Iron, lipocalin, and kidney epithelia



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发表于 2009-4-21 13:39 |显示全部帖子

作者：Jun Yang, Kiyoshi Mori, Jau Yi Li, and Jonathan Barasch作者单位：Department of Medicine and Anatomy and Cell Biology, College of Physicians and Surgeons of Columbia University, New York, New York 10032 , x0 d& F+ Z; k# U% P7 ^9 Y
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      【摘要】. F2 {: \1 R' }" f3 g: |9 f5 ?
   Brilliant new discoveries in the field of iron metabolism have revealed novel transmembrane iron transporters, novel hormones that regulate iron traffic, and iron's control of gene expression. An important role for iron in the embryonic kidney was first identified by Ekblom, who studied transferrin (Landschulz W and Ekblom P. J Biol Chem 260: 15580-15584, 1985; Landschulz W, Thesleff I, and Ekblom P. J Cell Biol 98: 596-601, 1984; Thesleff I, Partanen AM, Landschulz W, Trowbridge IS, and Ekblom P. Differentiation 30: 152- 158, 1985). Nevertheless, how iron traffics to developing organs remains obscure. This review discusses a member of the lipocalin superfamily, 24p3 or neutrophil gelatinase-associated lipocalcin (NGAL), which induces the formation of kidney epithelia. We review the data showing that lipocalins transport low-molecular-weight chemical signals and data indicating that 24p3/NGAL transports iron. We compare 24p3/NGAL to transferrin and a variety of other iron trafficking pathways and suggest specific roles for each in iron transport.
      【关键词】 metanephric mesenchyme induction neutrophil gelatinaseassociated lipocalin NGAL p transferrin embryo
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THE KIDNEY FORMS FROM THE interaction of an epithelial tubule called the ureteric bud and metanephric mesenchymal cells ( 103 ). The ureteric bud produces the collecting ducts, whereas the mesenchymal cells produce the glomeruli and tubules of the nephron. Mesenchymal cells convert into epithelia and form nephrons after receiving signals from the ureteric bud ( 15, 29, 42, 44, 61, 71, 103, 106, 115, 116, 119 ). The ureteric bud has been shown to control the growth of the mesenchyme (for example, by FGFs) ( 4, 92 ), the expression of the essential Wnt-4 gene (for example, by Wnt-6) ( 60 ), and the expression of cohorts of epithelial proteins [for example, by leukemia inhibitory factor (LIF)] ( 6, 95 ).

In this review, we describe a new ureteric bud protein called 24p3 (mouse) or neutrophil gelatinase-associated lipocalcin (NGAL; human) and review its relatives in the lipocalin superfamily. 24p3/NGAL induces epithelial development, but it neither targets the same mesenchymal cells as Wnt-6/Wnt-4 or LIF, nor does it activate the same type of signaling mechanism. We hypothesize that 24p3/NGAL is a novel signaling molecule and iron transporter, and we speculate that it is a new member of the non-transferrin-bound iron pool (NTBI). We describe general mechanisms of iron transport and the settings in which 24p3/NGAL and the NTBI pool are active. Last, we compare iron transport in the developing and adult kidney. Iron metabolism in fetal development is topical: 18% of human pregnancies in the industrialized countries are iron deficient, as are 38-55% of pregnancies in Africa and 62-88% in India, and worldwide the total affected births may near one billion ( 14 ). However, because little is known about embryonic mechanisms of iron handling, we have included a number of hypotheses and suggested experiments.7 r# F$ [0 t* w: H- V; X. z$ v
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24p3/NGAL INDUCES EPITHELIA2 `+ x- w! l+ x

To identify novel ureteric bud factors that activate the mesenchyme, we separated rat metanephric mesenchyme (for the mouse, see Refs. 6 and 60; other species, such as humans, have not been tested) from its ureteric bud ( E12-E14 ) and treated the mesenchyme with soluble ureteric bud proteins. The ureteric bud proteins were obtained from ureteric bud cell lines ( 3 ), and active proteins were purified to homogeneity by chromatography.

