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Laboratory of Human Physiology, Departament of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, Bahia Blanca, Argentina
Submitted 27 March 2008 ; accepted in final form 18 July 2008
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ABSTRACT |
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divalent metal transporter 1; anemia
Furthermore, it is known that control of iron in the body is mainly dependent on tight regulation of gastrointestinal (GI) uptake from the diet to balance iron loss by other ways (12). Renal excretion is central to the control of other divalent cations, but little is actually known about how the kidney handles iron. A few years ago Wareing et al. (34) reported not only that a significant amount of serum iron is available for glomerular ultrafiltration, but also that most of the filtered iron is reabsorbed in renal tubules. However, it is unclear whether this organ exerts any control over the amount of iron excreted (34).
The recent finding that hepcidin is present in renal cells in basal conditions provides us with a new window on the hepcidin-dependent role of the kidney in iron homeostasis (16).
In this context, identification and characterization of transmembrane iron proteins like Ferroportin and divalent metal transporter 1 (DMT1) have greatly advanced our knowledge of iron transport. Ferroportin, also known as Ireg1 or MTP1, is an iron export protein that acts as an iron efflux transporter in several tissues (8, 21). It was originally isolated from duodenal mucosa in basal conditions, where expression was high in basal membrane to facilitate iron transport from the enterocyte to the bloodstream (21), suggesting its involvement in maintaining body iron stores. Other sites of high expression are macrophages, particularly those in the red pulp of the spleen and Kupffer cells of the liver (18). There is evidence that MTP1 is also expressed in renal cells located on the glomerulus and proximal tubular cells of this tissue (1, 20). These latter results contribute to the hypothesis of its renal involvement in iron homeostasis regulation.
Nemeth et al. (22) reported a hepcidin-Ferroportin interaction which could be central in iron physiopathology, supporting the hypothesis that Ferroportin is the receptor for hepcidin.
Another key protein involved in iron metabolism is DMT1, identified in rat duodenum as being capable of transporting iron and other metal ions (13). DMT1 transport is pH dependent, optimal at acid pH (5.5 to 6.0), electrogenic, and associated with a proton cotransport (13). Alternative splicing of the DMT1 gene produces two isoforms distinguished by the presence (isoform I) or absence (isoform II) of an iron response element (IRE) in the 3' untranslated region, both being competent in iron transport. DMT1 has been studied in duodenum where modulation of dietary iron absorption is the main mechanism for regulating body iron balance. Expression of DMT1 is reduced in iron-enriched diets but increases when iron intake is restricted (5). The converse is true in liver, since this organ acts as a reservoir of iron. High levels of dietary iron produce an increase in DMT1 expression in hepatocytes, promoting iron acquisition, whereas low levels decrease hepatic DMT1 expression, causing a reduction in iron accumulation (26, 32). Although kidney is not usually associated with serum iron balance, several studies found renal DMT1 expression in mice and rats, suggesting that this organ may have a role in iron homeostasis (4, 10, 33). Furthermore, Wareing et al. (34) reported a decrease in urinary iron levels, suggesting iron reabsorption in renal tubules.
Chronic kidney disease patients gradually develop anemia, and despite maintenance erythropoietin therapy they frequently present functional iron deficiency. In cases such as these, persistently high levels of hepatic hepcidin expression, probably due to chronic inflammatory state, may explain why duodenal iron absorption remains inadequately low and the iron sequestration within cells of the reticuloendothelial system is high (19). In view of the relationship between hepcidin and the iron transporters Ferroportin and DMT1, an experimental model studying these proteins will serve to elucidate modifications in renal pathways in anemia associated with chronic kidney diseases.
Since renal involvement in iron homeostasis regulation remains poorly understood, the purpose of the present study was to determine renal expression of Prohepcidin in hemolytic anemia, and its relationship with the iron transporters DMT1 and Ferroportin.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Experimental design. After acclimatization, the animals were divided into two groups (16 mice per group): 1) nonanemic mice receiving saline solution intraperitoneally (0.9% NaCl) on days 0 and 2; and 2) anemic mice receiving phenylhydrazine (PHZ) dissolved in 0.5 ml of saline solution intraperitoneally on days 0 and 2. The PHZ concentration injected was 60 mg/kg body wt.
