Delayed hepcidin response explains the lag period in iron absorption following a stimulus to increase erythropoiesis

Abstract

Introduction: The delay of several days between an erythropoietic stimulus and the subsequent increase in intestinal iron absorption is commonly believed to represent the time required for body signals to programme the immature crypt enterocytes and for these cells to migrate to the villus. Recent data however suggest that signals from the body to alter absorption are mediated by circulating hepcidin and that this peptide exerts its effect on mature villus enterocytes.

Methods: We have examined the delay in the absorptive response following stimulated erythropoiesis using phenylhydrazine induced haemolysis and correlated this with expression of hepcidin in the liver and iron transporters in the duodenum.

Results: There was a delay of four days following haemolysis before a significant increase in iron absorption was observed. Hepatic hepcidin expression did not decrease until day 3, reaching almost undetectable levels by days 4 and 5. This coincided with the increase in duodenal expression of divalent metal transporter 1, duodenal cytochrome b, and Ireg1.

Conclusion: These results suggest that the delayed increase in iron absorption following stimulated erythropoiesis is attributable to a lag in the hepcidin response rather than crypt programming, and are consistent with a direct effect of the hepcidin pathway on mature villus enterocytes.

Figure 1

Haematological parameters in rats after phenylhydrazine (PHZ) induced haemolysis. Blood was obtained from rats at various time points following PHZ induced haemolysis, and haemoglobin concentration, haematocrit, and reticulocyte count were determined, as described in the methods. Reticulocytes are expressed as a percentage of total blood cells. Data represent the mean (SEM) of five animals. Statistical significance is shown relative to day 0: *p<0.05, **p<0.01.

Figure 2

Transferrin (Tf) species in rat serum after phenylhydrazine (PHZ) induced haemolysis. Serum was obtained from rats at various time points following PHZ induced haemolysis and analysis of the transferrin species present performed by urea gel electrophoresis and western blotting, as described in the methods. A representative gel from five replicates is shown.

Figure 3

Liver iron status in rats after phenylhydrazine (PHZ) induced haemolysis. Liver tissue was obtained from rats at various time points following PHZ induced haemolysis and hepatic iron concentration (A) was determined, as described in the methods. Liver tissue from day 0 and day 4 animals was fixed in formalin, sectioned, and subject to enhanced Perls’ stain (B), as described in the methods. A representative section demonstrating Kupffer cell (K) and hepatocyte (H) stainable iron is shown (n = 5). Data represent the mean (SEM) of five animals. Statistical significance is shown relative to day 0: **p<0.01.

Figure 4

Intestinal iron absorption in rats after phenylhydrazine (PHZ) induced haemolysis. Iron absorption was determined in rats at various time points following PHZ induced haemolysis using radioactive iron, as described in the methods. Absorption is presented as the percentage of the initial iron dose retained by the animals five days after dosing. Data represent the mean (SEM) of 4–10 animals. Statistical significance is shown relative to day 0: *p<0.05, **p<0.01.

Figure 5

Gene expression in rats after phenylhydrazine (PHZ) induced haemolysis. Duodenal enterocytes and liver tissue were isolated from rats at various time points following PHZ induced haemolysis. Total RNA was extracted and gene expression determined by ribonuclease protection assay (RPA) using 5 μg of RNA, as described in the methods. Representative RPAs are shown for each gene (A). Band intensities were quantitated by densitometry, corrected for loading using GAPDH as a control, and graphed as a proportion of GAPDH (B). Data represent the mean (SEM) of five animals. Statistical significance is shown relative to day 0: *p<0.05, **p<0.01. Dcytb, duodenal cytochrome b; DMT1, divalent metal transporter 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 6

Divalent metal transporter 1 (DMT1) protein expression in rats after phenylhydrazine (PHZ) induced haemolysis. Duodenal enterocytes were isolated from rats at various time points, as described in the methods. Protein was extracted from enterocytes and analysed by western blotting using antibodies specific to DMT1 and actin. A representative blot is shown above. Band intensities were quantitated by densitometry, corrected for loading using actin as a control and graphed as a proportion of actin. Data represent the mean (SEM) of five animals. Statistical significance is shown relative to day 0: *p<0.05, **p<0.01.

Figure 7

Ireg1 protein expression in rats after phenylhydrazine (PHZ) induced haemolysis. Duodenal enterocytes were isolated from rats at various time points, as described in the methods. Protein was extracted from enterocytes and analysed by western blotting using antibodies specific to Ireg1 and actin. A representative blot is shown (A). Band intensities were quantitated by densitometry, corrected for loading using actin as a control and graphed as a proportion of actin. Duodenal sections from day 0 and day 4 animals were analysed by immunofluorescence using antibodies specific for Ireg1, as described in the methods. Representative sections are shown indicating the apical (a) and basal (b) side of the epithelium (B). Data represent the mean (SEM) of five animals. Despite the increase in Ireg1, no statistically significant difference was observed between any treatment group and day 0 animals.