When metanephric mesenchyme is treated with ureteric bud proteins, the cells undergo three to five cycles of proliferation ( 133 ) and rapidly express epithelial E-cadherin, zonula occludens-1, and laminin- 5 (by 48 h of the addition of ureteric bud proteins). By 2-4 days of treatment, morphological epithelia appear, and by 7-9 days tubules and finally segmented nephrons have formed. This response was due to two sets of ureteric bud proteins. The first set maintained competent epithelial progenitors for days-week in culture (e.g., "mesenchymal growth TGF- tissue inhibitor of metalloproteinases-2) ( 4, 5, 67, 92 ) but did not cause extensive growth or any evidence of epithelial conversion. The second set of molecules had no survival activity but, when combined with one of the growth factors, induced proliferation followed by tubulogenesis (e.g., LIF, TGF- ) ( 95, 132, 133 ).
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One ureteric bud protein induced mesenchymal growth and tubulogenesis with an unusual delay, suggesting the selection of an undocumented subset of mesenchymal cells. We purified this activity and identified a member of the lipocalin family, called 24p3 [homologue of human neutrophil gelatinase-associated lipocalcin (NGAL)]. Cloned versions of 24p3 and NGAL confirmed the inductive activity, and a variety of techniques showed that 24p3/NGAL was expressed by the ureteric bud and not by metanephric mesenchyme in vivo ( 133 ). This identification was notable because there have been very few functional data for lipocalin-type proteins. In addition, 24p3/NGAL did not acutely stimulate signal transducers and activators of transcription factors (STATs) or -catenin activity (Barasch J, unpublished observations), typical of other kidney inducers, such as LIF and the Wnts ( 95 ), indicating that it activates other types of signaling pathways.

EXPRESSION OF 24p3/NGAL BY ADULT EPITHELIA1 M# l2 ^8 _1 z6 c) U5 g- D6 o# u
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24p3 Was first identified in mouse kidney cells transfected with SV40 T antigen ( 56 ), and NGAL was detected as a protease resistant monomer, homodimer, and metalloproteinase-9-linked heterodimer ( 68, 131 ) from neutrophils. These proteins are very highly expressed by adenocarcinoma of bowel and breast ( 87, 111 ) and by epithelia of diverticulitis, ulcerative colitis, and Crohn's disease. The expression of 24p3/NGAL was related to inflammation, because epithelia lying outside of the inflammatory zone did not express this protein. Additional correlations between 24p3/NGAL and inflammation have been reviewed ( 10, 128, 129 ) and should be contrasted with the limited expression of these proteins in the normal adult and embryonic animal ( 24, 38, 39, 111, 135 ).- l% N# w6 y, [, J2 g
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An explanation for the overexpression of 24p3/NGAL as a result of inflammation or malignancy has been lacking. One additional experiment showed that lipocalins can "enhance" the phenotype of adult epithelia. Introduction of a lipocalin called glycodelin into human breast carcinoma MCF-7 cells resulted in the de novo expression of E-cadherin, -catenin, and cytokeratins 8 and 18 ( 64 ), suggesting that a variety of lipocalins might regulate critical epithelial proteins in an autocrine manner. Glycodelin is also expressed by a subset of epithelia (exocrine glands and placental endothelia) ( 108 ), suggesting that different lipocalins regulate different types of epithelia. These data support a role for 24p3/NGAL as a regulator of the epithelial phenotype in embryo and adult., k& M/ D/ u3 j% w/ v
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However, the observation that 24p3/NGAL is highly expressed during the involution of the mammary gland and the uterus ( 100 ) suggested the alternative hypothesis that 24p3/NGAL mediates apoptosis. Furthermore, the withdrawal of interleukins from pro-B cells ( 26 ) resulted in the marked expression of 24p3, as well as the onset of apoptosis. 24p3 Was proximate to cell death because purified NGAL produced the same response as the withdrawal of the interleukin, including changes in BAX phosphorylation and the repression of activating transcription factor (ATF)x (ATF-5) ( 93 ) by proteosome-dependent degradation. ATF-5 is activated during cell-cell signaling, and it regulates cAMP-responsive elements. These data suggest that epithelia express 24p3/NGAL to reduce the population of inflammatory cells or perhaps to regulate their own demise, rather than to induce or maintain epithelial polarity. Further analysis of these data requires the identification of the 24p3/NGAL receptor and its signaling mechanisms (see below) and an analysis of the ATF family in embryonic kidney. In addition, to determine whether 24p3/NGAL accelerates or inhibits any of these processes, inspection of a 24p3/NGAL-deleted animal might be useful.2 ?: y! n, p" _/ C& F) Q
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LIPOCALINS TRANSPORT SMALL-CHEMICAL SIGNALS
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What is a lipocalin? A family of over 20 small, soluble proteins has been defined predominately on the basis of a highly conserved 3-dimensional folding pattern. The structure is a continuous eight-stranded, anti-parallel -sheet that forms a squat -barrel enclosing a cavity. In addition, outside of the -barrel there is a 3-10 helix at the NH 2 terminus and a -helix at the COOH terminus. Two or three short segments located in parts of the -barrel and helices are conserved throughout the family, whereas overall identity between these proteins is quite low ( 20%).3 N- b+ K, ]$ Z! N1 Q

