Comparison of proteolytic enzymes and glutathione S-transferase levels in non-heart-beating donors' (NHBD) kidney perfusates

In order to identify biochemical markers of kidney damage prior to transpla ntation. we determined the levels of activity of a range of proteolytic enz ymes in kidney perfusate samples from non-heart-beating donor (NHBD) cases. Urinary protease activities have been described as indices of kidney damag e in renal disease; their potential as markers of tissue damage in kidneys before transplantation has not been assessed. In an attempt to identify add itional/improved biochemical markers, the present study compared the levels of total glutathione S-transferase (GST) with corresponding levels of seve ral proteolytic enzymes in perfusate fluid from machine perfused NHBD kidne ys. 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 determin ed by fluorometric assays and comparison made with total GST activity. Indi vidual proteases were analysed in the transplanted and non-transplanted kid ney 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 tran splanted group, it was possible to set nominal upper limits of activity for alanyl-arginyl- and dipeptidyl IV-aminopeptidase (AP). In the non-transpla nted kidney group protease levels were increased above "normal" upper limit s for the same enzyme types. By the use of alanyl AP it was possible to dis criminate 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 sam ples could be related to differences in cellular localisation, suggesting t hat assays of alanyl AP may give complimentary biochemical information rela ting to kidney tissue damage. Quantification of alanyl AP in machine perfus ate samples may be a valuable additional independent biomarker of tissue da mage.