Orchestrated regulation of iron trafficking proteins in the kidney during iron overload facilitates systemic iron retention

The exact route of iron through the kidney and its regulation during iron overload are not completely elucidated. Under physiologic conditions, non-transferrin and transferrin bound iron passes the glomerular filter and is reabsorbed through kidney epithelial cells, so that hardly any iron is found in the urine. To study the route of iron reabsorption through the kidney, we analyzed the location and regulation of iron metabolism related proteins in kidneys of mice with iron overload, elicited by iron dextran injections. Transferrin Receptor 1 was decreased as expected, following iron overload. In contrast, the multi-ligand hetero-dimeric receptor-complex megalin/cubilin, which also mediates the internalization of transferrin, was highly up-regulated. Moreover, with increasing iron, intracellular ferritin distribution shifted in renal epithelium from an apical location to a punctate distribution throughout the epithelial cells. In addition, in contrast to many other tissues, the iron exporter ferroportin was not reduced by iron overload in the kidney. Iron accumulated mainly in interstitial macrophages, and more prominently in the medulla than in the cortex. This suggests that despite the reduction of Transferrin Receptor 1, alternative pathways may effectively mediate re-absorption of iron that cycles through the kidney during parenterally induced iron-overload. The most iron consuming process of the body, erythropoiesis, is regulated by the renal erythropoietin producing cells in kidney interstitium. We propose, that the efficient re-absorption of iron by the kidney, also during iron overload enables these cells to sense systemic iron and regulate its usage based on the systemic iron state.


Introduction
The kidneys are extremely sensitive to heme and hemoglobin exposure during hemolytic anemias [1][2][3][4]. In contrast, they are rarely mentioned amongst tissues that are damaged by a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 FPN internalization and breakdown [30,31]. This reduces dietary iron uptake by intestinal epithelial cells and iron efflux by many cells, including macrophages after erythrophagocytosis and hepatocytes that function as long-term iron stores. Other iron export pathways such as ferritin-or heme-secretion may play a role in iron trafficking through kidney epithelium [32,33]. In physiologic conditions, ferritin localizes near the apical membrane of the tubule in the polarized kidney epithelium [32,34].
In this research we studied proteins involved in iron re-absorption through the kidney during Parenterally induced systemic Iron Overload (PIO). PIO was elicited by iron dextraninjections, that mimic parenteral administration of iron supplements. We found that TfR1 is indeed down-regulated. In contrast, cubilin is highly up-regulated during PIO. We further show evidence that the cubilin-regulation by iron is post-transcriptional and may be mediated by an iron-dependent stabilization of cubilin through the transcriptional up-regulation of megalin. In epithelial cells, the intracellular ferritin distribution is shifted towards the basolateral membrane, close to the interstitium. In the interstitium, iron and ferritin accumulate mainly in macrophages, and more prominently in the medulla than in the cortex. This stands in contrast to iron absorbed through the diet, where iron accumulates mainly in the renal epithelium.

Animals and PIO
Animal experiments were done according to protocols approved by the Technion Animal Ethics Committee, Haifa, Israel. All mice were on C57Bl/6J background. PIO was elicited in 3 month-old male mice by 5 daily intra-peritoneal injections of 100μl iron-dextran solution (90mg iron/ml, Sigma-Aldrich). Three days after the last injection mice were sacrificed, kidneys were collected, frozen in liquid nitrogen and stored at -80˚C.

Histological methods
Kidneys and spleens were collected and fixed in 10% neutral buffered formalin or 4% PFA, respectively. The kidneys were trimmed mid-longitudinally, to include the cortex, medulla and papilla, and then embedded in paraffin, sectioned to a thickness of approximately 5 microns and stained with Hematoxylin & Eosin (H&E) for histology and Perl's Prussian blue (PPB) for ferric iron detection. Histopathological changes were analyzed and scored using a semi-quantitative grading of five grades (0-4), taking into consideration the severity of the changes (0 = no lesion, 1 = minimal change, 2 = mild change, 3 = moderate change, 4 = marked change). Spleens were prepared and analyzed similarly.