The cavity within the -barrel is thought to bind and transport a variety of ligands ( 36 ). The best example of a carrier function comes from the study of purpurin ( 105 ) and the retinol binding protein (RBP) ( 84 ). These two lipocalins deliver retinol, the precursor of retinoic acid. Confirmation that RBP transports and delivers retinol comes from the deletion of RBP, which resulted in a deficiency of retinol in the eye ( 122 ). However, the receptor for RBP in the eye is not yet certain. In the proximal tubule of the kidney, megalin acts as an apical RBP receptor, capturing filtered RBP by endocytosis ( 22, 78, 109 ). The deletion of megalin resulted in the appearance of retinol-RBP and retinol-RBP-transthyretin in the urine and their depletion from the proximal tubule, verifying that RBP traffic in the kidney is regulated by megalin. These studies demonstrate that lipocalins are carriers of low-molecular-weight chemicals that in some cases are delivered to cells by receptor-mediated endocytosis.

A second example of a carrier function for a lipocalin comes from the nitrophorins 1-4, which are produced in the salivary glands of Rhodnius prolixus, the vector of Chagas disease. These molecules sequester nitric oxide with a Fe 3  -heme prosthetic group and deliver nitric oxide to induce local vasodilation in the victim of a bite ( 81, 126 ). The Fe 3  -heme site also binds and sequesters histamine in exchange for nitric oxide, and this might limit the inflammatory reaction to these vector proteins. Nitrophorins are the first known nonglobin, heme-containing proteins, and like RBP, appear to play a role in local transfers of important low-molecular-weight chemicals.
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A more complex function is suggested for the major urinary proteins (MUPs) of mice. These proteins are thought to deliver volatile pheromones ( 13, 82 ). However, they may induce mating behavior by a second mechanism as well, because peptides from MUPs are active. This suggested that these lipocalins are bifunctional proteins ( 59 ). This type of analysis is not available for other members of the lipocalin family.( i$ C1 m% F, X$ e3 E

Taken together, these data show that the lipocalins function in intracellular chemical signaling, delivering low-molecular-weight cargo. However, the identification of ligands is still ongoing and is of great interest for 24p3/NGAL, grasshopper lazarillo ( 102 ), which is expressed by neuronal growth cones and tip cells of the Malphigian tubule, and for glycodelin, which is readily endocytosed by placental endothelia ( 136 ). Apo-lipocalins may also signal, but the data are presently limited to one experiment ( 59 ).
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HYPOTHESIS: 24p3/NGAL LIPOCALINS TRAFFIC IRON

We hypothesize that 24p3/NGAL transports iron. Transport of iron might require a cofactor, because bacterially cloned NGAL contains enterobactin, a small ringed structure that binds iron (siderophore) ( 83 ). These data were obtained by X-ray crystallography and molecular modeling ( 41 ). In fact, expression of the protein in bacteria inhibited their growth until additional iron was added to the cultures, suggesting that the cloned 24p3/NGAL bound siderophore-iron in situ ( 41 ).

