This study has explored the possibility of employing a new stain (CD 10 antibody) and a test of tissue viability (formazan based colorimetry) to study and quantifying renal ischaemia.
CD 10 (also called neutral peptidase, neprilysin or CALLA), is a 100 KD cell surface metalloproteinase. It inactivates a variety of biologically active peptides thus limiting the response of cells following stimulation by peptide hormones. It was originally identified as a common acute lymphoblastic leukaemia antigen (CALLA) and initially thought to be tumour specific. However, it is now known to be present in other haematological malignancies. The utility of the CD 10 monoclonal antibody in the diagnosis of these malignancies is now well established . Recent studies have shown that CD10 is expressed on the surface of a wide variety of normal and neoplastic cells. In particular, there is high expression on the brush border of normal renal tubules and glomerular capillary walls (McIntosh et al., 1999) and on the cytoplasmic cell membranes of human renal cell carcinoma [6, 7].
Renal ischaemia disrupts the cytoskeletal architecture resulting in relocation of apical and basolateral membrane-specific proteins such as ankyrin, fodrin and Na+ K+ -ATPase. In particular, the loss of polarity and blebbing of the brush border can be demonstrated after as little as 5 minutes ATP depletion . Since the CD 10 monoclonal antibody localises to the brush border, we felt that it might be useful to study its distribution in renal ischaemia. This monoclonal antibody had already been optimised for routine diagnostic use in our histopathology department for lymphoma typing.
The MTT reduction assay relies on the reduction of this compound by dehydrogenase enzymes, principally NADP and NADPH, in the mitochondria of viable cells and tissue. This indirectly measures the integrity of glucose metabolism through glycolysis and the Krebs cycle and has been shown to be a useful measure of tissue viability .
In this study, cell injury was accompanied by a redistribution of CD10 antibody into the lumen and the cell cytoplasm, and a reduction in formazan formation from the biopsy cores. There was a significant correlation between scores of ischemic damage following routine histology, biochemical scores of viability and CD 10 antibody staining. The use of biochemical tests and CD 10 antibody staining offers the possibility of complementing routine histology in the diagnosis of acute ischaemic renal damage.
Formazan based colorimetry has been used by one of the authors for several years to assess damage to parasitic helminths following trials of chemotherapeutic agents . Furthermore, prior to these studies, we had demonstrated that formazan production from isolated kidney tissue is reduced in a time and temperature dependent fashion ; in keeping with predictions from other experimental work . We therefore decided to use this assay to confirm the proof of principle in this pilot study. Nevertheless, it remains important to carry out further studies comparing formazan colorimetry with kidney function assessed by glomerular filtration rate. Additionally, there are many other biochemical assays (radioactive glucose uptake, leucine uptake, CO2 evolution, adenine uptake and leakage and lactate output) which have been employed in other scientific disciplines to assess the viability of cells or microbes [2, 3]. In future studies, it will be essential to compare predictions offered by these different techniques to whole organ function.
Alternate methods of assessing kidney viability prior to transplantation are being researched including the use of flow characteristics combined with enzyme leakage from machine perfused kidneys [10, 11]. The tests we have described may also be helpful in predicting the suitability of borderline kidneys particularly from non-heart beating donors prior to renal transplantation, compared to current methods mainly involving visual inspection.
CD10 antibody offers the possibility of complementing routine histology in the diagnosis of acute ischaemic renal damage, although we would see it as being probably more relevant in research applications. CD10 stains the tubular brush border more intensely than PAS and might lend itself to quantification by image analysis, either in immunoperoxidase or fluorescent preparations. The value of redistribution of other relevant proteins such as ankyrin and fodrin might be explored in the future.