Increased duodenal iron uptake and transfer in a rat model of chronic hypoxia is accompanied by reduced hepcidin expression

Abstract

Background: Despite the requirement for increased iron delivery for erythropoiesis during hypoxia, there is very little information on how duodenal iron uptake and its transfer to the blood adapts to this condition.

Aims: To assess the effects of 30 days of chronic hypoxia in rats on luminal iron uptake and transfer of the metal to blood, together with gene expression of hepcidin, a proposed negative regulator of iron transport.

Methods: 59-Fe uptake by isolated duodenum and its transfer to blood by in vivo duodenal segments was measured after exposure of rats to room air or 10% oxygen for four weeks. Liver hepcidin expression was measured by real time reverse transcription-polymerase chain reaction. The effects of hypoxia on hepcidin gene expression by HepG2 cells was also determined.

Results: Hypoxia did not affect villus length but enhanced (+192.6%) luminal iron uptake by increasing the rate of uptake by all enterocytes, particularly those on the upper villus. Hypoxia promoted iron transfer to the blood but reduced mucosal iron accumulation in vivo by 66.7%. Hypoxia reduced expression of hepcidin mRNA in both rat liver and HepG2 cells.

Conclusions: Prolonged hypoxia enhances iron transport from duodenal lumen to blood but the process is unable to fully meet the iron requirement for increased erythropoiesis. Reduced secretion of hepcidin may be pivotal to the changes in iron absorption. The processes responsible for suppression of hepcidin expression are unknown but are likely to involve a direct effect of hypoxia on hepatocytes.

Figure 1

Effects of hypoxia on duodenal iron uptake. 59-Fe²⁺ uptake by isolated everted duodenum after incubation of the tissue in iron containing buffer for five minutes. Iron was present in the uptake buffer at a concentration of 0.2 mM. Data are means (SEM); n = 6 and 7 for control and hypoxia respectively. *p<0.05.

Figure 2

Autoradiography of iron uptake. Autoradiographs (upper panel) show rat duodenal villi in tissue from control (A, B) and hypoxic (C, D) rats. Intestinal sections were mounted on slides, dipped in photographic emulsion, and developed one day later. The underlying tissue was stained with Light Green. Silver grains representing iron uptake were most clearly seen using dark field photography (B, D). Light fields (A, C) showed underlying tissue histology. The lower panel shows the positional dependence of 59-Fe²⁺ uptake by villus attached enterocytes under control and hypoxic conditions. Data were obtained from 18 villi (three villi from each of six control or hypoxic rats). Values are means (SEM). *p<0.05, **p<0.001. Scale bar = 50 μm.

Figure 3

Time course of 59-Fe²⁺ appearance in blood after instillation of buffer containing 59-Fe²⁺ into closed duodenal loops under control and hypoxic conditions. Iron was present in the uptake buffer at a concentration of 0.2 mM. Data are means (SEM); n = 8 per group. *p<0.05 compared with control values.

Figure 4

Effects of hypoxia on expression of liver hepcidin mRNA. Real time polymerase chain reaction analysis of total RNA isolated from liver samples removed from control and hypoxic rats. Data were normalised to levels of actin, expressed as arbitrary units (AU). Means (SEM) of liver samples from each of three (control) and four (hypoxia) animals. *p<0.01.

Figure 5

Effects of 24 or 48 hours of hypoxia on expression of hepcidin mRNA in HepG2 cells, normalised to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), expressed as arbitrary units (AU). Means (SEM) of samples from each of three (control and 24 hour hypoxia) and four (48 hour hypoxia) cells, *p<0.02.