Iron homeostasis and its disruption in mouse lung in iron deficiency and overload.

NEW FINDINGS: What is the central question of this study? The aim was to explore the role and hitherto unclear mechanisms of action of iron proteins in protecting the lung against the harmful effects of iron accumulation and the ability of pulmonary cells to mobilize iron in iron deficiency. What is the main finding and its importance? We show that pulmonary hepcidin appears not to modify cellular iron mobilization in the lung. We propose pathways for supplying iron to the lung in iron deficiency and for protecting the lung against iron excess in iron overload, mediated by the co-ordinated action of iron proteins, such as divalent metal transporter 1, ZRT-IRE-like-protein 14, transferrin receptor, ferritin, haemochromatosis-associated protein and ferroportin. Iron dyshomeostasis is associated with several forms of chronic lung disease, but its mechanisms of action remain to be elucidated. The aim of the present study was to determine the role of the lung in whole-animal models with iron deficiency and iron overload, studying the divalent metal transporter 1 (DMT1), ZRT-IRE-like protein 14 (ZIP14), transferrin receptor (TfR), haemochromatosis-associated protein (HFE), hepcidin, ferritin and ferroportin (FPN) expression. In each model, adult CF1 mice were divided into the following groups (six mice per group): (i) iron-overload model, iron saccharate i.p. and control group (iron adequate), 0.9% NaCl i.p.; and (ii) iron-deficiency model, induced by repeated bleeding, and control group (sham operated). Proteins were assessed by immunohistochemistry and Western blot. In control mice, DMT1 was localized in the cytoplasm of airway cells, and in iron deficiency and overload it was in the apical membrane. Divalent metal transporter 1 and TfR increased in iron deficiency, without changes in iron overload. ZRT-IRE-like protein 14 decreased in airway cells in iron deficiency and increased in iron overload. In iron deficiency, HFE and FPN were immunolocalized close to the apical membrane. Ferroportin increased in iron overload. Prohepcidin was present in control groups, with no changes in iron deficiency and iron overload. In iron overload, ferritin showed intracytoplasmic localization close to the apical membrane of airway cells and intense immunostaining in macrophage-like cells. The results show that pulmonary hepcidin does not appear to modify cellular iron mobilization in the lung. We propose the following two novel pathways in the lung: (i) for supplying iron in iron deficiency, mediated principally by DMT1 and TfR and regulated by the action of FPN and HFE; and (ii) for iron detoxification in order to protect the lung against iron overload, facilitated by the action of DMT1, ZIP14, FPN and ferritin.

Physiological focus on the erythropoietin-hepcidin-ferroportin axis.

To analyze the interconnection between erythropoiesis and iron metabolism, one of the issues raised in this study was to know iron bioavailability under physiopathological conditions. Our aim was to understand the functional axis response composed of erythropoietin (Epo)-hepcidin-ferroportin (FPN), when 2 dysfunctional states coexist, using an animal model of iron overload followed by hypoxia. FPN and prohepcidin were assessed by immunohistochemistry using rabbit anti-mouse FPN polyclonal and prohepcidin monoclonal antibodies. Goat-labeled polymer - horseradish peroxidase anti-rabbit EnVision + System (DAB) was used as the secondary antibody. Epo levels were measured by ELISA. Tissue iron was studied by Prussian blue iron staining. Erythropoietic response was assessed using conventional hematological tests. Iron overload increased prohepcidin that remained high in hypoxia, coexisting with high levels of Epo in hypoxia, with or without iron overload. In hypoxia, FPN was clearly evident in reticuloendothelial macrophages, more than in hypoxia with iron overload. Interestingly, duodenal FPN was clearly identified on the basolateral membrane in hypoxia, with or without iron overload. Our data indicate that 2 signals could induce the cell-specific response as follows: (i) iron signal, induced prohepcidin, which reduced reticuloendothelial FPN and reduced iron availability; and (ii) hypoxia signal, stimulated Epo, which affected iron absorption by stabilizing duodenal FPN and allowed iron supply to erythropoiesis independently of store size.

Immunolocalization of ferroportin in healthy and anemic mice.

Ferroportin (FPN), the only iron exporter identified to date, participates in iron release from enterocytes and macrophages, regulating its absorption and recycling. We used a murine model of experimental hemolytic anemia to study adaptive changes in the localization of FPN in duodenum, liver, and spleen. FPN was assessed by IHC in healthy and anemic mice using rabbit anti-mouse FPN polyclonal antibodies. Goat-labeled polymer-horseradish peroxidase anti-rabbit Envision+System (DAB) was used as secondary antibody. Tissue iron was studied by Prussian blue iron staining. Anemia evolution and erythropoietic recovery was assessed using conventional hematological tests. Healthy mice showed mainly supranuclear expression of FPN in enterocytes and a weak basolateral expression, whereas in anemic mice, the expression was detected mainly at the basolateral membrane (days 4 and 5). Red pulp macrophages of healthy mice showed FPN-hemosiderin colocalization. In the liver of healthy mice, FPN was mainly cytoplasmic, whereas in anemic mice, it was redistributed to the cell membrane. Our findings clearly show that anemia induces adaptive changes in FPN expression, contributing to anemia restoration by increasing available iron. FPN expression in the membrane is the main pathway of iron release. Our data indicate that iron homeostasis in vivo is maintained through the coordinated expression of this iron exporter in both intestinal and phagocytic cells.

