Am J Physiol Renal Physiol 291: F22–F36, 2006;
doi:10.1152/ajprenal.00385.2005.
Invited Review
The kidney in vitamin B12 and folate homeostasis: characterization
of receptors for tubular uptake of vitamins and carrier proteins
Henrik Birn
Department of Cell Biology, Institute of Anatomy, University of Aarhus, Aarhus, Denmark
megalin; cubilin; folate-binding protein; proximal tubule; endocytosis
organ, in which serum constituents
are specifically sorted either for urinary excretion or for conservation within the body. During this process, the kidneys
must handle a large number of different endo- and exogenous
substances, many of which are essential to life. Vitamins
constitute such important substances normally required from
food sources and essential to fundamental biological reactions.
Vitamin B12 and folate both belong to the group of watersoluble B vitamins. While structurally different, they interact in
common biochemical reactions and reveal a very similar picture of megaloblastic anemia when deficient. They are present
in serum either free or bound to carrier proteins and are filtered
in the renal glomeruli. Estimations of the filtration fraction
suggest that efficient renal tubular reabsorption of both is
essential to prevent excessive urinary losses in both humans
and animals. Animal studies within recent years have established close interaction and a functional relationship between
the structurally very different receptors involved in the renal
tubular uptake of these vitamins and their carrier proteins. The
present review will focus on the receptors and molecular
mechanism mediating renal uptake of folate and B12 and of
their carrier proteins. The common features of these processes,
THE KIDNEY IS AN EXCRETORY
Address for reprint requests and other correspondence: H. Birn, Dept. of
Cell Biology, Institute of Anatomy, Univ. of Aarhus, Bldg. 234, DK-8000
Aarhus C, Denmark (e-mail: [email protected]).
F22
involved also in the kidney uptake of a number of other
vitamins, hormones, and carrier proteins, will be discussed.
FOLATE
Folate is a water-soluble vitamin recognized in the 1930s as
a hematopoietic factor present in liver and yeast extracts. It was
isolated from 4 tons of spinach leaves in 1941 and reported to
be concentrated in liver and kidney. Chemically, it is made of
a pteridine core ring linked to p-aminobenzoic acid. Additional
glutamate residues are attached to the p-aminobenzoic acid,
forming mono- or polyglutamate forms. Mammals are able to
synthesize the pteridine ring but are unable to couple it to other
compounds and are thus dependent on either dietary intake or
bacterial synthesis within the intestine. The term folic acid
generally is used for the synthetic pteroylglutamic acid representing fully oxidized folate. The biologically active form is
reduced tetrahydrofolate (THF)1, serving as an essential cofactor in methylation reactions, including the vitamin B12-dependent formation of methionine from homocysteine, and as a
carrier of one-carbon units involved in the synthesis of purines
and pyrimidines (Fig. 1). Whereas intracellular folate accumulates in the polyglutamate form, folate in serum is in the
monoglutamate form, the only form that is transported actively
1
The present review will apply the term “folate” when referring to all
different forms of the vitamin that may be converted into the active form.
0363-6127/06 $8.00 Copyright © 2006 the American Physiological Society
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Birn, Henrik. The kidney in vitamin B12 and folate homeostasis: characterization of receptors for tubular uptake of vitamins and carrier proteins. Am J Physiol
Renal Physiol 291: F22–F36, 2006; doi:10.1152/ajprenal.00385.2005.—Over the
past 10 years, animal studies have uncovered the molecular mechanisms for the
renal tubular recovery of filtered vitamin and vitamin carrier proteins. Relatively
few endocytic receptors are responsible for the proximal tubule uptake of a number
of different vitamins, preventing urinary losses. In addition to vitamin conservation,
tubular uptake by endocytosis is important to vitamin metabolism and homeostasis.
The present review focuses on the receptors involved in renal tubular recovery of
folate, vitamin B12, and their carrier proteins. The multiligand receptor megalin is
important for the uptake and tubular accumulation of vitamin B12. During vitamin
load, the kidney accumulates large amounts of free vitamin B12, suggesting a
possible storage function. In addition, vitamin B12 is metabolized in the kidney,
suggesting a role in vitamin homeostasis. The folate receptor is important for the
conservation of folate, mediating endocytosis of the vitamin. Interaction between
the structurally closely related, soluble folate-binding protein and megalin suggests
that megalin plays an additional role in the uptake of folate bound to filtered
folate-binding protein. A third endocytic receptor, the intrinsic factor-B12 receptor
cubilin-amnionless complex, is essential to the renal tubular uptake of albumin, a
carrier of folate. In conclusion, uptake is mediated by interaction with specific
endocytic receptors also involved in the renal uptake of other vitamins and vitamin
carriers. Little is known about the mechanisms regulating intracellular transport and
release of vitamins, and whereas tubular uptake is a constitutive process, this may
be regulated, e.g., by vitamin status.
Invited Review
THE KIDNEY IN B12 AND FOLATE HOMEOSTASIS
F23
across cell membranes. Folate deficiency is associated with
homocysteinemia, megaloblastic anemia, leuco- and thrombocytopenia, cardiovascular disease, embryonic defects, in particular neural tube defects, and, possibly, malignancies (135).
Dietary folate is found in vegetables, fruits, grain, yeast, and
dairy products. Total body content of folate has been estimated
to be 38 –96 mg (86 –165 ␮mol), being slowly catabolized or
excreted mainly by the fecal route.
AJP-Renal Physiol • VOL
Following ingestion, folate polyglutamate is converted into
monoglutamate by intestinal brush-border glutamylcarboxy
conjugase and absorbed most likely by a carrier-mediated
mechanism involving the reduced folate carrier (RFC) (86).
The dominant folate form in serum is 5-methyl-tetrahydrofolate (5-MTHF) present either free, bound to high-affinity folate-binding protein (FBP), or loosely associated with other
serum proteins including albumin (61, 132). The fraction of
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Fig. 1. Metabolic pathways involving folate and vitamin B12. The major folate form in serum is 5-methyltetrahydrofolate (5-MTHF), in part bound to
folate-binding protein (FBP) or associated with albumin. Folate is trapped within the cell by polyglutamination of THF, which cannot be transported across the
cell membrane. Various folate derivatives, i.e., 5,10-methylene-THF, 10-formyl-THF, and 5-MTHF, serve as 1-carbon donors in biochemical reactions involving
the synthesis of purines, thymidine, and the conversion of homocysteine to methionine. The interconversion between various folate forms involves intermediate
forms and reactions not shown. The major source of 1-carbon units for folate metabolism in mammalian cells is the conversion of serine to glycine. Serum B12
is normally coupled to the binding proteins transcobalamin (TC) and haptocorrin (HC); however, most cells take up only TC-B12. Following internalization, B12
may be metabolized into the biological active forms methylcobalamin and 5-deoxyadenosylcobalamin by reactions involving reduction of the cobalt atom
(indicated by 2⫹ and 1⫹) and various enzymes not shown. 5-Deoxyadenosylcobalamin serves as a cofactor for the mitochondrial methylmalonyl-CoA-mutase
catalyzing of the conversion of methylmalonyl-CoA into succinyl-CoA, important for the oxidation of odd-chain fatty acids and for the degradation of certain
amino acids. Methylcobalamin, initially formed by methylation of enzyme-bound B12, serves as a cofactor for cytosolic methionine synthase. This enzyme
transfers a methyl group from 5-MTHF to homocysteine, forming methionine, the single known metabolic pathway common to folate and B12 (blue arrows).