Mammalian-expressed 24p3/NGAL also captures iron. The data ( 133 ) include 1 ) the copurification of iron and ureteric bud 24p3 in immunoprecipitations and column chromatography; 2 ) the time- and temperature-dependent, saturable accumulation of iron in many cell lines incubated with 24p3; 3 ) the upregulation of ferritin protein and the downregulation of transferrin receptor-1 protein in cells incubated with 24p3/NGAL, indicating the delivery of sufficient quantities of iron to regulate iron-dependent genes; 4 ) the delivery of 24p3 iron by endocytosis into compartments containing the divalent metal transporter; and, last, 5 ) the induction of the metanephric mesenchyme by iron-24p3/NGAL but not by gallium-24p3/NGAL ( 133 ). It is also notable that another lipocalin, the tear lipocalin (LCN-1), is inducible by iron ( 75 ), and many of these findings are also typical of transferrin ( 28, 120, 130 ) and perhaps lactoferrin ( 11, 12 ) but distinguish these proteins from the melanotransferrin pathway ( 37 ). The missing data from this list are an identification of a mammalian homologue of the bacterial siderophore. However, there was a reddish low-molecular-weight substance in the initial steps of purification of 24p3/NGAL, and there are older reports of low-molecular-weight siderophores produced by mammalian cells ( 25, 34, 62 ).- E" Z+ p. V' G

These data support the idea that 24p3/NGAL participate in iron transport and could explain various phenomena associated with this lipocalin. Iron trafficking is thought to be essential for epithelialization of the developing kidney ( 30, 73, 74 ), and lipocalin, produced in situ by the ureteric bud, might provide an alternate to the transferrin pathway. This idea would be most strongly validated by analysis of animals without the transferrin receptor. Second, 24p3/NGAL might be a member of the nontransferrin pool of iron carriers. In particular, 24p3/NGAL could redirect iron acquisition from bacteria to mammalian cells and traffic iron in states of iron overload. These speculations are not exclusive of other types of signaling by 24p3/NGAL, and we await identification of the 24p3/NGAL receptor to further test these possibilities.
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MANY SOLUTIONS TO THE PROBLEM OF IRON TRANSPORT

Iron must be bound to another molecule to be transported from one site to another. This is because in its common oxidized form (ferric or Fe 3  ), iron is entirely insoluble (Ksp 10 -17 M), whereas reduced iron (ferrous or Fe 2  ) is soluble (Ksp 10 -1 M) ( 69 ) but reactive (Fenton reaction) ( 49 ). Hence, iron traffic must start with the oxidation of Fe 2  to Fe 3  by ceruloplasmin or hephaestin (a duodenal homologue of ceruloplasmin) ( 123 ), or else iron overload in basal ganglia and liver (Wilson disease) ( 88 ) results from defective mobilization.
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After iron is oxidized, it is sequestered by transferrin ( 51 ). In fact, essentially all circulating iron in the adult is associated with transferrin, because of a very high affinity for iron at neutral pH (10 -20 M), and transferrin is usually only partially saturated ( 96 ). Transferrin-Fe 3  is imported by nearly all cells after binding transferrin receptors 1 (ubiquitous) and 2 (in hepatocytes), which then carry transferrin into endosomes ( 97 ). Transferrin has a unique pH sensitivity for Fe 3 , and this allows the release of iron but the recycling of apo-transferrin from specialized endosomes ( 28, 120, 130 ). As a result, transferrin efficiently transports the majority of iron in the adult ( 77 ).

However, startling new data show that transferrin is not the only molecule that can traffic iron. hpx/hpx Mice essentially lack transferrin ( 25, 58, 117 ) and as a result are severely anemic, but their Kupffer cells contain 10 times more iron than those of their littermates ( 80 ). This observation suggests that iron exit may require transferrin (or another desaturated acceptor protein) but that iron delivery does not. Atransferrinemic and hypotransferrinemic humans also exist ( 52, 53 ). They too have severe defects in hematopoesis, but most epithelial organs are said to be "normal," including gross kidney functions (personal communication, Dr. Y. Wada, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan; 53). In fact, mice lacking TRR1, the major transferrin receptor, can at least initiate epithelial organogenesis before succumbing to the effects of anemia, further suggesting that nontransferrin iron carriers are sufficient for nonhematopoetic tissues ( 76 ). These data graphically demonstrate the multiplicity of iron transport mechanisms.# S4 E. P0 O9 S* \& k3 |5 ~5 l

The alternative to transferrin-iron is called NTBI. The pool has been documented in a variety of iron overload syndromes when transferrin is saturated ( 101 ), including hemochromatosis (69% of patients) and end-stage renal disease (22% of patients) ( 16, 17, 50 ). Most interestingly, 18% of umbilical cord blood samples contained NTBI ( 31, 50 ), probably because transferrin is less abundant in the human fetus than in the mother (see below) and as a result of incomplete interchange of these pools. Hence, NTBI is known to mediate some component of iron delivery, particularly in iron overload, possibly in gestation, and, we speculate, also in deletions of the transferrin pathway., q% W% ?; e% ~7 b( |  ~2 U/ r

