Nephrol Dial Transplant (2013) 28: 274–281
doi: 10.1093/ndt/gfs565
Advance Access publication 4 January 2013
Full Review
From bowel to kidneys: the role of cubilin in physiology and disease
Erik I. Christensen, Rikke Nielsen and Henrik Birn
Section of Cell Biology, Department of Biomedicine, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark
Correspondence and offprint requests to: Erik Ilsø Christensen; E-mail: eic@ana.au.dk
Abstract
Cubilin is a large endocytic receptor serving such diverse
functions as the intestinal absorption of the intrinsic
factor–B12 complex and the renal proximal tubule reab-
sorption of filtered proteins including albumin, transferrin,
vitamin D-binding protein and other important plasma
carriers. Cubilin is a structurally unique, peripheral mem-
brane protein, which depends on the membrane protein
amnionless (AMN) for correct apical translocation. In
addition, AMN appears important for efficient internaliz-
ation of intrinsic factor–B12 in the intestine, whereas in
the proximal tubule cubilin interacts with another endocy-
tic receptor, megalin, for effective reabsorption. The
importance of cubilin has been demonstrated in several
animal models of cubilin deficiency as well as in a variety
of human diseases. Recent demonstration of cubilin in po-
docytes from various species awaits further clarification
with respect to the functional role as well as its role
in pathology.
Keywords: albuminuria; Imerslund-Gräsbeck syndrome; low-molecular-
weight proteinuria; vitamin B12; receptor-mediated endocytosis
Introduction
Receptor-mediated endocytosis is a general process by
which cells can selectively and actively internalize bio-
logical substances. It is essential to normal life and occurs
in almost all tissues. It is tightly regulated by intracellular
mechanisms involving pathways common to many differ-
ent cell types. In some cases, receptors have evolved
serving very different physiological functions in different
tissues. The endocytic receptor cubilin has very important,
yet very different functions, mediating the uptake of in-
trinsic factor–vitamin B12 in the intestine as well as the
uptake of filtered proteins, including albumin, in the
kidneys (Figure 1). Since then, the importance of cubilin
in normal renal physiology has been established and
new evidence suggests a role of cubilin in the develop-
ment and progression of chronic kidney disease. In this
paper, the fascinating history leading to the identification
of cublin will be summarized followed by a review of
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current knowledge on the structure, function and patho-
physiological role of this receptor.
In 1926 the therapeutic effect of orally administered
liver on pernicious anaemia was demonstrated [1], and the
studies by Castle et al. [2] showed that a gastric substance
(intrinsic factor, IF) acted together with the extrinsic
factor, vitamin B12, to prevent this disease. Receptor-
mediated uptake of IF–vitamin B12 complexes in the
small intestine was suggested by Herbert in 1959 [3] and
several groups attempted to purify the receptor (reviewed
by Seetharam and Alpers in 1982 [4]). In 1960 a much
more rare, inherited disease characterized by juvenile
onset and intestinal malabsorption of vitamin B12 was de-
scribed simultaneously by Imerslund [5] and Gräsbeck
et al. [6]. This disease, known as Imerslund-Gräsbeck
syndrome (IGS), is often associated with proteinuria and
it was noted that the anaemia was corrected by intramus-
cular injections of the vitamin B12; however, the protei-
nuria was not.
In the 1980s Verroust et al. [7–9] purified and charac-
terized a protein (gp280) localized in the clathrin-coated
luminal membrane domains in renal proximal tubules and
yolk sac epithelia. These observations indicated that
gp280 might be an endocytic receptor. Simultaneously,
Seetharam et al. [10] isolated an IF–B12-binding, renal
brush border protein that appeared to be identical to the
small intestinal IF–B12 receptor and later shown to be a
460-kDa protein identical to gp280 [11, 12]. The protein
was cloned from rats [13] and humans [14] and shown to
be a peripheral membrane protein, named cubilin.
In 1999 mutations in the cubilin gene CUBN were
identified as the cause of IGS in a group of Finnish
patients [15]. Prior to this, Fyfe et al. [16] described a
family of giant schnauzers with an inborn, intestinal IF–
B12 malabsorption similar to the human IGS [17];
however, with no apparent mutations in cubilin [18]. It
was shown that these dogs had a reduced brush border
expression of cubilin [16]. In 2003 Tanner et al. [19]
identified mutations in the AMN gene encoding the
protein amnionless (AMN) in Norwegian and Tunesian
IGS families. Similarly, the dogs were shown to harbour a
mutation in AMN [20, 21] These observations led to the
demonstration that AMN and cubilin form a complex
 Cubilin in bowel and kidneys 275

Fig. 1. The physiological role of cubilin as an endocytic receptor in the intestinal and renal proximal tubule epithelial cells.