Immunohistochemistry (IHC) and Immunofluorescence (IF)
Paraffin-embedded kidney sections from iron overloaded and control mice were heated in the microwave for 10 minutes for antigen retrieval with 6M urea in PBS (for all proteins) or with 0.01M citrate buffer (for H-ferritin) and rinsed with ddH 2 O. For IHC, quenching of endogenous peroxidase was performed by incubating the slides in 3% H 2 O 2 in a humid chamber following by ddH 2 O washes. Next, sections were blocked with 10% normal goat/donkey serum (Jackson) in PBS containing 0.1% bovine serum albumin (BSA, Sigma) that was chosen according to the animal in which the secondary antibody was raised. Then, sections were incubated overnight (ON) at room temperature (RT) in a humidified chamber with the following primary antibodies; polyclonal goat-anti-mouse cubilin (diluted 1:200, Santa Cruz Biotechnology), monoclonal mouse-anti-human TfR1 (diluted 1:200, Zymed), polyclonal affinity purified rabbit-anti-mouse H-or L-ferritin (a kind gift from Prof. A. M. Konijn, Hebrew University, Jerusalem; diluted 1:200) and polyclonal goat-anti-mouse CD68 (diluted 1:200, Santa Cruz Biotechnology). For IHC staining, sections were washed with PBS and incubated for 1 hour at RT with biotinylated goat-anti-rabbit or rabbit-anti-goat antibodies (diluted 1:500, Vector). Then, sections were incubated with Vectastain ABC kit following manufacturer's instructions. The immunohistochemical reaction was visualized using DAB (Sigma). Sections were dehydrated and mounted with Eukitt resin (Sigma). For IF staining, donkey-anti-goat 488, donkeyanti-rabbit 568 and goat-anti-mouse 488 were used as secondary antibodies and incubated for 1 hour at RT (diluted 1:1000, Invitrogen). Next, slides were washed with PBS and mounted with VECTASHIELD mounting medium containing DAPI (Vector laboratories). Negative controls were incubated with secondary antibodies only.

Ferric iron stain
Paraffin-embedded kidney sections from iron overloaded and control mice were blocked with peroxidase blocking solution (3% H 2 O 2 in PBS), stained with PPB solution (2% K 2 Fe(CN) 6 , 2% HCL mixed freshly in a 1:1 ratio) for 1 h and washed several times with ddH 2 O. Positive staining appeared as a blue color under light microscope.

Quantitative PCR (qPCR)
Total tissue RNA was isolated from kidneys using Trizol reagent (Invitrogen). Samples were treated with DNase I recombinant, RNase free kit (Roche) according to the manufacturer's instructions. cDNA was synthesized using total RNA (1 μg) by qScript cDNA synthesis kit (Quanta biosciences), and was amplified using SYBER Green (Quanta biosciences) in AB 7300 (Thermo Fisher Scientific).

Correlative microscopy with air-SEM and EDX detector
Paraffin-embedded kidney sections were fixed on Superfrost microscope slides. High iron kidney slides were H&E stained and left uncovered for airSEM (B-nano LTD) analysis. First, slides were imaged with an optical microscope for sample orientation and selection of region of interest, images were acquired using 20X, 40X and 100X objectives, then the sample was moved automatically to the SEM, where matching fields were imaged using backscattered electron imaging as described. [35,36] Elemental analysis and mapping were carried out by an EDX detector placed on the same optical axes of the SEM.

No morphological kidney damage due to parenteral systemic iron overload
To study how iron transport is regulated during PIO, we caused PIO in mice by injecting iron dextran into the peritoneum. Histological analysis of kidney and spleen sections of these mice showed completely normal kidney morphology with well-defined glomeruli and tubules. In the kidney, no iron accumulation can be detected in H&E staining, without specific visualization of iron ( Fig 1A-1D). In contrast, in the spleens of the same mice we detected hemosiderosis (brown haze, Fig 1F), a mild depletion of lymphocytes in the periarteriolar lymphoid sheath of the white pulp, and the boundaries of the white pulp were not well defined (Fig 1E and 1F).