The transfer of a methyl to homocystein causes reduction of methionine synthase-bound B12 and is followed by remethylation using a methyl group donated by
5-MTHF. Methionine may be converted into adenosyl-methionine, an important methyl donor in a number of different reactions. It is clear from the diagram
why both folate and vitamin B12 deficiency may lead to elevated homocysteine due to inhibited conversion of this into methionine. Vitamin B12 deficiency may
also lead to elevated methylmalonic acid (MMA) as a result of accumulation of methylmalonyl-CoA which is converted to MMA. Because the conversion of
5,10-methylene-THF to 5-MTHF is essentially irreversible, folate accumulates as 5-MTHF when the methionine synthase is blocked due to B12 deficiency. This
results in a functional folate deficiency despite elevated 5-MTHF-levels. Thus both folate and vitamin B12 deficiency may lead to inhibited synthesis of
nucleotides for RNA and DNA, causing megaloblastic anemia. Vitamin B12 deficiency is also associated with neurological disturbances, which may be explained
by decreased methylation of myelin basic protein, an important constituent of myelin, due to interruption of methionine formation. In contrast, neurological
symptoms are normally not associated with folate deficiency. This may be explained, in part, by the ability of nerve tissue to concentrate folate significantly above
serum levels. Enzymes appear in italics. Vitamin-B12 as an enzyme-bound cofactor is indicated in red. Dotted arrows indicate that the processes may involve
several transfer and/or biochemical steps not shown. The processes are reviewed in Refs. 25, 110, and 123. DHF, dihydrofolate.
Invited Review
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THE KIDNEY IN B12 AND FOLATE HOMEOSTASIS
folate bound to high-affinity serum proteins varies between
species, being high in pigs (83) and relatively low in rodents
such as mice and rats (127). The concentration of FBP in
human serum is estimated to be 0.6 nM (61) compared with a
normal serum folate concentration of 5–20 nM. The affinity of
FBP for folate in serum is ⬃0.1 nM, suggesting that serum
FBP is fully saturated. The amount of folate associated with
serum proteins has been estimated to be ⬃20% in rats within
a wide serum folate concentration range (127). In humans, the
fraction of protein-bound folate has been estimated to be from
⬃20 (156) to ⬃65%, being constant within a wide concentration range.
VITAMIN B12
FOLATE, VITAMIN B12, AND THE KIDNEY
The renal uptake of both folate and vitamin B12 involves
glomerular filtration followed by tubular reabsorption. Significant amounts of vitamins are filtered daily, and because
urinary excretion of B12 and intact folate is low, both are
reabsorbed within the renal tubular system to prevent urinary
loss. Furthermore, tubular uptake may result in kidney accumulation and possibly metabolism of B12. In addition to the
physiological relevance, interest in the molecular mechanisms
regulating these processes is stimulated by the clinical focus on
folate and vitamin B12 in relation to renal disease. Epidemiological studies show an association between elevated levels of
homocysteine and an increased risk of cardiovascular disease
(25) also in renal patients (5, 32). Folate supplementation is
able to reduce elevated homocysteine levels in renal patients
(32), and it was suggested that additional supplementation with
vitamin B12 may further lower homocysteine levels even in
patients with normal serum B12 levels (37). The metabolic
changes underlying elevated homocysteine levels in renal disease are not fully understood; however, the use of high-dose
folate and/or vitamin B12 supplementation in renal patients
attracts interest to the way these vitamins are handled by the
kidney.
Glomerular Filtration of Vitamins and Carrier Proteins
2
The term “vitamin B12” is chemically restricted to cyanocobalamin;
however, the present review will refer to all potential biologically active
cobalamins as vitamin B12 or B12. These include the coenzyme forms methylcobalamin and 5-deoxyadenosylcobalamin, as well as hydroxycobalamin
and cyanocobalamin, all found in serum with methylcobalamin as the dominant form (45).
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The molecular mass of folate (⬃440 Da) suggests that free
folate is freely filtered in the glomeruli. Due to the association
of folate with serum proteins, the filtration of folate in humans
is estimated to be 50 – 65% of that of inulin (46), suggesting a
filtered load of free folate of ⬃1 mg/24 h in humans (127). A
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Vitamin B122 was originally identified as the antianemic,
extrinsic factor present in liver and liver extract, reversing the
classic symptoms of megaloblastic anemia. In 1948, vitamin
B12 was isolated from liver as a red crystalline substance
causing clinical remission in cases of pernicious anemia.
Chemically, it contains a corrin ring consisting of four reduced
pyrrole rings surrounding a central cobalt atom. The different
chemical groups attached to the central cobalt atom classify the
type of cobalamin derivative. Vitamin B12 serves as a cofactor
in two essential biochemical reactions involving 1) the formation of methionine from homocysteine by cytoplasmic methionine synthase using 5-MTHF as one carbon donor, and 2) the
interconversion of methylmalonyl-CoA to succinyl-CoA by
mitochondrial methylmalonyl-CoA mutase (Fig. 1). Thus B12
deficiency is associated with accumulation of homocysteine,
methylmalonic acid (MMA), and, in some cases, also 5-MTHF
as a result of the trapping of 5-MTHF due to decreased
methionine synthase activity. Clinically, B12 deficiency results
in megaloblastic anemia, indistinguishable from that caused by
folate deficiency, and neurological dysfunction. The total body
content of vitamin B12 varies; however, most estimates are
⬃2–3 mg, with 1–1.5 mg in the liver, classically considered the
major organ for B12 accumulation (1, 45). Mammals are unable
to synthesize vitamin B12 and thus rely on dietary intake;
however, they are able to convert the different forms of vitamin
B12 into the active forms, methylcobalamin and 5-deoxyadenosylcobalamin. Vitamin B12 is synthesized by microorganisms, particularly in the gastrointestinal tract of herbivorous
animals, and absorbed. Thus the major food sources of the
vitamin are liver, meat, fish, dairy products, eggs, and shellfish.
After ingestion, vitamin B12 is released from dietary protein
by the acidic environment and peptic digestion in the stomach,
followed by binding to haptocorrin (HC), a ⬃65-kDa glycoprotein secreted in saliva and gastric juice and favored by the
acidic environment. In the duodenum, pancreatic secretion
raises intraluminal pH and facilitates degradation of HC by
pancreatic proteases, causing vitamin B12 to bind to intrinsic
factor (IF; molecular mass ⬃50 kDa) secreted by the parietal
cells of the gastric mucosa and by the pancreas (125). The
IF-B12 complex is absorbed in the ileum by binding to the
intestinal IF-B12 receptor, the cubilin-amnionless (AMN) complex (40, 126). The binding to the receptor depends on B12
binding to IF (11). Following absorption, B12 is released from
IF within the enterocyte (113). In serum, B12 is bound to
transcobalamin (TC), a 45-kDa nonglycosylated serum protein,
and HC. Normal B12 concentration in human serum is estimated to be ⬃0.3 nM (102) compared with ⬃1.1 nM in
normally fed laboratory rats (7). The concentration of TC in
normal human serum is ⬃1 nM, of which ⬃10% is saturated,
depending on vitamin intake (15), whereas the concentration of
HC is ⬃0.4 nM (93), of which ⬃75% is saturated (101). Thus
normally no free B12 is present in the circulation. Although TC
binds only ⬃25% of the circulating vitamin, it is responsible
for the delivery of B12 to most tissues. Indeed, whereas inherited TC deficiency is associated with severe megaloblastic
anemia, patients with HC deficiency may be asymptomatic
with normal levels of MMA despite low total serum B12 (110).