Cubam and that AMN is essential for normal apical

expression of cubilin [22], see also Coudroy et al [23].

Cubilin structure

The cloning of cubilin revealed a protein different from

all other known receptors at the time [13, 14]. The name
of the receptor refers to the 27 CUB [complement c1r/
C1s, Uegf (epidermal growth factor-related sea urchin
protein) and bone morphogenetic protein 1 (BMP)]
domains found in the C-terminal part of the protein
(Figure 2). They represent ∼88% of the glycosylated 460-
kDa-sized protein each composed of ∼110 amino acids
[13, 14]. The CUB domains are also identified in several
developmental regulated proteins such as BMP-1, tolloid
and TSG6 [24]. In the N-terminal region eight Ca2
+
-binding epidermal growth factor repeats and a terminal
region involved in trimerization of the protein are situated
[13, 14]. In cubilin the CUB modules act as ligand-
binding domains and the intrinsic factor–B12 complex
binds domains 5–8 [25, 26], which has recently been
crystallized [27].
Cubilin harbours no transmembrane domain and it
is thus dependent on additional factors for membrane an- Fig. 2. Graphic presentation of cubilin and AMN. The illustration
choring and for endocytosis of the receptor-bound depicts known domains of the receptors. The CUB domains are ligand
ligands. Initially, cellular translocation of cubilin was binding, but as cubilin is a peripheral membrane protein lacking
intracellular motifs it is dependent on other proteins for endocytosis. In
believed to rely solely on megalin, constituting an addi- the intestine AMN is believed to assist cubilin in absorption of IF–B12,
tional, multi-ligand receptor expressed in the proximal whereas in the proximal tubule endocytosis by this complex is limited.
tubule [28]. This was based on subcellular co-localization Here megalin (not shown) is required for internalization of cubilin
of the receptors in the proximal tubule, and a direct inter- ligands. AMN is furthermore needed for membrane localization of
action of the receptors through CUB domains 12–17, 22– cubilin.
27 and the N-terminus of cubilin [8, 11, 13, 26, 29, 30].
Furthermore, megalin knockout mice as well as megalin-
deficient patients (DB/FOAR patients; OMIM # 222448) believed to drive internalization of the complex [28]. In
fail to reabsorb endogenous cubilin ligands [31, 32] addition, megalin is a multi-ligand receptor harbouring an
supporting the importance of megalin for normal endocy- extensive extracellular domain which includes four
tic activity of cubilin in the proximal tubule. The cyto- binding clusters responsible for binding multiple, diverse
plasmic tail of megalin contains two endocytic motifs ligands [28].