Mice of both groups were subject to blood controls on day 0, before PHZ or saline solution administration. Anemia evolution and erythropoietic recovery were assessed using conventional hematological tests on days 3, 4, and 5. Animals were anesthetized with ketamine (50 mg/kg)-xylazine (20 mg/kg) for blood collection. For each time point, samples from three individual mice from each group were collected from the retro-orbital venous plexus (25 µl each).
PHZ preparation. PHZ (Sigma Chemical) in 0.1 M potassium phosphate buffer pH 7.4 was prepared on day 0. The solution was sterilized by filtration before use and administered by intraperitoneal injection on days 0 and 2.
Hematology. The following tests were performed on EDTA samples: hemoglobin (Hb), hematocrit (HCT), reticulocytes, and Heinz body counts. Reticulocytes were counted in blood smears stained with Brilliant Cresyl Blue. Each reticulocyte count was based on a count of 1,000 red blood cells (RBC). Differential Heinz body RBC count was obtained by counting 1,000 cells in a Brilliant Cresyl Blue-stained preparation. Blood smears were prepared immediately after blood sample collection and were stained with May Grünwald-Giemsa. Cell count was performed three times for each time point and for each group.
Tissue harvestation at necropsy. On days 0, 3, 4, and 5, a subset of each group (n = 3) was anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt) to perfuse kidneys. Immediately after the animals were killed by cervical dislocation, renal tissue of both control and PHZ-treated mice (60 mg/kg) was removed under sterile conditions by abdominal incision and separated from the surrounding organs. The tissues were kept in sterile saline solution. Histological techniques were followed using Ringer solution plus heparin (0.25% wt/vol) and Bouin solution as perfusion liquids. Approximately 25 ml of solution were perfused in each organ to wash out the red cells.
Antibodies. DMT1 immunodetection was performed using an affinity-purified rabbit anti-mouse polyclonal antibody directed against the peptide VKPSQSQVLRGMFV, which corresponds to amino acids 229-242, common to isoforms I and II (kindly provided by S. Gauthier, Canada). For Ferroportin detection, an affinity-purified rabbit anti-mouse polyclonal antibody directed against COOH-terminal segments (kindly provided by B. Galy, Germany) was used. Prohepcidin immunodetection was performed with an anti-mouse Pro-Hepcidin (Alpha Diagnostic). Goat-labeled polymer-horseradish peroxidase anti-rabbit Envision+System (DAB; Dako Cytomation) was used as secondary antibody.
Immunohistochemistry technique. Kidney samples from control and PHZ-treated mice (60 mg/kg) were postfixed by immersion in fresh fixative solution (10% neutrally buffered formalin, pH 7.2) and embedded in paraffin. Sections of 5 µm were obtained and mounted on glass slides. Before labeling, sections were deparaffinized in xylene and rehydrated in a graded series of ethanol baths. Endogenous peroxidase activity in deparaffinized sections was blocked with 3% H2O2. Sections were then incubated in PBS for 30 min at room temperature. Incubation with the primary antibody diluted into PBS (pH 7.1) was carried out in a wet chamber for 1 h at room temperature for DMT1 and MTP1, and overnight at 4°C for Pro-Hepcidin immunodetection. Dilutions of antibodies were as follows: anti-DMT1 (1:1,000), anti-MTP1 (1:500), and anti-Prohepcidin (1:100). Following incubation with the primary antibody, the tissues were washed with PBS, incubated with a goat anti-rabbit IgG peroxidase-coupled secondary antibody for 30 min at room temperature, and then rewashed with PBS. Subsequent localization of proteins was revealed by reaction with 3'-diaminobenzidine tetrahydrochloride (DAB) in solution. Sections were then counterstained with Harris hematoxilin. Finally, sections were dehydrated in ethanol and xylene and mounted on coverslips. Negative controls included incubation with PBS without the primary antibody. Immunostaining was analyzed with an Olympus BX51 microscope using x10 and x40 dry objectives and a x100 oil immersion objective. Digital images were obtained with an Olympus C7070 camera.
Tissular iron. To determine iron store, Prussian blue iron staining was performed in sections of mouse kidney, following the manufacturer's instructions. The technique is based on the ability to release ferric iron from protein-bound tissue deposits, which in the presence of ferrocyanide ions is precipitated as the highly colored and highly water-insoluble complex, potassium ferric ferrocyanide, Prussian blue.