The largest gap in these data is the identification of the components of NTBI. Low-molecular-weight non-transferrin iron carriers have been partially purified but never clearly identified ( 25, 34, 62 ). Candidates include iron salts, such as iron ascorbate, citrate, or nitrilotriacetate, which display saturable uptake in cell lines ( 46, 63, 66, 112 ). Other candidates have been defined by an affinity for iron that is relatively less than transferrin ( 16, 17, 50 ). Typically, low-affinity binding is measured by iron absorption on ion-exchange beads ( 55 ) or chelators such as oxalate or EDTA, followed by transfer to a fluorescent or DNA binding molecule, or transfer through an ultrafiltration membrane. These chelators distinguish the NTBI pool because they do not remove iron from transferrin. This definition is inclusive of abundant serum molecules like albumen, but whether it also includes 24p3/NGAL is not yet clear.  @5 ?* R  e; w, B) x
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How does iron enter the cell cytoplasm? Divalent metal transporter (DMT)-1, 3 -integrin, and stimulator of iron transport (SFT) can transport iron from transferrin and from NTBI. DMT-1 is a divalent metal transporter that is located on the apical membrane of the duodenal enterocyte and in endosomes of most cells. It captures Fe 2  that has been reduced from dietary Fe 3  in the duodenum ( 19, 45, 80 ) and from Fe 3  -transferrin and apparently from Fe 3  -NTBI in endosomes ( 35, 43, 104, 113 ). Because DMT1 is an Fe 2  -H symporter, the recovery of iron by DMT1 is stimulated by the acidity of the duodenal contents and by the activity of vacuolar H -ATPase ( 45 ). Conversely, the capture of iron is blocked by the alkalinization of endosomes ( 40 ). These findings stress the complexity of iron capture, because intracellular DMT1 must colocalize with a ferrireductase, the vacuolar H -ATPase, and soluble carriers of iron. Precise localization of these components in different organs is a central aim of research in iron metabolism ( 113 ). In fact, the intracellular itinerary of DMT1 is not fully established, including whether it traffics to the cell surface of nonenterocytes or whether it is present in more than one population of vesicles. The importance of the DMT1 pathway is established by two naturally occurring mutations, the mk mouse and the Belgrade rat ( 19, 35, 45 ), that feature severe anemia.9 _! i. w7 O8 _+ K! x% b

Ferric iron can also be transferred to the cytoplasm by binding a complex composed of 3 -integrin, mobilferrin, and a flavin monooxygenase. Evidence for this complex includes (see also Ref. 7 ) the specific blockade of ferric iron transport by antibodies to 3 -integrin and the unique specificity for Fe 3 , rather than Fe 2  or other divalent metals that characterize DMT1 ( 23 ). However, DMT1 might be part of the integrin- 3 -mobilferrin pathway ( 118 ), suggesting that it may modify DMT1 specificity. Whether these components transport iron from the cell surface or from endosomes is presently not clear, because most of these proteins are intracellular ( 107 ).

A third pathway for iron transfer is SFT protein ( 47 ). SFT is highly expressed in apical plasma membranes of the intestine, lung, and kidney, as well as the brain ( 70 ) and in transferrin-recycling endosomes. SFT has six membrane-spanning domains and a cytoplasmic iron-binding motif (REXXE), which is necessary for iron transport ( 134 ). In addition, iron transport requires binding and hydrolysis of ATP, despite the absence of a Walker motif or any requirement for a transmembrane gradient pH or potential ( 48 ). Transfection of SFT cDNA enhances both transferrin and NTBI transport but not the transport of other divalent metals. All of these characteristics demonstrate that SFT is distinct from DMT1, and hence it is plausible that SFT provides an alternative route for iron transport that mitigates the lethality of defects in DMT1. In fact, SFT can dimerize and mimic the 12 membrane-spanning structure of DMT1. The importance of SFT is also suggested by its upregulation in the setting of iron depletion ( 8 ). However, there is more to learn about this iron transport pathway because some data show that ferrous and not ferric iron is the transported species ( 134 ), perhaps indicating some shared mechanisms with DMT1.