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 276
In addition to the endocytic apparatus of the small
intestine [33], and the proximal tubule [7–9], cubilin is
also expressed in podocytes [34], placenta [35], inner ear
[36] and the visceral yolk sac [8].
Amnionless
As mentioned, cubilin was shown to associate with AMN
[22], a 38–50-kDa protein essential for amnion and primi-
tive streak development in mice [19, 37, 38]. AMN is a
transmembrane protein with a cytoplasmic tail containing
two NPXY motifs and a cystein-rich stretch as the only
known characteristic feature of the extracellular domain
(Figure 2) [22, 38]. The domain shares similarities with
cysteine-rich domains found in bone BMP-binding pro-
teins and may play a role in modulation of BMP signal-
ling. Cubilin and AMN, in complex also known as
CUBAM [22], co-localize closely in the kidneys [22],
intestine [19] and yolk sac of mice [38] and AMN is es-
sential during biosynthesis and trafficking of cubilin. The
two proteins assemble, through the EGF domains in
cubilin, early in the translational machinery, and muta-
tions in the AMN gene in both mice and dogs cause re-
tention of cubilin in intracellular compartments, thereby
eliminating apical membrane localization [21–23, 39].
The proteins are mutually dependent, which is also
evident in IGS patients with the disease-causing mutation
present in cubilin and in cubilin-deficient mice, where
AMN is retained intracellularly [31, 40]. In vitro the
cubilin–AMN complex is endocytically active driven by
the intracellular endocytic motifs in the tail of AMN [22,
41]. Despite the possible expression of megalin in the
intestine [12] AMN appears to facilitate the internalization
of IF–B12 bound to cubilin in the intestine as evidenced
by the absence of megaloblastic anaemia in megalin-
deficient patients. In the kidneys, however, efficient
uptake of filtered proteins requires a high-capacity endo-
cytic system and in this setting CUBAM is unable to re-
absorb filtered cubilin ligands in the absence of megalin
as evidenced both in the megalin knockout mice and in
megalin-deficient patients [31, 32]. These observations
indicate that the molecular mechanisms for intestinal ab-
sorption of intrinsic factor–B12 are different from the
proximal tubular reabsorption of protein. This does not
exclude that AMN is able to mediate endocytosis of
cubilin independently of megalin in the proximal tubule,
as observed when IF–B12 is injected in megalin-deficient
mice; however, the uptake is significantly reduced [31].
Cubilin function
Serving as an endocytic receptor cubilin mediates the
uptake of proteins and protein-bound substances both in
the intestine and in the kidneys (Figure 3). Intrinsic factor,
synthesized by the gastric parietal cells and complexed to
B12 in the duodenum, is the sole, known physiological
ligand for cubilin, in the terminal ileum. In the kidneys
proximal tubule cubilin mediates the uptake of several fil-
tered proteins [42] and the combined action of cubilin and
megalin essentially clears the ultrafiltrate of filtered
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E.I. Christensen et al.
Fig. 3. Endocytosis and cellular processing of cubilin and cubilin-bound
ligands. The anchoring and intracellular trafficking of cubilin rely on the
association with AMN and/or megalin within the membrane. In the
kidney proximal tubule epithelial cells, efficient endocytosis of cubilin is
dependent on megalin. In other organs, such as the intestine, AMN may
play a similarly important role, while little is known about the
mechanisms regulating sorting and recycling of cubilin. Cubilin-bound
ligands, including protein–vitamin complexes such as IF–B12 or DBP–
vitamin D, dissociates from the receptor as a result of acidification of the
endocytic compartment. This is followed by lysomal degradation of the
protein, while vitamins and other nutrients are released from the
basolateral cell surface into the circulation. This may be preceded by
biologically important, intracellular metabolism, as in the case of 1α-
hydroxylation and activation of vitamin D by the kidney proximal tubule
cells.
proteins, lipids, vitamins and hormones resulting in an
almost protein-free final urine [28]. Albumin was among
the first ligands to be identified showing the importance
of cubilin for urinary albumin excretion [43, 44]. It is
now evident from the complete lack of endocytic uptake
of albumin in cubilin-deficient mice that cubilin with the
assistance of megalin is the only physiologically impor-
tant albumin receptor in the proximal tubule [31, 45]
(Figure 4). Additional ligands include vitamin carrier pro-
teins, lipoproteins, other carriers, immune- and stress-
related proteins and drugs [42, 46] (Table 1).
The identification of these ligands indicated the mol-
ecular basis of the low-molecular–weight (LMW) protei-
nuria often observed in IGS patients [5, 6]. Proteinuric
IGS patients were recognized to hold mutations abolish-
ing membrane localization of cubilin, whereas patients
with mutations affecting only the intrinsic factor–B12-
binding site do not present with LMW proteinuria (un-
published results) as originally demonstrated for vitamin
D-binding protein (VDBP) [47].
Cubilin ligands may be divided into ligands that bind
only to cubilin and ligands that bind to both megalin and
cubilin (Table 1). Most ligands have been identified by
in vitro-binding studies, and the identification has been
established by urinary excretion studies in mouse [31, 45]
and dog models [43, 47–49] as well as in patients suffer-
ing from IGS [40].
 Cubilin in bowel and kidneys 277

absence of megalin in humans [32], which is not the case

in mice [31, 50]. The role of cubilin in fetal development
apparently also differs in mice and man as it has been re-
ported that the deletion of cubilin in mice is incompatible
with survival [51], whereas the receptor is anticipated to
be less important or at least complemented by other
factors in humans as evident from the survival of cubilin-
deficient patients [40].