To carry out the stain, sections were deparaffinized and hydrated to distilled water. Slides were incubated with 10% potassium ferrocyanide for 4 min followed by incubation with potassium ferrocyanide-hydrochloric acid for 12 min. Sections were then rinsed well in distilled water, counterstained with nuclear fast red for 1 and 30 min, and finally rinsed in distilled water, dehydrated, cleared, and mounted.
Proximal tubule identification. Proximal and distal tubules are two of several structures contained in the renal cortex. The following criteria were used to identify proximal tubules (4): 1) their predominance in outer medullary rays, 2) the fact that they are most abundant in the cortex, and 3) their epithelium is taller and the lumen much narrower. Different segments of proximal tubules were distinguished as follows: 1) S1 and S2 segments are in the cortical zone close to the glomeruli, whereas S3 segments lie in outer strip of the outer medulla and 2) S1 and S3 segments have taller epithelium and narrower lumen than S2 segments.
Statistical analysis. At least four individuals were tested in each mouse experiment. Data were analyzed by one-way ANOVA followed by the Tukey multiple comparison test. The level of statistical significance was set at P < 0.05. All values are expressed as means ± SD.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Prohepcidin. The immunohistochemical study of medullar tissue in the basal condition (nonanemic mice) showed Prohepcidin expression with intracytoplasmic localization in the inner medulla (Fig. 1A). Under severe hemolysis, a decrease in staining was seen in the inner medulla compared with the basal condition (Fig. 1B). The outer medullar zone was negative for Prohepcidin expression both in the basal and anemia conditions (data not shown).
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Ferroportin. The study of MTP1 distribution in renal medullar tissue in basal conditions revealed weak immunoreactivity in tubules of the inner medulla (Fig. 2A). Specific immunostaining was observed mainly in the intracellular zone in these tubules and none in the basal membrane (Fig. 2B). Outer medulla only showed MTP1 expression in bundles of vessels and in some tubules, being weak and intracytoplasmic (data not shown).
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In nonanemic mice, we detected MTP1 expression in structures like proximal tubules (Fig. 2E), with intracytoplasmic localization near to the basal membrane as well as in apical cytoplasm (Fig. 2F). Other tubular structures not clearly identifiable also showed positive. In a more detailed study, we observed intense immunostaining in S2 segments, weaker staining in S1 segments, and a total absence of staining in S3 segments (Fig. 2E). Other cortical structures were negative for this iron transporter.
Along the anemic state, we observed the main differences in MTP1 expression on days 4 and 5. The cortical study showed an increase in MTP1 immunostaining in the same renal tubular structures as those seen in control tissue. S2 and S1 segments showed more intense staining, whereas S3 segments were negative for Ferroportin expression (Fig. 2G). In addition, subcellular localization of this protein remained mainly intracellular near to the basal membrane, although MTP1 staining can be observed over the brush border (Fig. 2H). Other cortical structures remained negative.
DMT1. In nonanemic mice, we detected intracellular DMT1 expression with homogeneous distribution through renal medulla (Fig. 3A) with intracellular localization (Fig. 3B). The pattern of DMT1 expression was studied along the instauration of the anemic state and after the restitution of erythropoietic activity. On day 3, we detected DMT1 mainly in large diameter tubules of the inner medulla with intracellular localization (Fig. 3, C and D). As in the case of basal conditions, no changes were observed in outer medulla staining (data not shown).
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The renal cortical zone of nonanemic mice revealed an abundance of this iron transporter in structures like proximal tubules, where DMT1 localization was intracellular, near to the apical membrane (Fig. 3F). Upon more detailed study to identify which specific segments of proximal tubules were stained, we detected more intense immunoreactivity in S2 and S3 segments and weaker immunoreactivity in S1 segments. We did not detect any DMT1 immunostaining in the parietal Bowman's epithelium, blood vessels, and glomeruli (Fig. 3E).
The same pattern of DMT1 distribution was observed on day 3 as in control tissue, with specific immunoreactivity in proximal tubules being localized in cytoplasm near to apical membrane (Fig. 3H). The staining detected in S3 had the same intensity as in the control. S2 segments on the other hand showed an increment in DMT1 expression with respect to the basal state, with S1 segments maintaining a weak DMT1 expression (Fig. 3G). No changes were observed in the renal cortical zone of anemic mice on day 4 with respect to basal conditions. On day 5, as in the basal state, we observed DMT1 expression in the renal cortical zone and in medulla.