This is also reflected in the half-life of the two carrier proteins,
being ⬍2 h for TC compared with several days for HC. HC
binds to the asialoglycoprotein receptor, and in adults it is
probably only taken up in significant amounts in the liver (24).
The function of HC in adults is not fully understood, although
a role in the clearance of cobalamin analogs from the circulation has been suggested (39). Vitamin B12 is a highly conserved vitamin, and the daily losses in humans is estimated to
be 0.1– 0.2% of total body content (1). The highest losses occur
through feces, and B12 is secreted in bile, although to a large
extent reabsorbed when supported by sufficient secretion of IF
and a functional ileal-absorptive apparatus (48).
Invited Review
F25
THE KIDNEY IN B12 AND FOLATE HOMEOSTASIS
Renal Tubular Reabsorption of Filtered Vitamin and
Carrier Proteins
The major pathway for the uptake of filtered macromolecules in the proximal tubules is by receptor-mediated endocytosis. This involves the specific binding of a ligand to a
receptor in the apical plasma membrane. The receptor-ligand
complex is internalized by invagination of the plasma membrane caused by adaptor molecule-mediated formation of a
cytoplasmic clathrin coat (122). Internalization is followed by
dissociation of the invaginations from the plasma membrane,
forming vesicles. While the coat detaches, vesicles may fuse
with other newly formed vesicles, or with an existing pool of
larger vesicles, followed by acidification of the intravesicular
lumen and the dissociation of the ligand from the receptor. The
ligand may be further transported into lysosomes for degradation, or possibly storage, or into the cytosol for further processing/transport. The receptor is recycled back to the luminal
membranes through a recycling compartment; however, it may
also be transported to lysosomes for degradation. In addition to
the formation of a clathrin coat, other mechanisms of internalization have been established, including noncoated endocytosis
and internalization by caveolae involving the protein caveolin
(63). It has been proposed that glycosylphosphatidylinositol
(GPI)-anchored proteins may by internalized by caveolae
rather than by clathrin-coated pits (3).
Several different receptors mediating endocytosis of filtered
ligands have been identified in the kidney proximal tubule cell
(reviewed in Ref. 28). The receptors, megalin and cubilin, and
the folate receptor (FR) have been implicated in the uptake of
folate, vitamin B12, and their carrier proteins. The structure,
renal expression, regulation, and mutual interaction of these
receptors will be reviewed, followed by a discussion of their
possible role in the renal handling of B12 and folate.
Megalin. Megalin is a multifunctional, endocytic receptor
binding a number of structurally and functionally different
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Table 1. Ligands for megalin and cubilin
Megalin
Cubilin
Vitamin carrier proteins
Transcobalamin-vitamin B12
Vitamin D-binding protein
Retinol-binding protein
Folate-binding protein
Intrinsic factor-vitamin B12
Vitamin D-binding protein
Other carrier proteins
Albumin
Myoglobin
Hemoglobin
Lactoferrin
Neutrophil-gelatinase-associated lipocalin
Oderant-binding protein
Transthyretin
Liver-type fatty acid-binding protein
Sex hormone-binding globulin
Albumin
Myoglobin
Hemoglobin
Transferrin
Lipoproteins
Apolipoprotein
Apolipoprotein
Apolipoprotein
Apolipoprotein
Apolipoprotein
B
E
J/clusterin
H
M
Apolipoprotein Al
High-density lipoprotein
Hormones, hormone precursers, and signaling proteins
Parathyroid hormone
Insulin
Epidermal growth factor
Prolactin
Thyroglobulin
Hedgehog protein
Angiotensin II
Leptin
Bone morphogenic protein 4
Enzymes and enzyme inhibitors
Plasminogen activator inhibitor-type 1
Plasminogen activator inhibitor-type
1-urokinase
Plasminogen activator inhibitor-type
1-tissue plasminogen activator
Pro-urokinase
Lipoprotein lipase
Plasminogen
␣-Amylase
␣1-Microglobulin
Lysozyme
Immune- and stress-response related proteins
Immunoglobulin light chains
Pancreatitis-associated protein 1
Advanced glycation end products
␤2-Microglobulin
Immunoglobulin light chains
Clara cell secretory protein
Receptors and transmembrane proteins
Cubilin
Heavy metallotionein
Cation-independent mannose-6-phosphate
receptor
TCII-B12 receptor
Megalin
AMN
Drugs and toxins
Aminoglycosides
Polymyxin B
Aprotinin
Trichosantin
Others
Receptor-associated protein
Ca2⫹
Cytochrome c
Receptor for seminal vesicle secretory
protein II
AMN, amnionless.
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small fraction of folate is bound to serum FBP. The molecular
mass of ⬃35 kDa suggests that this protein to a large extent is
filtered, and FBP has been detected in human urine at a concentration of 0.5– 4 nM (50). Most protein-bound folate in serum is
loosely associated with albumin (132). The fraction of filtered
albumin is traditionally considered to be low; however, recent
evidence has suggested that a much larger amount is filtered in the
normal kidney (117), although this is yet to be established.
The glomerular filtration of vitamin B12 similarly is dependent on serum protein binding and thus on the concentration of
B12 in serum. In humans, no urinary excretion of B12 was
detected at concentrations ⬍1.1 ng/ml (⬇800 pM), whereas at
concentrations ⬎12 ng/ml (⬇8 nM) B12 is excreted at a rate
similar to the glomerular filtration rate (147). Thus unbound
B12 is freely filtered and may in fact serve as a marker to
estimate glomerular filtration rate (99). At normal serum B12
concentration, the filtered load of the vitamin in humans has
been estimated to be 1.5 ␮g (79). The molecular mass of TC
suggests that the TC-B12 complex is filtered, supported by the
demonstration of small amounts of B12-binding proteins, including TC, in human urine (13, 79, 143). Most circulating HC,
originating from myeloid cells, is heavily glycosylated (39),
probably limiting filtration of this protein, and the origin of
urinary HC remains to be established.
Invited Review
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THE KIDNEY IN B12 AND FOLATE HOMEOSTASIS
dependent and favored by cationic sites on the ligands (91).
However, many ligands are anionic proteins, indicating that the
distribution of charge rather than the overall isoelectric point is
important for binding.
RENAL EXPRESSION. Megalin is heavily expressed in the kidney proximal tubule brush border and all components of the
luminal endocytic apparatus (Fig. 4) (26, 30, 69). In addition,
smaller amounts of immmunoreactive megalin have been identified in lysosomal structures. Megalin has also been identified
in the glomerular podocytes of Lewis rat kidney (70). In
addition to the kidney, megalin is expressed in a number of
other epithelia (reviewed in Ref. 27), including the ileum (11,
154) and the rodent yolk sac (119).
Decreased renal megalin expression has been demonstrated
in some diseases characterized by proteinuria. These include
Dent’s disease, which is caused by mutations in the renal Cl
channel ClC-5. Disruption of ClC-5 impairs proximal tubule
endocytosis and causes a reduction in megalin expression in
some knockout mouse models and in some reports of human
cases (29, 105, 108), although the findings in humans remain to
be confirmed.
