The generation of reactive oxygen species (ROS) by metal oxidants such as iron, represents a potential damage mechanism in diabetic nephropathy and altered iron homeostasis is observed in diabetic humans and animal models (Shah, 2001). If true, then the proteins responsible for iron uptake and handling could contribute to this process and their relative expression levels could be relevant in diabetic nephropathy. One such protein is the divalent metal transporter, DMT1, which we have previously shown to be differentially expressed along the nephron (Ferguson et al. 2001). Here, we report chronic down-regulation of DMT1 in the kidneys of diabetic rats.

Experiments were performed in accordance with the UK Animals (Scientific Procedures) Act and rats were killed humanely at the end of the experiments. Experiments were performed on two series of rats, Sprague-Dawley (series 1) and Wistar-Kyoto (series 2) (Charles River Laboratories, Kent). Rats were rendered diabetic with streptozotocin (STZ; Series 1, 60 mg kg^-1 I.P. in citrate buffer; Series 2, 55 mg kg^-1 I.P. in sterile saline). After 2 weeks, Series 1 rats were either killed for tissue collection or perfusion-fixed with 4 % paraformaldehyde under anaesthetic (sodium thiopentone I.P.) for immunohistochemistry. For Series 2, animals were killed after 12 weeks of diabetes either in the absence or presence of (a) insulin replacement (interscapular subcutaneous 14 % porcine insulin implant (Linplant; LinShin Canada Inc., Toronto, Canada) under halothane anaesthesia) or (b) treatment with the aldose reductase inhibitor, sorbinil (20 mg kg^-1 day^-1 P.O. for the final 5 weeks). Kidneys were collected and particulate (crude membrane) protein fractions prepared by differential ultracentrifugation. DMT1 expression was studied by semi-quantitative immunoblotting and immunofluorescence.

In Sprague-Dawley rats, renal DMT1 expression is reduced by 62 ± 9 % (mean ± S.E.M.; P < 0.01 by unpaired t test; n = 4) following 2 weeks of STZ-diabetes. These changes were confirmed by immunofluorescence, which revealed a clear reduction in DMT1 expression from the proximal tubules to the collecting ducts. To exclude the possibility that DMT1 down-regulation is short-lived, we studied the renal DMT1 content of Wistar rats following 12 weeks of STZ-diabetes. Here, renal DMT1 expression was reduced by 34 ± 4 % (P < 0.01 by ANOVA; n = 5), whereas in insulin-replaced diabetics, DMT1 expression was returned to control levels (-10 ± 7% P < 0.05 vs. untreated diabetic, n.s. vs. non-diabetic; n = 5). Treatment with sorbinil for 5 weeks failed to significantly alter renal DMT1 expression in the STZ-diabetic Wistar rats, although a trend towards control was observed (Diab, -41 ± 2% sorbinil/diabetic, -19 ± 5 %, n.s., n = 3). We conclude that expression of the iron transporter DMT1 is significantly reduced in two strains of rat with chronic diabetes. Further work will be required to determine if renal DMT1 down-regulation contributes to iron-mediated damage in diabetic nephropathy, either by enhancing it or protecting against it.

This work was supported by grants from The Wellcome Trust and National Kidney Research Fund. D.T.W. is an NKRF Career Development Fellow.