Tissular iron. Renal tissue of anemic mice showed abundant iron deposits in the cortical zone (Fig. 3J), whereas there were no iron deposits in the basal condition (Fig. 3I). Medullar structure showed an absence of tissular iron in anemic and nonanemic mice (data not shown).
The histological examination on day 3 of hemolytic anemia revealed the presence of iron in structures whose morphology was similar to that of proximal tubules. A more detailed assessment showed strong iron deposits in S1 and S2 segments but none in S3 segments (Fig. 2J). There was a clear intracellular localization of iron in these structures. The monitoring of iron deposits on days 4 and 5 in this experimental design showed morphological changes similar to those occurring on day 3.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Renal Prohepcidin expression. In hemolytic anemia, Prohepcidin immunostaining decreased significantly in renal inner medulla with respect to basal conditions. This finding is crucial to understand renal involvement in iron metabolism.
The distribution pattern in the inner medulla of healthy mice was found to coincide with that observed by Kulakzis et al. (16), although under our assay conditions we were unable to identify the protein in the outer medulla.
Our findings in hemolytic anemia demonstrate a clear decrease in the intensity of cortical Prohepcidin expression. Although Kulakzis et al. (16) found no evidence of proximal Prohepcidin expression in healthy mice, in our study we observed intense immunostaining in S2 segments of proximal tubules.
It is important to note that Prohepcidin localization was strictly associated with basal membrane, possibly suggesting a close interaction with MTP1, the molecule proposed as the hepcidin receptor (22).
Our findings suggest that the high Epo levels present in hemolytic anemia may be responsible for the reported negative regulation of hepatic hepcidin (24) and for the low renal expression of this peptide found by us. Several hypotheses could explain our findings. First, it is known that the kidney is closely associated with erythropoiesis since low levels of oxygen present in hemolytic anemia induce increases in HIF-1, which in turn plays an important role in the expansion of committed erythroid progenitors and in the terminal differentiation of erythroid cells (31). This could explain hematological restoration to hemolytic anemia (6, 14).
Although hemolytic anemia causes hypoxemia, it is not clear whether hepcidin regulation by oxygen is independent of the pathway via anemia. Nor is it clear as yet whether hypoxia or other systemic signals are involved in the synthesis and expression of Prohepcidin. Second, although there is abundant blood flow to the kidney (9), it is known that cortical tissue is very sensitive to hypoxic conditions and that peritubular fibroblasts in this zone produce erythropoietin under hypoxia (17). Moreover, some studies reported that hepatic synthesis of hepcidin is suppressed in anemia induced by hemolysis (24) and that a potent downregulation of hepcidin expression in the liver is observed after erythropoietin administration (25). Finally, Dallalio et al. (7) showed that there is a significant decrease in colony formation in the presence of hepcidin, suggesting an additional mechanism by which hepcidin can inhibit erythropoiesis.
Although the relationship between Epo and hepcidin has been demonstrated, the specific mechanisms that regulate Prohepcidin renal expression in hemolytic anemia, a state with high Epo levels, are not known. It is possible that direct and/or indirect pathways are involved.
Renal Ferroportin expression. Another objective was to study the relationship between Prohepcidin and renal MTP1 expression in hemolytic anemia. A recent study reports MTP1 expression and the mRNA of this protein in renal tissue of healthy mice (22). However, little information is available in the literature on the subcellular localization of this exporter in healthy mouse kidney, and even less on immunohistochemical changes in hemolytic anemia. The main contribution of the present study is therefore the reported evidence of the presence of MTP1 in renal tissue under severe anemia.
The increased MTP1 immunostaining seen on S1 and S2 segments of proximal tubules under hemolytic stimuli suggests its involvement in the movement of iron into the bloodstream to increase iron availability and prevent loss of iron through urine.
The strong intracellular expression observed in the basal cytoplasm suggests that as reported by Oates and Thomas (27) in intestinal cells, MTP1 can transport iron into organelles to internalize iron in the cell. This could allow the subsequent export of iron to the plasma, since it is known that high iron demand is common in anemia. However, the reason for this behavior in renal tissue requires further study.
It is important to note that in healthy mice Ferroportin was seen both in basal as well as in apical cytoplasm of proximal tubules. The basal localization of Ferroportin is consistent with its role as iron exporter, which is enhanced in anemic state. The finding about apical localization in healthy state is in accordance with the reports by Oates and Thomas (28) on duodenum and it could be indicating a modulation of DMT1 activity by Ferroportin on apical surface.