Much of our knowledge on the functions of megalin is based
on data recovered from the study of megalin-deficient mice.
These mice, produced by gene targeting, exhibit severe forebrain abnormalities as well as lung defects (150). Most of them
die perinatally; however, some survive to adulthood, constitut-
Fig. 2. History of the discovery and characterization of megalin, cubilin, and the folate receptor (FR) in the kidney. Modified from Ref. 27 with permission from
Nature Reviews Molecular Cell Biology (2002), Macmillan Magazines. RAP, receptor-associated protein; AMN, amnionless; GPI, glycosylphosphatidylinositol.
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ligands (Table 1). Ligands for megalin that may be filtered in
the glomeruli include vitamin D-binding protein (DBP), retinol-binding protein (RBP), TC-B12, FBP, parathyroid hormone, insulin, epidermal growth factor (EGF), prolactin, albumin, hemoglobin, myoglobin, ␤2- and ␣1-microglobulin, apolipoprotein H, lysozyme, cytochrome c, and ␣-amylase.
STRUCTURE. Megalin was originally identified as the antigen
in Heymann nephritis, a rat model of membranous glomerulonephritis (Fig. 2) (69). It is a 600-kDa protein (Fig. 3) with a
large NH2-terminal extracellular domain, a single transmembrane domain, and a short COOH-terminal cytoplasmic tail
(57, 98, 120). The protein belongs to the LDL receptor family,
sharing common features with, in particular, the LDL receptorrelated protein (LRP). The extracellular domain is composed of
four clusters of cysteine-rich complement-type/LDL receptor
class A repeats separated by 17 EGF-like repeats. The latter
contain YWTD motifs involved in pH-dependent release of
ligands (36). Megalin binds Ca2⫹ very strongly, constituting
⬃40% of all Ca2⫹-binding activity in the renal cortex (30). The
ligand-binding type A repeats are negatively charged Ca2⫹binding protein domains, and Ca2⫹ is important for most
ligand binding to megalin. An important role of megalin in
Ca2⫹ metabolism has also been proposed (27, 57, 151). Sitedirected mutations of basic amino acid residues in aprotinin, a
6-kDa proteinase inhibitor and a ligand for megalin, decrease
the affinity for the receptor, suggesting that binding is charge
Invited Review
THE KIDNEY IN B12 AND FOLATE HOMEOSTASIS
F27
ing a model for the study of megalin function. Additional
evidence has been recovered from mice with targeted kidneyspecific knockout of the megalin gene (77). Megalin-deficient
proximal tubule cells are characterized by a loss of endocytic
invaginations, vesicles, and the membrane-recycling compartment, dense apical tubules (DAT) (31). So far, no significant
changes in transport of water, electrolytes, glucose, or amino
acids have been described in megalin-deficient mice; however,
an increased amount of a number of low-molecular-weight
serum proteins has been identified in urine. Analyses of megalin knockout mice and patients with low-molecular-weight
tubular proteinuria, including Dent’s disease, have shown several analogies (78, 105).
Little is known about the regulation of megalin gene expression in vivo; however, receptor-associated protein (RAP), a
45-kDa protein modulating posttranslation protein processing
(22, 23, 149, 152) and shown to bind megalin (30) as well as
other members of the LDL-receptor family, is required for
normal expression and subcellular distribution of megalin in
kidney proximal tubules (12). RAP inhibits binding of almost
all ligands to megalin, constituting an important tool for the
study of megalin function. A HNEL endoplasmic reticulum
(ER) retention signal has been identified in RAP (22) targeting
it mainly to the ER, where it recycles between the ER and the
cis-Golgi (22). RAP serves as a chaperone protecting newly
synthesized LRP from the early binding of ligands, which are
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also synthesized by the cells (Fig. 5) (22, 23, 149, 152). Such
premature binding of ligands within the ER may cause receptor
aggregation and retention. In addition, RAP may be involved in
folding of the receptors (Fig. 5) (23). RAP deficiency is
associated with a reduction in megalin expression to ⬃25% of
normal and a change in subcellular distribution, causing a
reduction in brush-border expression and an accumulation of
megalin in intracellular compartments including the ER and the
paramembranous ER (12). This strongly suggests that RAP
serves a similar chaperone function for megalin. RAP-deficient
mice are viable and fertile with no overt abnormalities in renal
function (149). In contrast to megalin-deficient proximal tubule
cells, no changes in the ultrastructural appearance of the
endocytic apparatus were observed in RAP-deficient mouse
kidneys (12). Detailed analysis including two-dimensional gel
electrophoresis showed increased urinary excretion of specific
proteins, including DBP and ␣-amylase (12). Thus RAPdeficient mice, having a ⬃75% reduction in megalin expression, exhibit low-molecular-weight proteinuria and have been
used to study megalin function in vivo.
Cubilin. Cubilin is a multiligand endocytic receptor. The
first ligand for cubilin was not identified until 1997, when it
was recognized as the intestinal IF-B12 receptor (126). Since
then, a number of other ligands have been described (Table 1),
of which many may be filtered in the renal glomerulus and
reabsorbed by cubilin-mediated endocytosis, including albu-
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Fig. 3. Schematic representation of the structure of megalin, cubilin, AMN, and the FR. Megalin is an ⬃4,600amino acid transmembrane protein with a nonglycosylated
molecular mass of ⬃517 kDa. The larger extracellular
domain is composed of 4 cysteine-rich clusters of LDL
receptor type A repeats constituting the ligand binding
regions, separated and followed by a total of 17 EGF-type
repeats and 8 spacer regions containing YWTD repeats.
The single transmembrane domain is followed by the
cytoplasmic tail containing 2 NPXY sequences, 1 VENQNY sequence, involved in apical sorting (137), and several Src homology 3 and 1 Src homology 2 recognition
sites possibly involved in signal transduction. Cubilin is a
⬃3,600-amino acid protein with no transmembrane domain and a nonglycosylated molecular mass of ⬃400 kDa.
The extracellular domain is composed of 27 bone morphogenic protein-1 (CUB) domains responsible for interaction
with a multiple of other proteins. The CUB domains are
preceded by a stretch of 110 amino acids followed by 8
EGF-type repeats. The NH2-terminal region contains a
potential palmitoylation site and an amphipatic ␣-helix
structure with some similarity to the lipid-binding regions
of apolipoproteins. AMN is a 434-amino acid, singletransmembrane protein with a nonglycosylated molecular
mass of ⬃46 kDa. It has no known closely related proteins,
but a cysteine-rich stretch of 70 amino acids in the extracellular domain is similar to modules present in a small
group of proteins serving as bone morphogenic protein
inhibitors. GPI-anchored FR isoforms are ⬃210-amino
acid glycoproteins with a nonglycosylated molecular mass
of ⬃25 kDa. Human FR␣ and FR␤ show 68% homology,
whereas almost 100% homology is observed between
human FR␣ and soluble human milk FBP lacking the GPI
anchor. FRs also reveal some homology with riboflavinbinding protein. The GPI-anchored FRs are attached to the
membrane at the amidated COOH-terminal serine or aspargine, through a phosphodiester linkage of phosphoethanolamine to a tri-mannosyl glucosamine core. The glucosamine is linked to phosphatidylinositol anchored to the
cell membrane.
Invited Review
F28
THE KIDNEY IN B12 AND FOLATE HOMEOSTASIS
Fig. 4. Electron micrograph showing the distribution of megalin, FR, and
cubilin in the apical parts of a cryosectioned, rat kidney proximal tubule cell.