Cubilin is found in the medulla of iron-overloaded mice
To understand how Tf-iron uptake systems are regulated in the kidney during iron-overload, we studied the expression of these systems, which are known to be expressed at the apical epithelial membrane. Both cubilin and TfR1 play a role in Tf and iron uptake to the epithelium and thus we localized both candidates (Fig 2). At the same time we planned to use cubilin as a marker for the apical membrane of the cortex as described [37]. As expected, we detected cubilin expressed apically in the cortex, but surprisingly, in iron-overloaded mice, it was not only highly upregulated in the cortex, it was also detectable in the medulla (Fig 2A-2F). This suggested an iron mediated up-regulation of cubilin. In control mice, TfR1 was expressed in the cortex as described earlier [10], but was also found in the medulla, further supporting that the medulla is playing an important role in the re-absorption of iron from the primary urine. In the kidney sections of iron overloaded mice, TfR1 was under detection limits, consistent with the instability of TfR1 mRNA during iron overload (Fig 3A and 3C).

Cubilin and TfR1 are regulated in opposite directions by PIO
As expected, TfR1 protein and mRNA levels were decreased in the kidneys of iron overloaded mice (Fig 3A and 3C, respectively). In contrast, cubilin-protein was highly up-regulated in the kidneys of these mice (Fig 3B), supporting the observation of the immunofluorescence experiment (Fig 2A-2F). The cubilin protein elevation could not be explained by a transcriptional regulation of cubilin, as mRNA levels were not elevated in PIO mice (Fig 3D).

Megalin mRNA is elevated in kidneys of iron overloaded mice
To further investigate the elevated cubilin levels in kidneys of iron overloaded mice we wondered if megalin, that is part of the megalin-cubilin complex, is affected by iron as well. Indeed, megalin mRNA expression was highly elevated in the kidneys of iron overloaded mice ( Fig  3E), suggesting a role for megalin in the iron mediated upregulation of cubilin.

Epithelial ferritin undergoes an iron-mediated intracellular redistribution
In renal epithelial cells of control mice, ferritin has a polarized appearance near the apical brush-border of the PT [32,34]. We noticed that in the PT epithelium of iron overloaded mice, this polarization was completely lost and ferritin was distributed throughout the epithelial cells (Fig 4). This suggested that cellular iron level might regulate intracellular ferritin distribution. To test this hypothesis, we analyzed epithelial localization of ferritin in Irp2-/-mice, which suffer from a functional iron deficiency [38][39][40]. Indeed, in the Irp2-/-mice ferritin was strongly polarized in the apical pole of the epithelial kidney cells (Fig 4), further supporting a regulated ferritin distribution within this polarized cell-type, with an apical enrichment of ferritin in cells with lower iron concentration. PIO did not cause morphologic kidney damage, but affected the spleen. Fixed kidney and spleen sections from control and iron-loaded mice were histologically stained (H&E) and imaged. No damage was observed in kidney sections of iron-loaded mice (B and D) compared to the control sections (A and C). However, iron accumulation caused damage to the spleen as can be evaluated by moderate degree of hemosiderosis (seen as brownish pigment in the red pulp of section F compared to E), and by a mild depletion of lymphocytes in the white pulp (indicated by red arrowheads); the inserts in the left corner of E and F are higher magnifications of these sections. Scale bar 100 μm, n = 3 for control and iron-loaded mice each. https://doi.org/10.1371/journal.pone.0204471.g001

Iron export from renal epithelial cells
To further study the route of iron through kidney epithelium, we asked whether the ferrous iron exporter FPN was regulated also in the kidney by the iron status of the mouse. We found that FPN was not reduced by PIO in the kidney, but was significantly reduced in the spleen of the same mice (Fig 5). In mice with a targeted deletion of IRP2, spleen FPN was slightly reduced and FPN in the kidney slightly elevated. These data suggested that in the kidney, iron is exported efficiently from cells that express FPN also during PIO.

Parenterally administered iron accumulates in interstitial macrophages, mainly in the medulla
To test where iron accumulates in the kidney during iron overload, we compared iron accumulation in kidneys from parenterally and dietary iron-overloaded mice. Iron was first visualized by PPB staining and was significantly enriched in the medullar interstitium of PIO mice (Fig 6A). In these mice, iron was also detected in the cortical interstitium near the PT and in and near the glomerulus, but not near the distal tubules (Fig 6A). The epithelial cells were spared from iron overload (Fig 6A and 6B). Interestingly, following dietary iron overload, iron accumulated mainly in the proximal tubule epithelial cells of the cortex (Fig 6C), whereas there was little staining in medulla, suggesting that different mechanisms are behind the handling of dietary or parental administration of iron overload. In order to better characterize and quantify iron content per compartment, correlative microscopy (epi-fluorescence and SEM) was used to image both optical-, and backscattered electron-images by airSEM and metal content was analyzed by EDX (Fig 6D-6F). This verified the iron accumulation in the medullar interstitium and quantification of the interstitial areas in cortex and medulla showed two times more iron in the medullar than in the cortical interstitium (Fig 6G). To identify the celltype in which iron accumulates, we co-stained kidneys from iron-overloaded mice with ferritin and the macrophage-marker CD68. PPB stain visualizes ferric iron (Fe 3+ ), which is mainly found in ferritin and hemosiderin. Indeed, ferritin immune-staining presented a very similar pattern to PPB stain and localized predominantly to the interstitium in the kidneys of PIO mice. Most of the ferritin co-localized with the CD68-macrophage-marker (Fig 7) indicating that iron is mostly accumulated in interstitial macrophages.