We observed a decrease in MTP1 staining in the inner medulla of anemic mice compared with healthy mice. This could be the consequence of coordinated activity in the different renal zones, some structures having weak activity and others playing a major role in the process of iron reabsorption in renal tissue. Possibly, these changes allow iron reabsorption in anemia in certain areas of the kidney closely associated with iron homeostasis. However, the mechanisms responsible for variations in Ferroportin expression in different zones of the kidney are not known.
Interestingly, we found an inverse relationship between hepcidin and Ferroportin in basal conditions. High Prohepcidin expression was observed in conjunction with weak MTP1 expression throughout a downregulation mechanism.
In support of these findings, a recent study indicated that Ferroportin is regulated by hepcidin through the direct binding of hepcidin to Ferroportin on cell membranes, causing its internalization and degradation inside the cell (22).
Renal DMT1 expression. Renal DMT1 is another iron protein that to date has not been studied in depth in hemolytic anemia. Our results showing intense DMT1 expression in medulla in anemia are in agreement with those reported in animals on an iron-restricted diet, a condition of increased iron demand (33). The DMT1 expression observed in inner and outer medulla in healthy mice is similar to that reported by other authors (10, 13). It is important to note that changes in DMT1 expression during development of anemia, more intense under high iron demand, indicate a marked involvement of renal tissue in iron metabolism disease.
Medullar DMT1 localization could be explained by the interdependence known to exist between pH and DMT1 activity (13). Since DCT and collecting ducts are colocalized with a H+-ATPasa (3, 10), their luminal pH can be acidified by the presence of transport coupled to H+. The acid pH in these tubules therefore correlates well with the functional requirements of DMT1.
The most important finding on cortical DMT1 expression in healthy and anemic mice is its localization along the proximal tubule. DMT1 immunostaining, mainly in S2 segments in both healthy and anemic mice, suggests a specific tubular function. In this kidney zone iron transport by DMT1 is also pH dependent, being more active at acid pH; one can therefore expect to find DMT1 in proximal tubules, where the pH of the glomerular filtrate is acid (15).
Coordinated action between Prohepcidin and iron transporters. In view of the relationship between iron metabolism and erythropoiesis described in the literature, the present study sought to further elucidate renal involvement in hemolytic anemia, a condition for which the expression of regulatory peptides in kidney has not as yet been described in depth.
Our initial hypothesis was that in hemolytic anemia, with increased erythropoietic activity, DMT1, MTP1, and Prohepcidin expression is regulated in a coordinated manner (Fig. 4). In this context, we observed a direct relationship between DMT1 and MTP1 in S2 segments under hemolytic crisis, concomitant with reduced Prohepcidin expression in this tubular segment. The regulatory relationship between hepcidin and iron transporters has been widely studied by other authors in several tissues (2, 7, 11). Interestingly, the changes in protein expression observed in renal segments were accompanied by an increase in iron deposits. In this sense, the proximal tubule could be the main renal structure responsible for regulating systemic iron balance in intense erythropoietic demand (Fig. 4).
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The presence of Prohepcidin in several renal zones both in healthy and anemic mice is an important finding and suggests an intrinsic synthesis of this peptide in kidney, as hypothesized by Kulakzis et al. (16) in healthy mice.
Although the latter authors propose a luminal release of this peptide, the intracellular localization closely associated with basal membrane observed in our study suggests a possible release of the peptide to the bloodstream. Moreover, the behavior of this protein in the anemic state suggests the regulation of hepcidin production, which depends on systemic iron demand.
Since it has been reported in the literature that iron-restricted diets are associated with high renal DMT1 expression and a decrease in urinary iron (34), our evidence of the coincident localization of iron proteins and iron deposits under anemia might indicate the reabsorption of iron from glomerular ultrafiltrate when iron demand is high. The purpose of this mechanism could be the provision of iron to renal cells for their metabolic activity and to help maintain iron homeostasis, emphasizing the importance of renal physiology in hematological processes.
In conclusion, the findings of our study indicate an intrinsic renal synthesis of Prohepcidin in basal conditions which is reduced in anemia and acts in a coordinated manner with MTP1 and DMT1. This behavior, associated with the increment of renal tissular iron, could help to elucidate possible renal involvement in restoration of the hemolytic state.
It will therefore be necessary to carry out further studies to discover the regulatory mechanisms at work between Prohepcidin and iron transporters in the renal tubular system under severe hemolysis.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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