Megalin, FR, and cubilin are identified by triple-labeling immunocytochemistry using polyclonal sheep anti-megalin (1:25,000), polyclonal rabbit anti-FR
(1:2,000), and monoclonal mouse anti-cubilin (1:5,000) followed by 3 different
gold-conjugated secondary antibodies (1:50): donkey anti-sheep (10 nm),
donkey anti-rabbit (18 nm), and donkey anti-mouse (6 nm). Megalin (filled
arrowheads), FR (open arrowheads), and cubilin (arrows) colocalize in the
microvilli (MV), endocytic invaginations (INV), endocytic vesicles (EV), and
dense apical tubules (DAT) constituting the membrane-recycling compartment
in the kidney proximal tubule. Insets: selected structures, marked by rectangles, enlarged for better identification of gold particles. The rat kidney was
fixed by perfusion with 4% paraformaldehyde, infiltrated with sucrose, and
frozen before cryosectioning and immunolabeling. Bar ⫽ 0.2 ␮m.
min, immunoglobulin light chains, apolipoprotein A-I, transferrin, DBP, hemoglobin, and myoglobin.
STRUCTURE. Cubilin was originally identified as the target of
teratogenic antibodies in rats (Fig. 2). It is a 460-kDa protein
with little structural homology to other known endocytic receptors (Fig. 3) (72, 92). Cubilin has no transmembrane doAJP-Renal Physiol • VOL
Fig. 5. Role of RAP in the processing of megalin. RAP binds to the newly
synthesized receptor in the endoplasmic reticulum (ER), thus preventing early
binding of ligands also present in the ER. As megalin is transported through the
Golgi, RAP dissociates and is retrieved to the ER by vesicular transport
following binding to specific receptors. It is suggested that ligand binding to
megalin in the Golgi and later transport vesicles is prevented by a lower pH in
these compartments. Furthermore, the binding of RAP may assist formation of
intradomain disulfide bounds and proper folding of the receptor in the ER, as
suggested for LDL receptor-related protein (LRP). From Ref. 27 with permission from Nature Reviews Molecular Cell Biology (2002), Macmillan Magzines, as modified from Refs. 23 and 148.
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main, and it can be released from renal cortical membranes by
nonenzymatic and nonsolubilizing procedures (92). It is composed of a NH2-terminal 110-amino acid region necessary for
membrane anchoring of the receptor (74) followed by eight
EGF-like repeats and 27 complement subcomponents C1r/C1s,
Uegf, and bone morphogenic protein-1 (CUB) domains. The
structure of the CUB domains has been studied in spermadhesin. They are organized as barrel-like structures containing
two, five-stranded ␤-sheets connected by surface-exposed
␤-turns (115), which may be arranged so that the less-conserved surface of the ␤-turns is externally exposed for ligand
binding. The many CUB domains in cubilin support the ability
of the receptor to bind a variety of ligands. Binding sites for
selected ligands have been partially localized. A binding site
for IF-B12 has been located within CUB domains 5– 8, whereas
the binding site for RAP has been located within CUB domains
13–14 (74). Other studies have suggested additional IF-B12 as
well as albumin binding sites in a 113-residue NH2 terminus,
(155). Although IF-B12 inhibits binding of albumin to cubilin,
the binding sites for the two proteins do not appear to be
identical within this domain (155). Finally, megalin appears to
bind to the NH2-terminal region including CUB domains 1 and
Invited Review
THE KIDNEY IN B12 AND FOLATE HOMEOSTASIS
AJP-Renal Physiol • VOL
Whether this was due to a direct role of RAP in the expression
of cubilin, or reflects the reduced expression of megalin,
remains to be established.
FRs. FRs are membrane-anchored proteins binding folate
with high affinity. They were originally identified as a soluble
FBP in milk (Fig. 2) (44), and later their presence was established in both serum and tissues (4, 54). The term folate
receptor has been introduced to indicate its function in cellular
folate uptake (4).
STRUCTURE. FRs are glycosylated ⬃40-kDa proteins binding
folate with high affinity (Kd ⬃1 nM) (4, 54, 67). At least four
FR isoforms have been identified and characterized in humans.
FR␣ and FR␤ are membrane-associated, GPI-linked proteins
(Fig. 3) (75, 141) expressing different affinities for different
stereospecific folate analogs (145). They may be enzymatically
released from the membranes (38, 75, 82). FR␥ is specific for
hematopoietic cells and present in serum (130). The fourth
human FR gene (FR␦) predicts a 27.7-kDa protein with a
unique expression pattern in both adult and embryonic tissues
(133). Whereas at neutral pH FRs bind most naturally occurring folates, dissociation is rapid at low pH. Binding of folate
to the FR induces conformational changes, increasing stability
and decreasing hydrophobicity of milk FBP (64).
RENAL EXPRESSION. FRs are heavily expressed in kidney
proximal tubule brush-border membranes (Fig. 4) (9, 58, 59,
66, 129) and have been identified in the mouse glomerulus (14)
and in human urine (50). Immunocytochemical studies have
localized the FR to the proximal tubule brush border, endocytic
invaginations, including coated pits, endocytic vesicles, and
DAT (9, 58). Identification of mRNA suggests that the adult
human kidney expresses both FR␣ and FR␤ (116). In addition
to the kidney, FR␣ is present in other, predominantly epithelial, cells, whereas FR␤ is expressed at low to moderate levels
in several different tissues (4, 54, 75, 116).
Targeted gene knockout of folbp1 and folbp2 (the mouse
equivalents to FR␣ and FR␤) has confirmed a role of the FR in
folate metabolism (107) and renal folate transport (10). Deletion of folbp1 is lethal and associated with changes in serum
folate and embryonic defects that can be rescued by supplementing the dams with folate (107, 134).
The expression of FRs is regulated by extracellular folate
concentration. In vitro studies suggest that FRs are upregulated
when cells are grown under low-folate conditions (55, 68),
whereas in the kidney FRs are downregulated in mice and rats
fed a low-folate diet (35, 43).
Interaction among megalin, cubilin, and FBP. A metabolic
interaction between folate and B12 is well established. Recent
studies have indicated additional interaction between proteins
involved in the uptake of these vitamins, showing binding of
both cubilin and FBP to megalin. A high-affinity, Ca2⫹dependent, and partially RAP-inhibitable binding between
megalin and cubilin has been demonstrated in vitro (92).
Megalin deficiency is associated with reduced brush-border
expression of cubilin (6), and in megalin-deficient mice the
cubilin ligand transferrin is accumulated at the proximal tubule
luminal membranes, revealing defective uptake (71). Also, the
uptake of another cubilin ligand, HDL, is inhibited in vitro by
anti-megalin antibodies as well as by megalin anti-sense oligonucleotides (49). This indicates that megalin may mediate
the cointernalization and possibly recycling of cubilin. In
addition, megalin and cubilin share a number of ligands,
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2 (154). The NH2-terminal region contains a furin-cleavage
site, a potential cysteine palmitoylation site, and a putative
amphipathic helix structure similar to the lipid-binding regions
of apolipoproteins (74).
RENAL EXPRESSION. Cubilin is highly expressed in the renal
proximal tubule (Fig. 4) (119). As with megalin, immunoreactive cubilin can also be identified in lysosomes (126). Although
the extrarenal expression of cubilin seems more restricted than
megalin, the two receptors are coexpressed in a number of
epithelia, including the ileum and the rodent yolk sac (27).