Role of the kidney in iron homeostasis: renal expression of Prohepcidin, Ferroportin, and DMT1 in anemic mice.

It is known that renal tissue plays a role in normal iron homeostasis. The current study examines kidney function in iron metabolism under hemolytic anemia studying renal expression of Prohepcidin, Ferroportin (MTP1), and divalent metal transporter 1 (DMT1). The relationship between these proteins and iron pigments was also investigated. Immunohistochemical procedures to study renal expression of Prohepcidin, MTP1, and DMT1 were performed in healthy and anemic mice. Renal tissue iron was determined by Prussian blue iron staining. To assess anemia evolution and erythropoietic recovery, we used conventional tests. In healthy mice, Prohepcidin expression was marked in proximal tubules and inner medulla and absent in outer medulla. Cortical tissue of healthy mice also showed MTP1 immunostaining, mainly in the S2 segment of proximal tubules. Medullar tissue showed MTP1 expression in the inner zone. In addition, S2 segments showed intense DMT1 immunoreactivity with homogeneous DMT1 distribution throughout renal medulla. The main cortical findings in hemolytic anemia were in S2 segments of proximal tubules where we found that decreased Prohepcidin expression coincided with an increment in Ferroportin and DMT1 expression. This expression pattern was concomitant with increased iron in the same tubular zone. However, in medullar tissue both Prohepcidin and MTP1 decreased and DMT1 was detected mainly in larger diameter tubules. Our findings clearly demonstrate that in hemolytic anemia, renal Prohepcidin acts in coordination with renal Ferroportin and DMT1, indicating the key involvement of kidney in iron homeostasis when iron demand is high. Further research is required to learn more about these regulatory mechanisms.

Immunohistochemical studies on duodenum, spleen and liver in mice: distribution of ferroportin and prohepcidin in an inflammation model / Estudios inmunohistoquímicos en duodeno, bazo e hígado de ratón: distribución de ferroportina y prohepcidina en un modelo de inflamación

Duodenum, spleen and liver have a crucial role in iron balance on the whole organism and are the major sites of Ferroportin (FPN) expression. Specific regulations between FPN and hepcidin are responsible for changes seen in physiopathological conditions such as inflammation. We studied in vivo effects of turpentine oil-induced acute inflammation on FPN expression, and its relation with prohepcidin and iron mobilization. Immunohistochemical procedures were performed using rabbit anti-mouse FPN and prohepcidin antibodies with goat-labeled polymer-HRP anti-rabbit (DAB) as secondary antibody. Plasma and tissular iron were also studied. Our results showed a notable expression and redistribution of duodenal FPN to basolateral membrane in turpentine-treated mice, compared with supranuclear and the weak basolateral expression observed in healthy mice. Red pulp macrophages of healthy mice showed FPN-hemosiderin co-localization, compared with turpentine-treated mice which showed lack of FPN. In liver of healthy mice, FPN was seen in Kupffer cells, whereas in turpentine-treated mice decreased. In addition, we observed an increment of hepatic pro-hepcidin with a significant hypoferremia. Our findings demonstrated that acute inflammation induced a differential distribution of FPN, showing a cell type specific response. In macrophages, increased hepatic prohepcidin induced degradation of FPN, resulting in hypoferremia. In enterocytes, the redistribution observed of duodenal FPN reflects a different regulation in this tissue. The observed response of the proteins studied may be part of a cyclical pattern of systemic effects of acute inflammation on mouse tissue.

El duodeno, bazo e hígado desempeñan un rol clave en el balance de Fe del organismo y son los mayores sitios de expresión de ferroportina (FPN). Regulaciones específicas entre FPN y hepcidina son las responsables de los cambios observados en condiciones fisiopatológicas como la inflamación. Nuestro objetivo fue estudiar los efectos in vivo de la inflamación aguda inducida con turpentina sobre la expresión de FPN y su relación con prohepcidina y la movilización de hierro. Los procedimientos inmunohistoquímicos fueron desarrollados utilizando anticuerpos anti FPN y prohepcidina de ratón, desarrollados en conejo y un polímero conjugado con anticuerpos secundarios anti conejo desarrollado en cabra (HRP-DAB). Se evaluaron los niveles de Fe plasmático y tisular. Nuestros resultados mostraron una clara expresión y redistribución de FPN duodenal hacia la membrana basolateral en ratones tratados con turpentina, con respecto a la expresión perinuclear y leve expresión basolateral observada en ratón sano. Macrófagos de la pulpa roja esplénica mostraron co-localización de FPN y hemosiderina, comparado con la ausencia de expresión en ratón tratado con turpentina. En hígado de ratón sano, se observó expresión de FPN en células de Kupffer, mientras que en ratón tratado con turpentina la expresión fue menos evidente. Además, observamos un aumento en la expresión de prohepcidina hepática con una hipoferremia significativa. Nuestros resultados demostraron que la inflamación aguda indujo una distribución diferencial de FPN, mostrando una respuesta específica del tipo celular. En macrófagos, el aumento de prohepcidina hepática indujo degradación de FPN, resultando en hipoferremia. En enterocitos, la redistribución observada de FPN duodenal, refleja una regulación diferente en este tejido. La respuesta observada de las proteínas estudiadas podría ser parte de un patrón cíclico de efectos sistémicos de la inflamación aguda en tejidos murinos.