Fig 4. Ferritin distribution in renal epithelial cells is regulated by the iron status: Kidney sections from control, iron-loaded and Irp2-/-mice were stained with H-ferritin antibody.
Ferritin was apically polarized in kidneys from Irp2-/-mice and to some extent also in wild-type control mice (arrow heads). In contrast, in iron overloaded mice ferritin was distributed throughout the cells and also found in basolateral regions (arrows). Scale bar represents 50μm.
https://doi.org/10.1371/journal.pone.0204471.g004 Iron transport through the kidney during iron overload  Iron transport through the kidney during iron overload

Discussion
Several regulatory mechanisms protect the mammalian organisms from iron overload, including control of iron uptake, recycling and storage by hepcidin, the IRPs, hypoxia-inducible factor (HIF) and NCOA4 [41][42][43][44][45]. In contrast to most cell types, where this regulation inhibits cellular iron uptake during iron overload, we show that kidney epithelial cells have all the tools to re-absorb most iron from primary urine regardless of systemic iron status. Thus, it can be assumed that a considerable amount of iron traffics through the kidney during systemic iron overload.
Together with albumin and many other plasma proteins, also a fraction of plasma-transferrin is filtered through the glomerulus, and re-absorbed from the primary urine into kidney epithelial cells. TfR1 is found apically on kidney epithelium [10] (and Fig 2) and takes up holo-Tf, in an iron regulated manner. It is strongly down-regulated during PIO (Fig 3), thus it is not likely for the Tf-TfR1 system to be the main route for Tf and iron uptake from primary urine of iron overloaded mice. In contrast, upregulation of megalin/cubilin during PIO suggests that Tf-iron is mainly reabsorbed by cubilin under these conditions, which explains why Tf reabsorption through the kidney is not limited during systemic iron overload and none of this protein is found in urine. In a rat kidney cell model, megalin regulation by iron and the functional competition between TfR1 and megalin was suggested [46]. Recently also cubilin upregulation was observed in a mouse-model for hemolytic anemia and this was accompanied by increased function of the megalin/cubilin complex [47]. A patient with two mutations in the megalin gene, which led to a mostly intracellular location of megalin and absence of membrane megalin, had elevated urinary levels of cubilin and type 3 carbonic anhydrase due to shedding of these proteins [48]. Thus cubilin, which has no trans-membrane domain and depends on megalin for its membrane location and internalization, may possibly be stabilized by megalin during PIO.
Once in the epithelial cells, iron needs to be transported across the cells. We have previously shown a possible role for ferritin in both intra-and intercellular iron trafficking in the Sertoli cells of the testis [49]. There we suggested, that iron that is taken up apically by Sertoli cells may traffic within ferritin to the basolateral pole of these epithelial cells, where ferritin is secreted in a regulated way. In macrophages, much intracellular ferritin is found in membrane bound vesicles of the endo-lysosomal system and manipulation of the endo/lysosomal trafficking machinery affects ferritin secretion [50]. The distinct distribution of ferritin in renal epithelial cells of iron overloaded mice and of mice suffering from a functional iron deficiency (Irp2-/-mice) suggests that iron status regulates the trafficking of ferritin containing vesicles, which are located near the apical membrane, in iron deficient cells, and are dispersed throughout the cell and near the basolateral membrane in iron overloaded cells. Similarly, transferriniron has been shown to be involved in the regulation of endosomal trafficking in erythroid cells [51,52]. This finding further suggests that ferritin may be secreted basolaterally and contribute to the iron flux through renal epithelial cells. Yet, in a cell-model of proximal tubule cells, no ferritin was detected in the basolateral compartment, in the first 4-7 hours of apical iron exposure [23].