Normal expression of cubilin is dependent on AMN, a
⬃45-kDa transmembrane protein (Fig. 3) identified as an
important factor for the normal development of the middle
portion of the primitive streak in mice (65). Previously, a 40to 45-kDa protein of unknown nature was shown to coelute
with cubilin when purified from kidney by IF-affnity chromatography (11). Later studies suggested this protein to be AMN
(40). Cubilin and AMN colocalize in kidney proximal tubule.
Cotransfection of cubilin fragments and AMN into Chinese
hamster ovarian (CHO) and Madin-Darby canine kidney cells
shows that AMN interacts with the EGF-type repeats of cubilin
and is essential for normal translocation of the cubilin-AMN
complex from the ER to the plasma membrane and for the
subsequent endocytosis (34, 40). Furthermore, dogs with a
mutation in the AMN gene (52, 53) as well as AMN-deficient
mouse epithelial cells reveal defective apical insertion of cubilin (6, 41, 136). Mutations in either the cubilin or the AMN
gene have been identified in Imerslund-Gräsbeck disease (2,
138), a rare inherited vitamin B12 deficiency syndrome characterized by defective intestinal absorption of IF-B12 (21, 144)
and an apparent geographical concentration in Scandinavia and
the Middle East (139). Cubilin gene mutations were identified
in Finnish and Arab families (2, 139). In two Finnish families,
this involved either a one-amino acid substitution in CUB
domain 8 affecting the binding of IF-B12 or a point mutation
expected to activate a cryptic intronic splice site causing an
in-frame insertion with several stop codons, predicting a truncation of the receptor in CUB domain 6 (2, 73). Whereas the
patient with the latter mutation has overt proteinuria, patients
with amino acid substitution reveal varying degrees of proteinuria ranging from little or no, to clear-cut (144). Affected
members of three Norwegian families, shown to have a nucleotide deletion causing the introduction of an early stop codon
in the AMN gene (138), were all previously characterized and
had proteinuria at the time of diagnosis. Other mutations have
been identified in the AMN gene; however, the renal phenotype of these patients is not clear (139). Based on the variety of
mutations shown to cause Imerslund-Gräsbeck syndrome, it
may by hypothesized that the difference in renal phenotype, in
particular the degree and type of proteinuria, reflects the degree
of inactivation of cubilin function, in particular whether the
mutation affects the multiligand properties or only the IF-B12binding site.
Like megalin, cubilin binds RAP (11); however, the function
of RAP binding to cubilin remains unclear. Unpublished observations in our laboratory showed that the expression of
cubilin was reduced in RAP-deficient mice, somewhat similar
to the changes in megalin expression, showing decreased
overall expression to ⬃25% of controls. In addition, increased
amounts of proteins (up to 140 kDa) reactive with anti-cubilin
antibodies were observed in the urine of RAP-deficient mice.
F29
Invited Review
F30
THE KIDNEY IN B12 AND FOLATE HOMEOSTASIS
Proximal Tubule Uptake of Folate and Vitamin B12
Proximal tubule uptakes of both folate and vitamin B12 were
recognized early and shown to be saturable processes (46). At
physiological serum concentrations, the estimated amounts of
vitamins filtered in the glomeruli exceed the recommended
daily intake, whereas the urinary excretion of both intact folate
and vitamin B12 is minimal. Thus proximal tubule receptormediated uptake efficiently prevents urinary losses. Recent
studies have provided significant information on the molecular
mechanisms responsible for this, involving the endocytic receptors presented above.
Vitamin B12. Megalin is essential for the proximal tubule
reabsorption of filtered TC-B12 (13), mediating endocytosis of
TC-B12 (90, 104). Megalin and TC colocalize within the
endocytic apparatus of rabbit kidney proximal tubule cells
(142). TC-B12 binds to megalin with an estimated affinity (Kd)
of ⬃183 nM when analyzed by surface plasmon resonance
(SPR) analysis, also showing a possible second binding site
(Kd ⬃1.4 ␮M). Binding to immobilized megalin in microtiter
trays revealed a half-maximum binding of 12.5 nM, indicating
that the affinity may be higher than estimated by SPR analysis.
The importance of megalin for the tubular reabsorption and
renal accumulation of TC-B12 was established using megalin
knockout mice (13). These revealed increased urinary excretion of B12 and a 28-fold increase in renal B12 clearance, along
with a 4-fold decrease in the B12 content of megalin-deficient
mice kidneys. Immunocytochemistry in wild-type mice
showed that most of the reabsorbed vitamin was located in the
very early part of the proximal tubule, indicating efficient
reabsorption (13).
An additional 62-kDa TC-B12 receptor has been identified in
the kidney and other tissues, including placenta, liver, and
intestine (16 –18). This receptor is a glycosylated 45-kDa
single polypeptide with a yet unknown primary structure. It is
normally present as a 124-kDa dimer in both apical and
basolateral membranes of kidney proximal tubule, however,
with a 90% distribution to the basolateral membranes when
estimated by membrane fractionation (17, 18). The expression
appears to be regulated by corticoids (17). Its role in TC-B12
uptake is supported by the low tissue B12 and apparent vitamin
B12 deficiency developing in rabbits following injection with
AJP-Renal Physiol • VOL
an antiserum against this receptor (16) and by the decrease in
renal uptake of orally administered labeled B12 in adrenalectomized rats (17). It was recently suggested that this ubiquitous
TC-B12 receptor binds to megalin and that this binding is
associated with increased binding of TC-B12 to the purified
receptors (153). It was also shown that the immunization of
rabbits with megalin was associated with decreased expression
of the TC-B12 receptor in purified apical kidney membranes,
leading to the suggestion that megalin is involved in the apical
targeting of this receptor. This proposed functional and structural interaction between megalin and the 62-kDa TC-B12
receptor awaits further clarification. Evidence suggests that the
major mechanism for renal accumulation of vitamin B12 is by
tubular reabsorption of filtered TC-B12. Thus the 10 –90%
distribution of the 62-kDa TC-B12 receptor in apical vs. basolateral renal membranes is puzzling; however, it may suggest
an alternative role for the 62-kDa receptor in the kidney, e.g.,
in basolateral uptake or secretion. The distribution of labeled
B12 injected during vitamin depletion as well as during vitamin
load fits a model of cellular TC-B12 uptake involving two
distinct receptors: a possibly regulated ubiquitous TC-B12
receptor mediating saturable cellular uptake, and a luminal
renal tubule receptor mediating constitutive high-capacity reabsorption of filtered TC-B12 (7, 90).
Although both megalin and cubilin mediate endocytic uptake of vitamin B12, and both are heavily expressed in proximal
tubule epithelial cells, a role for cubilin in kidney B12 uptake
has not been established. Cubilin purified from kidney binds
IF-bound B12, and uptake of IF-B12 in the kidney is inhibited
by anti-cubilin antibodies (11). Minute amounts of IF can be
detected in human serum and may be filtered, as IF has been
identified in urine (111, 143). However, due to the very small
amounts of B12 filtered in complex with IF, the significance of
cubilin for renal uptake of B12 is dubious.
