We have recently demonstrated that the rat kidney reabsorbs metabolically significant amounts of iron (Wareing et al. 2000), and that the DMT1 mRNA and protein are strongly expressed in the kidney (Ferguson et al. 2001). The Belgrade rat (b) is a rodent model that exhibits anaemia due to a mutation in the DMT1 gene. To investigate whether DMT1 expression is affected in the kidneys of Belgrade animals, we performed Western analysis and immunofluorescence microscopy in the kidneys of male Wistar, heterozygous (b/+) and homozygous (b/b) Belgrade rats using an affinity-purified rabbit polyclonal antibody against rat DMT1. Animals were maintained on a diet containing 220 mg kg^-1 iron for 3 weeks. At the end of the experimental period, animals were humanely killed according to UK legislation, their kidneys removed and dissected into cortex, outer medulla and inner medulla and processed for Western analysis. For semi-quantitative immunoblotting ANOVA with a Dunnet’s post-hoc test was used and data are expressed as means ± S.E.M. Results show that DMT1 immunoreactivity is unchanged in b/+ animals (105.3 ± 12.4 %, n = 4) compared with Wistar rats, whereas it is significantly reduced in the kidneys of b/b animals (26.9 ± 6.7 %, n = 4). Immunofluorescence microscopy was performed on rat kidney cryosections from paraformaldehyde-fixed animals. Rats (n = 4) were anaesthetized with Inactin (5-ethyl-5 (1Ì-methyl-propyl)-2-thiobarbiturate) at a dose of 100-110 mg kg^-1 I.P., the left kidney was flushed with phosphate-buffered saline (PBS) and perfusion-fixed with 4 % paraformaldehyde in PBS followed by 750 mosmol kg^-1 PBS/sucrose solution. Results show that DMT1-specific immunoreactivity is present in the proximal tubule and apically and intracellularly in the distal nephron in the kidneys of Wistar rats. A similar pattern is observed in the kidneys of b/+ rats, whereas in b/b rats DMT1 staining is absent in the distal nephron. Faint intracellular staining can still be seen in S3 segments. No signal is detected when the antibody is pre-absorbed with the specific antigenic peptide. Histological examination of b/b kidneys reveals the presence of increased cellularity in the mesangium and of dilated lacunae at the cortico-medullary junction. In addition, collecting duct epithelium and stromal component of the medullary epithelium appear in cortical regions. Our results indicate that DMT1 expression is strongly reduced in the kidneys of b/b, but not in those of b/+ rats. The lack of DMT1 protein in b/b animals could contribute to the severe anaemia observed in Belgrade animals.

This work was funded by The Wellcome Trust.