Ferrous iron can be exported through FPN, which is located basolaterally in kidney epithelium [27]. In our hands, FPN was strongly reduced in the spleen of the PIO mice and slightly reduced in Irp2-/-mice, as described [53]. Yet in the kidneys FPN levels were not reduced by PIO, suggesting that the different regulatory forces acting on FPN [30,54] are balancing it to remain unchanged in iron overload, in the kidney. The slight elevation of FPN in the kidneys of Irp2-/-mice further supports the notion that IRPs contribute to FPN regulation in the kidney. This implies that iron may not only be efficiently imported to kidney epithelial cells during iron overload but it may also be efficiently exported to the interstitium and the blood. Interestingly, in response to dietary iron overload, cortical epithelial cells were the major sites of iron accumulation, which stood in contrast to the pattern of iron accumulation in PIO ( Fig  6). This discrepancy may origin in a different ratio of Tf-bound iron and NTBI reaching the primary urine in the two ways of iron overload, which will affect the site of reabsorption along the tubule and subsequent handling of iron. In addition, we speculated that different hepcidin levels may offer an explanation for the differential iron distribution. However liver hepcidin levels increased about four-fold in both, dietary iron overload [55] and PIO (control 1.22±0.8; PIO 4.61±0.67; p<0.005 n = 3). It remains possible that in response to dietary iron administration, renal hepcidin production may increase more than in response to PIO and that this plays a role in the kidney iron distribution [56]. More research may clarify these hypotheses.
Iron chemistry is dominated by the inter-conversion of ferrous and ferric iron [57], which are maintained at equilibrium. The low oxygen conditions in the renal medulla (1.3-2.6% O 2 ) support an iron homeostasis with slightly higher concentrations of ferrous iron in solution than in the well-oxygenated cortex (6.6% O 2 ) [58,59]. Thus, ferrous iron transport may be facilitated across cellular plasma membranes in the medulla, which may permit the medulla to maintain a highly dynamic iron pool that is not used for long-term iron storage.
We can think of two biological functions for efficient iron re-absorption of the kidney also when systemic iron is high. 1) Erythropoiesis is the most iron consuming process in the body and is regulated by erythropoietin (epo), made in the kidney interstitium [60]. Epo is regulated mainly by HIF, which senses both oxygen and iron, thus integrating the systemic need for red blood cells and the systemic ability to make them. Hence, if the iron flux through the kidney represents systemic iron stores, rather than responds to-and regulates these stores, important information is convened to the regulatory system of erythropoiesis. 2) There is a tough competition for iron acquisition between host and pathogens [61]. The iron mediated up-regulation of the multi-ligand receptor complex megalin/cubilin facilitates not only the efficient reabsorption of Tf protein and its bound iron but also uptake of iron bound to other molecules. Thus, it may be part of a mechanism that prevents bacteria causing urinary tract infections [62] to thrive during systemic iron overload. On the other hand, with the megalin/cubilin complex being able to bind and internalize many other and potentially harmful molecules including carcinogens and drugs its upregulation may contribute to the toxicity of iron overload [48].
Taken together, we have evidence that during PIO, iron is transported efficiently across the epithelial barrier. TfR1 levels are low, but cubilin levels are high and the cubilin-megalin heterodimer likely plays a major role in iron transport from the primary urine back to the body. Ferritin is distributed throughout the epithelial cells and does not accumulate at the apical brush-border, suggesting that it may contribute to intra-and inter-cellular iron trafficking. FPN is not down-regulated by the high iron conditions and thus may export iron efficiently from the basolateral epithelium into the renal interstitium and also from interstitial macrophages. We suggest that the highly expressed cubilin-megalin complex mediates Tf-iron reabsorption during PIO both in the cortex and the medulla, where excess iron is stored predominantly in interstitial medullar macrophages. The strategy of shifting a significant part of iron re-absorption to the medulla during PIO may accelerate renal iron flux. In conclusion, we suggest that iron transport through the kidney epithelium is unique in its regulation, reabsorbing iron even when systemic iron is high. This may protect the host from uropathogenic bacteria and provide erythropoietin producing cells with important information on body iron stores.