Folate. A role of the FR in renal tubular folate uptake was
hypothesized when FRs were identified in the kidney proximal
tubule (66, 129) and further supported by kinetic studies
showing that the urinary clearance of folate derivatives was
inversely related to their affinity for the FR (127). The importance of the FR was established by the analysis of renal folate
handling in mice with targeted gene knockout of folbp1 and
folbp2 (10). Mice defective in folbp1 (equivalent to human
FR␣) reveal a significant increase in renal folate clearance at
both low-folate and normal-folate intakes, showing impaired
tubular uptake of filtered folate (10). It was calculated that the
amount of folate excreted in the urine of folbp1 null mice is
⬃100 times higher than in wild-type mice, indicating that the
tubular reabsorptive capacity related specifically to folbp1 in
the low-folate situation is ⬃4 nmol/24 h in mice. With an
estimated 20,000 nephrons in the mouse, this corresponds to a
transport rate of ⬃0.15 fmol䡠min⫺1 䡠nephron⫺1. Folbp2
(equivalent to human FR␤)-deficient mice revealed lower serum folate levels compared with wild-type but no significant
changes in urinary folate clearance (10). Thus, although
mRNA corresponding to both the human equivalents of folbp1
and folbp2 has been demonstrated in the human kidney (116),
the role of folbp2 in renal folate reabsorption remains unclear.
The rate of transtubular folate transport has been estimated
using several other approaches. Perfusion of isolated rabbit
proximal tubules revealed a reabsorptive capacity of 4.0
fmol䡠min⫺1 䡠nephron⫺1, which should be compared with an
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including DBP, immunoglobulin light chains, albumin, and
RAP (Table 1). Thus in epithelia coexpressing megalin and
cubilin, including kidney proximal tubule, megalin and cubilin
appear to act in concert, mediating endocytosis of the same
ligands. Megalin also binds milk FBP (14), providing a mechanism for the uptake of FBP-bound folate. Soluble milk FBP
structurally resembles GPI-linked FRs (60, 81) coexpressed
with megalin in the luminal plasma membranes of kidney
proximal tubule and other epithelia, i.e., the choroid plexus and
yolk sac (28). No direct interaction between megalin and
GPI-linked FRs in the plasma membrane has been demonstrated; however, it has previously been shown that the related
LRP mediates internalization of the GPI-anchored urokinase
receptor (33), indicating that a similar interaction between
megalin and FR is possible. This may lead to the hypothesis
that megalin is involved in intracellular translocation of FRs,
possibly regulating luminal expression of membrane FR by
internalization and degradation.
Invited Review
THE KIDNEY IN B12 AND FOLATE HOMEOSTASIS
AJP-Renal Physiol • VOL
mice can be rescued by folate supplementation of the dams.
These mice revealed defects in erythro- and lymphopoiesis
along with abnormalities in renal and seminiferous tubule
development (157). Additional organic anion transporters,
which may also transport folate, have been identified in the
kidney (86). Thus a carrier-mediated mechanism may be responsible for the transport of folate out of endosomes, as well
as for the exit of folate from the tubule cells.
In addition to the FR, megalin may also be involved in the
tubular uptake of folate. SPR analysis, autoradiography, and
uptake studies suggested that megalin mediates binding and
internalization of soluble FBP (14). Folate bound to serum FBP
is filtered as a complex. It may be estimated that ⬍1% of
filtered FBP is excreted (14), suggesting efficient tubular reabsorption by megalin-mediated endocytosis similar to other
vitamin carrier proteins (27, 31). Approximately 108 nmol or
⬃48 ␮g of FBP-bound folate may be recovered daily by
megalin-mediated uptake, which could be important in individuals with low-folate intake. Thus FBP is involved in folate
uptake both as a GPI-linked, membrane-associated receptor for
filtered free folate and as a filtered, soluble, folate carrier
protein binding to megalin.
A potential role for cubilin in the recovery of filtered folate
is suggested by the important role of this receptor for the
reabsorption of filtered albumin (6). Albumin binds to cubilin
with an estimated Kd of ⬃0.6 ␮m (6). Because albumin is a
carrier of folate, along with other vitamins, cubilin may be
involved in the uptake of folate, depending on the amount of
filtered albumin. The significance of this remains to be established both in the normal kidney and under pathological conditions characterized by increased filtration of albumin.
Postendocytic Processing in the Kidney
Following internalization into renal proximal tubule cells,
vitamins may be metabolized, stored, or released (Fig. 6). A
recent study has shown endocytosis and lysosomal accumulation of a fluorescent folate probe using in vivo two-photon
microscopy (121). Following intravenous injection of labeled
folate into rats, there is a transient increase in the accumulation
of label within the kidney at levels exceeding the liver; however, 24 h after injection, the amount of labeled folate within
the kidney is significantly reduced by redistribution into other
tissues (131), indicating that the kidney does not accumulate
large amounts of folate. The endocytosed free folate is transported to the cytoplasm, possibly by a carrier protein (109).
The reabsorbed folate may, in part, undergo metabolic transformation into other folate forms but is catabolized only to a
very small extent (88, 96). Alternatively, folate may be transported across the tubular cells in a vesicular compartment, as
suggested based on the finding of fluorescent-labeled folate in
basolateral vesicles of the proximal tubule (121). However, no
morphological evidence of such transport was observed following proximal tubule micropuncture with folate-gold particles (8) or radiolabeled folic acid (58).
Substantial amounts of B12 accumulate in the rodent kidney,
especially in states of vitamin load (7, 13, 47, 51, 124). In vitro
TC is degraded within 24 h, whereas B12 accumulates and is
only slowly released (79, 104). Studies on the distribution of a
single oral or parenteral dose of labeled B12 suggest that high
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estimated rabbit single-nephron filtration of folate ⬃3 fmol/
min (8). Micropuncture of single rat proximal tubules for 1 min
with [3H]folic acid demonstrated tubular uptake at a rate of
0.65 fmol/mm tubule or 4.5 fmol/proximal tubule (128), close
to the observed rate in isolated perfused rabbit tubules (8). In
contrast, in vitro studies using cultured human proximal tubule
cells suggested a specific apical-to-basolateral folate transport
rate of only 0.12 fmol䡠min⫺1 䡠cm⫺2 (89), considerably lower
than that observed with perfused rabbit proximal tubules,
indicating either lower activity of cells in vitro, possible effects
of flow rate in microperfusion studies, or species differences,
conceivably reflecting differences in serum folate levels. Studies using cultured human proximal tubule cells have suggested
bidirectional transport (95), but tubular secretion of folate in
vivo has not been established.
Uptake of folate by the FR is suggested to involve internalization of the FR-folate complex (129). Studies in monkey
kidney MA104 cells and other cell lines indicated that binding
is followed by invagination of the FR into caveolae rather than
the clathrin-coated pit pathway (3). As the folate-FR complex
clusters into caveolae, this is followed by acidification (76) and
dissociation of the ligand (3). The FR is recycled to the plasma
membrane by reopening of the caveolae. Later in vitro studies
have challenged this hypothesis by showing that GPI-linked
receptors concentrate in caveolae by cross-linking with antibodies, but not with folate (87, 106), that FRs can be endocytosed following stimulation (106), and that in KB cells derived
from a carcinoma of the human nasopharynx most internalized
FR bypasses caveolae (114). Experiments in CHO cells have
suggested that FRs are internalized into a distinct GPI-anchored, protein-enriched compartment independent of clathrin
(118). Thus FRs may be internalized by several different
endocytic pathways depending on cell type and on the stimulus
evoking internalization. Micropuncture of kidney proximal
tubules showed endocytosis of folate-gold particles into coated
pits and vesicles (8), and the FR has been localized to the
compartments of the classic endocytic and recycling pathway
(9, 14, 58). Also, caveolae are almost never observed in kidney
proximal tubule cells (20), making it unclear whether this
mechanism operates in these cells. Micropuncture with anti-FR
antibodies (9), as well as kinetic studies (128), suggests rapid
recycling of FRs to the plasma membrane for regeneration of
folate-binding sites.