In conclusion, while the transferrin pathway has physiological predominance in wild-type and DMT1 is the critical transmembrane transporter, there are many other routes for iron transfer that may dominate different settings and with different effects.

24p3/NGAL VS. TRANSFERRIN IN THE DEVELOPING KIDNEY: NOT THE SAME5 F0 Y/ p) N: j: V

If NTBI can substitute for transferrin in some developing organs, the important question is whether 24p3/NGAL is one of these substitutes. First, it would be valuable to know where 24p3/NGAL and its putative receptor are expressed in normal gestation and whether they are overexpressed in mice and humans with deletions of the transferrin pathway.
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Second, it would be valuable to test different iron carriers in explanted embryonic organs to see whether they can replace transferrin or whether they have distinct functions. For example, Fe 3  -transferrin must be added to minimal essential media to permit robust branching of the ureteric bud, conversion of mesenchyme, and growth of nephrons ( Fig. 1 ) ( 30, 73, 74 ). 24p3/NGAL can also rescue cultures that are iron poor, but not to the same extent as transferrin ( Fig. 1 ). In fact, in isolated metanephric mesenchyme these molecules are complementary rather than redundant. Both 24p3/NGAL and transferrin must be added to the explant to generate nephrons: tubulogenesis was possible when 24p3/NGAL was added before transferrin (by 24-48 h) but not after. This suggested that 24p3/NGAL rescued epithelial progenitors from apoptosis, whereas transferrin does not have this activity ( 132 ).) v  e0 r- }5 ?  g9 D& E

Fig. 1. Partial rescue of transferrin by 24p3/neutrophil gelatinase-associated lipocalcin (NGAL). Mouse kidneys were harvested at embryonic day 11.5 ( E11.5; ureteric bud was T-shaped) from animals expressing -galactosidase in the BF-2/Foxd1 locus (marking stromal cells) and were cultured in a low-iron media (DMEM) for 6 days. The media was supplemented ( 67 ) but without transferrin. A : while the stroma remained viable (blue; nuclear staining), there was limited branching of the ureteric bud and very limited expansion or development of the mesenchyme. B and C : when either iron-loaded 24p3/NGAL (doubly purified recombinant protein; 50 µg/ml) or holo-transferrin (50 µg/ml) was added to the media, the ureteric bud underwent branching morphogenesis and the metanephric mesenchyme expanded, resulting in the centrifugal displacement of stromal cells. However, the effect was more dramatic with transferrin than with 24p3/NGAL. The data suggest that both molecules deliver iron to the mesenchyme by no redundant pathways.4 h: ^3 P4 ]( W( M9 j

Functional dissimilarity between 24p3/NGAL and transferrin could result from activities other than iron delivery, which we have not ruled out. On the other hand, we found that the two proteins targeted different mesenchymal cells ( Fig. 2 ). 24p3/NGAL was taken up throughout the kidney, but most prominently at the cortical margin, which is composed of mesenchymal (early epithelial progenitors?) and stromal progenitors. In contrast, the transferrin domain was restricted to Pax-2  , WT1  , Wnt-4 mesenchymal cells and their progeny that surround the ureteric bud (late epithelial progenitors and nascent epithelia, respectively). These data suggest that 24p3/NGAL and transferrin act sequentially in lineally related cells.
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Fig. 2. Distinct targeting of embryonic mesenchyme by 24p3/NGAL and transferrin. Rat kidneys [ E13.5 ( A ) and E15 ( B )] were incubated with fluorescein-transferrin (green) and Alexa 568- 24p3/NGAL (red) for 3 h. A unique distribution of the 2 molecules is found. 24p3/NGAL is incorporated throughout the kidney, but especially at the cortical margin (contains stromal cells and early epithelial progenitors), whereas transferrin is incorporated only by late epithelial progenitors and their progeny that surround the ureteric bud.; z/ _8 F1 |6 _( @
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From these data we speculate that transferrin-iron and non-transferrin-iron delivery may overlap but are not fully redundant. For example, transferrin receptor 1 is generally induced at the commencement of cell proliferation (by stem cell factor through the c-MYC pathway in hematopoeitic cells) ( 65, 110 ), but is not expressed during the initial part of the lineage. In the kidney, proliferation and conversion of late-stage epithelial progenitors in vitro were supported by transferrin, whereas the initial part of the lineage did not require this protein ( 30, 73, 74, 114 ). Similarly, lactoferrin is transiently expressed by the nasopharynx, proximal gut, and airway ( 124 ), and uteroferrin by the uterus ( 32 ). These data confirm the idea that the delivery of iron is specialized to specific stages of development and suggest that heterogeneity of iron delivery is a general principal in organ development.
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HETEROGENEITY OF IRON TRANSPORT = VARIATION IN CYTOPLASMIC IRON?4 p! E8 [  e( g1 T! R. P7 }; |/ V

