We have recently demonstrated that the kidney reabsorbs metabolically significant amounts of iron (Wareing et al. 2000), and that DMT1 is strongly expressed apically and intracellularly along the distal nephron and intracellularly in the proximal tubule of Wistar rats (Ferguson et al. 2001). To investigate whether dietary iron intake could affect the distribution and/or the expression of DMT1 protein in rat kidney, we evaluated the effects of chronic dietary iron depletion and supplementation on DMT1 immunoreactivity using an affinity-purified rabbit polyclonal antibody against rat DMT1. For Western analysis animals were killed humanely according to UK legislation and kidney membrane particulate prepared as described elsewhere (Ferguson et al. 2001), using ANOVA and data are expressed as means ± S.E.M. Semi-quantitative immunoblotting shows that chronic (4 weeks) low (26 mg kg^-1) vs. high (5 g kg^-1) dietary iron significantly reduced DMT1-specific immunoreactivity from 48.0 ± 10.7 to 14.4 ± 2.0 (P < 0.05, n = 4). For immunofluorescence experiments animals were anaesthetised 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 PBS and perfusion-fixed with 4 % paraformaldehyde in PBS followed by 750 mosmol kg^-1 PBS/sucrose solution, and DMT1-specific immunoreactivity was assessed in 4 µm thick rat kidney cryosections.

Results from four animals for each experimental condition show that dietary iron deprivation enhanced luminal expression of DMT1, particularly in the thick ascending limb of Henle’s loop and the distal convoluted tubule. DMT1 immunoreactivity was also considerably stronger in S3 segments of kidneys from animals on low vs. high dietary iron. No signal was detected when the antibody was pre-absorbed with the antigenic peptide and no DMT1 immunostaining was found at the basolateral membrane of any nephron segment. Histological examination reveals the presence of an increased cellularity in the mesangium and enhanced cytosolic granularity in tubular structures in kidneys from animals on a high vs. low iron diet. These results indicate that dietary iron directly modulates the expression of DMT1 protein by the kidney. Given the ability of DMT1 to transport other metals, it should therefore be possible to modulate DMT1 expression during conditions such as iron-deficient anaemia or iron overload.

This work was funded by The Wellcome Trust.