Comparison of Proteolytic Enzymes and Glutathione S-Transferase Levels in Non-Heart-Beating Donors' (NHBD) Kidney Perfusates

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Abstract
In order to identify biochemical markers of kidney damage prior to transplantation, we determined the levels of activity of a range of proteolytic enzymes in kidney perfusate samples from non-heart-beating donor (NHBD) cases. Urinary protease activities have been described as indices of kidney damage in renal disease; their potential as markers of tissue damage in kidneys before transplantation has not been assessed. In an attempt to identify additional/ improved biochemical markers, the present study compared the levels of total glutathione S-transferase (GST) with corresponding levels of several proteolytic enzymes in perfusate fluid from machine perfused NHBD kidneys. Proteases were selected to represent factors that may influence enzyme efflux, such as intracellular localization or molecular size. Methods: Perfusate samples were obtained over an 8-hour period from machine-preserved NHBD kidneys. Protease activities in these samples were determined by fluorometric assays and comparison made with total GST activity. Individual proteases were analysed in the transplanted and non-transplanted kidney groups (discarded on the basis of other viability parameters). Results: A correlation between protease activity and total GST was obtained for only leucyl- and pyroglutamyl aminopeptidase. Furthermore, in the transplanted group, it was possible to set nominal upper limits of activity for alanyl-arginyl- and dipeptidyl IV-aminopeptidase (AP). In the non-transplanted kidney group protease levels were increased above “normal” upper limits for the same enzyme types. By the use of alanyl AP it was possible to discriminate 75% of unsuitable kidneys discarded by the use of other criteria. Conclusion: The lack of correlation between total GST and protease activity for most of the enzymes investigated and alanyl AP levels in perfusate samples could be related to differences in cellular localisation, suggesting that assays of alanyl AP may give complimentary biochemical information relating to kidney tissue damage. Quantification of alanyl AP in machine perfusate samples may be a valuable additional independent biomarker of tissue damage.