Not only might different carriers direct iron to different cells, but they also might deliver iron to different intracellular compartments ( 40 ). For example, incubation of erythroid cells with transferrin-Fe 3  resulted in the production of heme-Fe 3 , but incubation with non-transferrin-Fe 3  resulted in deposition of iron in a separate compartment (see Fig. 3 ). Perhaps, then, different iron transport systems produce different levels of cytoplasmic iron in lineally related cells. This quantity is important because it regulates transcription of many genes and the translation of others ( 69 ). In fact, ferritin ( Fig. 4 ), which is regulated by cytoplasmic iron, is most highly expressed in late epithelial progenitors adjacent to the ureteric bud, whereas peripheral cells had less expression. Eya, which is necessary for renal development ( 133 ), may also be regulated by iron and has a similar localization. The expression of different iron-dependent genes implies that late progenitors have a higher level of cytoplasmic iron than earlier cells, but measurement of this iron gradient awaits invention of a new type of probe ( 2 ).  [  A7 h  V7 L: A

Fig. 3. Distinct subcellular trafficking of NGAL (red) and transferrin (green) in kidney cells. Species-matched ligands and cells were used. Steady-state labeling shows that the 2 proteins occupy different organelles, with limited overlap (yellow arrows).) o! F: [3 t/ r1 v& O" |8 K$ O
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Fig. 4. Ferritin expression detected by immunocytochemistry in rat kidney ( E13.5 ). Late epithelial progenitors surrounding the ureteric bud (UB) express higher levels of ferritin than peripheral progenitors or stromal cells. A gradient of ferritin should (theoretically) reflect increasing levels of cytoplasmic iron ( 69 ).) f! v6 g. O" \! _8 ~, ^
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THE ADULT KIDNEY IS A PRINCIPAL ORGAN OF IRON TRANSPORT7 C5 M- i) v0 ]! _
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In rats, 0.5 mg/day of iron is filtered, but only 5 µg/day are excreted ( 125 ). In humans, urinary iron is not detectable ( 18 ). Iron reabsorption occurs in the thick ascending loop of Henle (TALH) and cortical collecting ducts ( 125 ). Indeed, adult rats express DMT1 on the apical cell surface of the TALH, distal convoluted tubule ( 33 ) intercalated cells of the cortical collecting ducts, and perhaps brush borders of the proximal tubule ( 20 ), whereas in other segments DMT1 is present only in endosomes.

There are complex kidney-specific mechanisms that regulate DMT1 expression and trafficking. Four forms of DMT1 are distinguished by two types of exon-1 and the presence or absence of an iron-responsive element (IRE) in the 3'-untranslated region that confers iron dependence on the half-life of mRNA. The duodenum mostly regulates the 1A  IRE transcript, whereas the kidney most highly expresses the 1B -IRE version ( 57 ), indicating much less flexibility in variation with iron load ( 57 ). Nonetheless, it would be interesting to measure urinary iron and DMT1 expression after changes in dietary iron ( 20 ). In addition, the DMT1-defective mouse (mk/mk) and rat (Belgrade) should also be examined to determine whether DMT1 is essential for iron recovery.( n8 d  M# F0 r$ A# i: O( P  e0 T0 z) L

However, the kidney has other mechanisms to recapture iron, in addition to DMT1. Cubilin and megalin are present on the apical membrane of the proximal tubule and recover filtered transferrin, routing it to lysosomes, where it is degraded ( 72, 89 ). This pathway is quite unusual in that transferrin is usually recycled from the basolateral domain. However, the apical pathway is important because deletion of cubulin or megalin leads to urinary loss of transferrin. In addition, a number of disease-inducing permeability defects in the glomerulus cause urinary loss of transferrin ( 98, 121 ).

