approach, we searched for useful biomarkers and potential therapeutic targets in the perfusate. Our program is unique in that all transplant kidneys (even living donor kidneys, LKD) are placed on machine cold perfusion prior to transplantation. MATERIAL AND METHODS Perfusates from donation after neurological and circulatory determination of death (DNDD and DCDD respectively) and LKD were collected (n=41) and analyzed for LDH, neutrophil gelatinase-associated lipocalin (NGAL), and matrix metalloproteinase-2 (MMP-2) as markers of injury. Perfusate from each kidney was subjected to 2-dimensional gel electrophoresis, then analyzed using software to identify those spots which are significantly different between the 3 groups. Mass spectrometry was used to identify the proteins and their identity was confirmed with Western blot. RESULTS The highest levels of MMP-2, LDH, and NGAL were seen for the DCDD kidneys, followed by the DNDD kidneys and then LDK. Peroxiredoxin-2, NGAL, and alpha-1-antitrypsin were identified as significantly different between the different types of donor kidneys, and their role and possible therapeutic strategies are discussed. Collagen fragments, albumin, and immunoglobulin were also identified as possible byproducts of the injury and may be useful is assessing the degree of injury. CONCLUSIONS Comparison of the perfusates from the different types of kidneys has allowed us to identify proteins that will be useful in future research into reducing injury in transplant kidneys.