Several other mechanisms have been implicated in renal
tubular folate transport, including dual-component transport
systems (96) or even nonspecific pathways (97). Folate uptake
has been studied in vitro using a number of different cell
systems (4, 54, 67). In certain cell types, including KB cells
and MA104 cells, folate uptake apparently depends on FRs.
Other cell lines, i.e., mouse L1210 leukemia cells, utilize a
high-capacity folate uptake system mediated by the reduced
folate carrier (RFC). Thus folate uptake may be mediated by a
receptor-mediated mechanism depending on the FR and/or
carrier-mediated mechanisms, of which the RFC has been most
extensively characterized (86). The RFC has been located to
basolateral membranes in kidney tubules (146), suggesting that
the RFC may be involved in basolateral folate uptake or in the
cellular exit of reabsorbed folate. RFC represents a highcapacity, low-affinity transport system for folate with a Ki for
5-MTHF on the order of 2 ␮M. Targeted gene knockout of
RFC is embryonically lethal; however, a limited number of
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Invited Review
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THE KIDNEY IN B12 AND FOLATE HOMEOSTASIS
doses of the vitamin are retained in the kidneys weeks after
injection (51). Although traditionally the liver is considered the
major storage organ for B12, it has been shown that the ratio of
injected labeled B12 to total B12, estimated as microbiologically active B12, is higher in the kidney than in other organs up
to 17 days after injection, suggesting that the injected B12 is
retained in the kidney for that long (47). Internalized vitamin
B12 accumulates in rodent proximal tubule lysosomes, predominantly as free vitamin (7, 13, 100), suggesting that this
organelle may serve a storage function. When injection of a
single dose of radiolabeled B12 was followed by injections of
large doses of B12, more vitamin was retained in the kidneys up
to 30 days after injection (51). This may indicate that the
release from the kidneys is negatively regulated by vitamin
status, but it may also reflect reabsorption of increased amounts
of filtered, labeled B12 by the constitutive high-capacity, megalin-mediated mechanism. Much less free vitamin is identified
in vitamin-depleted animals (7, 13), indicating that the transport out of lysosomes may constitute a rate-limiting, and
perhaps regulated, step in the transtubular vitamin transport. A
pH-sensitive, lysosomal membrane-associated B12 transporter
has been identified in liver, suggesting that B12 may be transported out of the lysosomes for further transport and/or proAJP-Renal Physiol • VOL
cessing in the cytosol (62). This is supported by the identification of an inborn error of cobalamin metabolism causing the
lysosomal accumulation of B12 (140), possibly due to a defective lysosomal membrane transporter. Studies have suggested
that injected and reabsorbed cyanocobalamin is metabolized
within the kidney (103, 104), possibly in the cytosol or the
mitochondria following transport out of lysosomes.
Alternatively, B12 may be transported along with TC across
the tubular cell. Megalin-mediated transcellular transport of
intact protein has been suggested for thyroglobulin (85) and for
RBP (84). So far, this has not been confirmed in vivo, and the
accumulation of free vitamin B12 along with rapid degradation
of internalized TC in cultured proximal tubule cells strongly
suggest that only the vitamin is transported through the cells.
The mechanism, by which reabsorbed B12 is released from the
tubular cells following reabsorption, is largely unknown. Both
TC and HC, and possibly IF, are synthesized in proximal
tubule cells in vitro (19, 104, 112). TC mRNA was identified
in adult porcine and human kidney, whereas HC mRNA could
not be identified in the adult porcine kidney (104). Whether B12
and carrier proteins are combined within in the cell before
secretion or are secreted separately remains to be established.
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Fig. 6. Schematic representation of a kidney proximal
tubule cell showing possible pathways by which megalin, cubilin, and FR are involved in endocytosis and
transcellular transport of folate and vitamin B12. Filtered
vitamins, free or complexed to carrier proteins, bind to
megalin and/or cubilin, or FR, followed by endocytosis.
AMN and/or megalin facilitates the endocytosis of cubilin. The ligands are released from the receptors by the
low pH in the endosomes, and receptors recycle through
a membrane-recycling compartment (DAT). The protein
component of carrier molecules is degraded in lysosomes, whereas the vitamin is transported across the
epithelial cell. A minor fraction of receptors may be
transported to lysosomes for degradation. Endo- or lysosomal membrane transporters for cobalamin and folate have been suggested. FR-mediated transcellular
transport (right) has been suggested in the kidney. Pathways or interactions yet to be established are indicated
by ?.
Invited Review
THE KIDNEY IN B12 AND FOLATE HOMEOSTASIS
CONCLUSIONS AND FUTURE PERSPECTIVES
ACKNOWLEDGMENTS
The author thanks Professor Erik I. Christensen, Professor Søren K.
Moestrup, and Professor Ebba Nexø for constructive and inspiring comments
regarding the manuscript.
GRANTS
This work was supported by the Danish Medical Research Council, the
University of Aarhus, the NOVO-Nordisk Foundation, Fonden til Lægevidenskabens Fremme, the Biomembrane Research Center, the Beckett Foundation,
the Leo Nielsen Foundation, the Ruth König-Petersen Foundation, and the Birn
Foundation.
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vitamin B12 in the live subject. Clin Sci (Colch) 39: 107–113, 1970.
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By mediating endocytosis of filtered vitamins and specific
carrier proteins, both megalin and the FR play an important
role in the conservation of vitamin B12 and folate. In addition,
cubilin may be involved by mediating uptake of albumin, an
additional carrier of folate. Functional interaction between
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metabolic relation between the vitamins. The unraveling of the
molecular basis for renal folate and B12 uptake has shown this
to be a constitutive process, similar to the uptake of other
vitamin carrier proteins, causing vitamins to locate in specific
vesicular compartments. Significant amounts of free B12 appear to accumulate in kidney lysosomes, suggesting that this
serves a storage function, and, in addition, vitamins are metabolized within the kidney, suggesting a role in vitamin
homeostasis.
The mechanism by which vitamins are further transported,
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unresolved (Fig. 6), although it may involve specific carriers
associated with intracellular vesicular and basolateral membranes, as well as proximal tubule synthesis and secretion of
carrier proteins. Further studies, including the use of inherited
and genetically engineered models of defective vitamin transport as well as transfection studies with fluorescent-labeled
carrier proteins, should provide clues to resolve these mechanisms and their possibly regulation. The cytoplasmic tail of
megalin contains potential signaling motifs as well as domains
interacting with cytosolic proteins (27, 42, 80, 94, 98, 158).
Future studies may reveal signaling pathways, as has been
implicated for other members of the LDLR family (56), possibly also involved in regulation of vitamin transport.
Most of the evidence establishing the mechanisms for renal
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animals, particularly in rodents. Studies in humans, including
patients with renal disease and proteinuria as well as patients
with inherited, specific receptor defects, should assess the
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humans.
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