It is also likely that 24p3/NGAL is filtered by the glomerulus and reabsorbed by the proximal tubule. 24p3/NGAL is present in the urine of normal animals and humans, but at low levels. In the setting of malignancy, the level of urinary lipocalin is increased, appearing as a 25- or 35-kDa monomer and a 50-kDa dimer, as well as higher molecular weight species cross-linked to matrix metalloproteinase-9 ( 131 ). One study using pathological specimens showed that NGAL is present in the proximal tubule in a patchy distribution, suggesting that the complex could be cleared (or synthesized) at this site ( 38 ). These studies introduce a second mechanism by which the kidney participates in the regulation of iron metabolism, the catabolism of iron-proteins.9 V+ S$ x* \* e6 G8 r- ^. v+ u
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There are likely to be other mechanisms of iron recovery in the kidney that have not been explored. SFT ( 70 ), basolateral iron transporter ferroportin ( 27, 80 ), and hephaestin (or ceruloplasmin) ( 123 ) are expressed, but localization in the kidney needs to be determined to indicate whether these proteins recover iron from the filtrate. Furthermore, it will be necessary to determine whether these molecules are regulated by hepcidin ( 85, 86, 94 ), a circulating hormone that negatively regulates iron absorption in the gut. Perhaps this hormone increases iron loss in the urine, an effect that would be reminiscent of the regulation of phosphate by parathyroid hormone, but this is entirely speculative. Last, it would be important to determine whether pharmacological therapy enhances iron loss. One idea is to measure urinary iron after diuretic therapy, when the tubular flow rate is increased and other cations, such as K  , are lost. In addition, changes in the transapical membrane potential should affect iron reclamation, given that transport of iron by DMT1 is electrogenic (e.g., Bartter's syndrome) ( 54 ).

In sum, given the glomerular filtration of iron and a variety of recovery mechanisms throughout the nephron, these data show that the adult kidney is a principal organ of iron traffic.

DIFFERENT IRON TRANSPORT IN ADULT AND EMBRYO?. @' ]8 B$ ?" R0 Q5 G  E' O
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The adult kidney provides a guide to the type of carriers and transporters that must be expressed by the time of birth. However, iron trafficking in the adult and embryonic kidneys must be quite different. First, the rodent mesenchyme expands from 10 4 cells to 10 5 nephrons in a 2-wk period, meaning that there is a dramatic requirement for iron absorption to support proliferation. Hence, the 1A  IRE DMT1 isoform may be expressed in embryo, rather than the non-IRE 1B form of the adult. Conversely, iron absorption by the developing nephron may be less stringent than in the adult because fetal urine is recycled through the amnion and the gastrointestinal tract ( 90, 91, 99 ). Third, neonatal blood contains NTBI ( 9, 31, 50 ), which is likely to traffic by pathways not found in the adult. In fact, because of low Po 2, high levels of ascorbate, and low levels of ceruloplasmin that have been found in the newborn, some of the circulating iron may be soluble Fe 2 .

The presence of NTBI in the embryo, and the absence of a dramatic disturbance of organogenesis in animals lacking components of the transferrin pathway, may indicate that local, rather than long-range, transfers of iron are important in the embryo. For example, many metanephric mesenchymal cells become apoptotic, and it is not clear what happens to their iron. Perhaps ureteric bud 24p3/NGAL is present to scavenge this iron and return it to viable cells. While local transport mechanisms might play a role in iron trafficking in the embryo, this may not be important in the adult, except in the setting of inflammation and cell death, where we speculate that epithelia recapitulate a local mechanism of iron recovery by reexpressing 24p3/NGAL./ F5 U7 m6 u& G3 @/ V/ q) T

ACKNOWLEDGMENTS4 X1 A# W8 h, x4 u
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J. Barasch is supported by Research Grant FY-00165 from the March of Dimes Birth Defects Foundation and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-55388 and DK-58872.
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Address for reprint requests and other correspondence: J. Barasch, Dept. of Medicine and Anatomy and Cell Biology, College of Physicians and Surgeons of Columbia Univ., 630 W 168th St., New York, NY 10032 (E-mail: jmb4{at}columbia.edu - g8 V# l* C$ I
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