Ablation of Na+/H+ exchanger‐3 prevents tissue iron loading in the Hfe mouse model of hereditary hemochromatosis

Abstract

Several common hereditary disorders are associated with toxic iron overload which leads to liver cirrhosis, cardiomyopathy, and endocrine disorders. Hereditary hemochromatosis arises from mutations in the genes coding for any of several proteins involved in iron sensing (e.g. Hfe), hepcidin production (e.g. hemojuvelin), or hepcidin action, resulting in elevated iron absorption relative to the iron need. Divalent metal‐ion transporter‐1 (DMT1) is a H+‐coupled Fe2+ transporter and the principal or only mechanism by which nonheme iron is taken up at the intestinal brush border [Shawki A et al (2015) Am J Physiol Gastrointest Liver Physiol 309, G635–G647]. DMT1 is a validated therapeutic target in treating iron overload. We have shown that Na+/H+ exchanger‐3 (NHE3) is required for adequate iron absorption, via its physiological role in generating at the intestinal brush border an acidic microclimate that energizes DMT1‐mediated H+‐coupled Fe2+ transport [Shawki A et al (2016) Am J Physiol Gastrointest Liver Physiol 311, G423–G430], so we have explored here the contribution of NHE3 to pathological iron loading. We tested the hypothesis that NHE3 activity is necessary for pathological iron loading. We examined the effect of ablating the SLC9A3 gene coding for NHE3 in the Hfe mouse model of hereditary hemochromatosis. We measured tissue iron levels, hematological and blood‐iron variables, and the hepatic expression of Hamp1 (coding for hepcidin) by using qPCR in male and female FVB/N mice, age ≍ 120 d, fed a normal diet (NIH‐07). We examined four genotypes in both sexes: wildtype mice, NHE3 knockout (NHE3−/−), Hfe knockout (hemochromatosis disease model, Hfe−/−), and double knockout (NHE3−/− | Hfe−/−) (n = 12–26 mice per group). We chose α = 0.05, and analyzed our data (mean, SD) by using multifactorial ANOVA. We found that ablation of NHE3 prevented the liver iron loading characteristic of the Hfe−/− mouse model, an effect that was independent of sex. We observed a similar effect on iron loading in the spleen. Ablation of NHE3 produced no sign of overt anemia. Hepatic expression of hepcidin was depressed in NHE3−/− and in Hfe−/− mice relative to wildtype mice, and remained low in the NHE3−/− | Hfe−/− mice, providing a plausible explanation for the observation that, despite correction of the tissue iron levels in NHE3−/− | Hfe−/− mice, iron levels remained high in the readily accessible serum iron pool (typically 0.1% of total body iron). We conclude that ablation of NHE3 prevents pathological tissue iron loading in the Hfe mouse model of hemochromatosis. Pharmacological blockade of NHE3 may offer a means of inhibiting iron absorption in hereditary hemochromatosis.