ARTICLE |
Correspondence to: Francesco Zappa, Dept. of Medical Oncology, Institute of Oncology of Southern Switzerland (IOSI), 6500 Bellinzona-TI, Switzerland. E-mail: fzappa@ticino.com
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Summary |
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NAD(P)H:quinone oxidoreductase 1 (NQO1; DT-diaphorase; DTD) is a cytosolic two-electron reductase, and compounds of the quinone family such as mitomycin C are efficiently bioactivated by this enzyme. The observation that DT-diaphorase is highly expressed in many cancerous tissues compared to normal tissues has provided us with a potentially selective target that can be exploited in the design of novel anticancer agents. Because of the relative lack of information about the cell-specific expression of DT-diaphorase, the purpose of this study was to map the distribution of this enzyme in normal human tissues. Fifteen tissue samples from normal human kidney were analyzed for expression of DT-diaphorase by immunohistochemistry (two-step indirect method). We found a specific high expression of DT-diaphorase in glomerular visceral epithelial cells (podocytes). These results suggest that a high expression of DT-diaphorase in podocytes could play a major role in the pathogenesis of renal toxicity and mitomycin C-induced hemolytic uremic syndrome, in which injury to the glomerular filtration mechanism is the primary damage, leading to a cascade of deleterious events including microangiopathic hemolytic anemia and thrombocytopenia. This observation has potential therapeutic implications because the DT-diaphorase metabolic pathway is influenced by many agents, including drugs, diet, and environmental cell factors such as pH and oxygen tension.
(J Histochem Cytochem 51:297302, 2003)
Key Words: DT-diaphorase, NQO1, kidney glomerulus drug effects, immunohistochemistry, P450-reductase
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Introduction |
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MITOMYCIN C (MMC) is an antitumor antibiotic of the quinone family, isolated in 1958 from Streptomyces caespitosus (
DT-diaphorase-rich cells are therefore more sensitive to MMC (
Although there are increasing numbers of reports of MMC-associated renal toxicity, the key events that initiate or precede the glomerular injury have not yet been identified, and the reason why the renal glomerulus is apparently the major target for MMC-induced renal toxicity is still unknown.
Here we report our findings on the immunohistochemical detection of DT-diaphorase in human kidney.
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Materials and Methods |
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Human Tissues
Archival samples of formalin-fixed, paraffin-embedded tissues were supplied by the Institute of Pathology of Southern Switzerland (Locarno, Switzerland). We analyzed 15 bioptic samples of normal human kidney taken from patients who underwent nephrectomy because of kidney carcinoma (hypernephroma). All these patients were chemotherapy-naive.
Methods
We performed an immunohistochemical analysis for detection of DTD on tissue sections using anti-DTD antibodies.
Antibodies and Reagents. Anti-DTD monoclonal antibody (IgG1)-secreting hybridomas (clones A 180 and B 771) were derived from a BALB-c mouse immunized with purified recombinant human DTD protein. All hybridoma cell lines were grown in spinner flasks in RPMI medium containing 50 U/ml penicillin, 50 µg/ml streptomycin, 1% L-glutamine, and 10% fetal bovine serum in 5% CO2 at 37C to a concentration of 106 cells/ml. Hybridoma tissue culture supernatants were prepared by centrifugation at 1800 rpm for 10 min and then stored at 80C.
Before use, supernatants were centrifuged at 14,000 rpm for 5 min. We demonstrated specificity of our proprietary antibody by preabsorbing it with antigen (human recombinant DTD) and showing that immunohistochemical staining was completely blocked. We also performed a Western analysis on tissue extracts to confirm immunohistochemical staining specificity.
Non-human reactive monoclonal mouse antibodies produced in tissue culture, subclass IgG1, were used as negative control reagent (Mouse IgG1 Negative Control Code No. X0931; DAKO, Glostrup, Denmark).
Immunohistochemical Method. Immunohistochemistry was performed on tissue sections (3 µm) cut from archival paraffin blocks. Sections were de-paraffinized in xylene and rehydrated through graded alcohol to running water, then placed in citrate buffer, pH 6.0, and microwaved for two 3-min cycles. Endogenous peroxidase activity was blocked by adding Peroxidase Blocking Agent (DAKO En Vision Kit; Carpinteria, CA). Nonspecific binding was blocked by adding 20% normal rabbit serum. Serial sections of each tissue sample were then incubated with either anti-DTD or control antibodies for 30 min at room temperature. The secondary antibody was added for 30 min (DAKOKIT, labeled Polymer HRP anti-mouse: peroxidase labelled polymer conjugated to goat anti-mouse immunoglobulins). Immunodetection was performed using a substratechromogen solution (DAKOKIT, hydrogen peroxide and 3,3'-diaminobenzidine chromogen). Slides were counterstained with hematoxylin, dehydrated through graded alcohols, mounted, and photographed.
Scoring of DTD Immunostaining.
The intensity of immunostaining of DTD (brown staining) was visually scored as 0 (negative), +1 (very weak), +2 (weak), +3 (strong), or +4 (very intense), as previously reported for human lung cancers (
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Results |
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All samples analyzed were suitable for immunohistochemical analysis of DTD expression. There was no immunostaining in control sections when nonspecific antibodies were used.
DTD expression was absent in intertubular stroma (score 0), weak in epithelial cells of proximal convoluted tubules (score +2), strong in epithelial cells of distal convoluted tubules and cortical collecting tubules (score +3), and very strong in glomerular visceral epithelial cells (podocytes) (score +4) of all samples analyzed. Mesangial cells and glomerular parietal epithelial cells did not show any DTD staining (Fig 1A1C).
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Discussion |
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We report a high expression of DT-diaphorase in human glomerular visceral epithelial cells (podocytes). This implies that DTD-directed compounds, such as MMC, might be selectively bioactivated in podocytes and might thus explain the histological feature of glomerular injury that is central to the pathogenesis of MMC-induced kidney toxicity and hemolytic uremic syndrome. Podocytes are highly differentiated and quiescent cells that play a crucial role in the physiology and pathology of the kidney glomerulus. These cells, which envelop the glomerular capillaries, are an important component of the glomerular filter. The glomerular filtrate must traverse capillary endothelium, basement membrane and the podocyte layer before reaching Bowman's space. Podocytes also produce vascular endothelial growth factor (VEGF), which regulates glomerular endothelial permeability (
If the high expression of DT-diaphorase in these cells is the key element in the pathogenesis of MMC-induced toxicity, other questions remain unanswered. Why is the risk for development of C-HUS after treatment with MMC between 4% and 15% and not higher? What is it that protects more than 85% of patients against this potentially lethal side effect? We propose the following mechanisms that might explain this individual susceptibility to MMC-related renal toxicity. (a) DT-diaphorase can be induced by a wide variety of compounds, including many dietary substances (
We postulate that genetic, dietary, and environmental cell factors may be responsible for the individualism of susceptibility to DTD-directed anticancer agents. We also propose that MMC-induced hemo-lytic uremic syndrome and kidney toxicity could be related to the point mutation in the NQO1 gene, encoding an inactive protein. This report represents an attempt to explain the selective toxicity of quinones on kidney glomeruli, and proposes exciting new fields for further research.
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Acknowledgments |
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Francesco Zappa is supported by an ESMO (European Society for Medical Oncology) grant.
Anti-DTD antibodies were a generous gift from Prof David Ross (Department of Pharmaceutical Sciences, School of Pharmacy and Cancer Center, University of Colorado Health Sciences Center; Denver, Colorado).
Received for publication February 27, 2002; accepted October 9, 2002.
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