Cubilin function and dysfunction in disease

As may be anticipated from the expression pattern and

functional studies of cubilin, receptor dysfunction has
been implicated in vitamin B12 deficiency due to defective
intestinal absorption and in renal dysfunction with tubular
proteinuria.

Imerslund-Gräsbeck syndrome
Fig. 4. Immunofluorescent demonstration of endogenous albumin
uptake in cubilin mosaic knockout mouse kidneys. Albumin (red) is seen Imerslund-Gräsbeck syndrome (IGS, hereditary megalo-
only in cells expressing cubilin (green). blastic anaemia, OMIM #261100) is usually diagnosed on
the basis of failure to thrive, recurrent infections, and
fatigue accompanied by megaloblastic anaemia develop-
Table 1. Ligands for cubilin
ing in early childhood. Once treated the prognosis of the
disease is favourable with stable proteinuria and kidney
Vitamin carrier proteins function [52], although, as mentioned the proteinuria is
Intrinsic-factor vitamin B12 Seetharam et al. 1997 [11] not corrected by B12 supplementation.
Vitamin D-binding proteina Nykjaer et al. 2001 [47] Several different mutations in CUBN and AMN have
Other carrier proteins
Albumina Birn et al. 2000 [43]
been identified as the cause of IGS [53]. Mutations in
Myoglobina Gburek et al. 2003 [82] CUBN dominate in Finnish patients, mutations in AMN
Haemoglobina Gburek et al. 2002 [83] dominate in Norwegian cases, while both have been
Transferrin Kozyraki et al. 2001 [49] identified in the Middle East [54, 55]. The common func-
Lipoproteins tional implication of these different mutations is insuffi-
Apolipoprotein AI Kozyraki et al. 1999 [48]
High-density lipoprotein Kozyraki et al. 1999 [48] cient ability to mediate intestinal absorption of IF–B12.
Enzymes and enzyme inhibitors This may be caused by (i) a single amino acid substitution
Recombinant activated factor VIIaa Seested et al. 2011 [88] within the IF–B12-binding site of the CUBN [15] or (ii)
Immune- and stress-related proteins
a mutations or gene deletions likely to result in absence of
Immunoglobulin light chains Batuman et al. 1998 [89]
Clara cell secretory protein Burmeister et al. 2001 [90]
expression of functional cubilin [15, 40, 56], which also
α1-microglobulina Storm et al. 2011 [40] includes mutations in AMN [21, 22]. The proteinuria ob-
Drugs and toxins served in some patients is due to the defective tubular re-
Aminoglycosidesa Tauris et al. 2009 [36] absorption of filtered proteins including albumin [40].
Receptors Increased urinary excretion of the cubilin ligand VDBP
Megalin Moestrup 1998 [13]
AMN Fyfe et al. 2004 [22] was identified in a Finnish patient with a mutation result-
Others ing in early truncation of the receptor, while this was not
a
Receptor-associated protein Birn et al. 1997 [12] observed in a patient with a single amino acid substitution
a
in the IF–B12-binding site [47]. This suggests that the pro-
Designate ligands shared with megalin. teinuria is related to specific mutations affecting the func-
tion of cubilin other than IF–B12 binding, which would
also include mutations in AMN (unpublished results).
Notable, cubilin [47], as well as megalin [50], bind and Although this relationship has not been consistently de-
mediate the proximal tubule uptake of vitamin D in scribed [55, 57], albuminuria with no reported B12
complex with VDBP which is essential for the final, renal deficiency has recently been associated with a single-
hydroxylation and activation of the vitamin [50].The role base, homozygous deletion in CUBN located differently
of cubilin in the renal handling of ligands is comparable from the known IF–B12 binding site [58], supporting the
in mouse and man, although small differences have been notion that the proteinuria may be dependent on the type
observed. In proteinuric patients with IGS, increased of mutation. Varying degrees of proteinuria have been re-
urinary excretion of albumin, Apo AI and VDBP is ob- ported in IGS with protein/creatinine ratios ranging from
served [40], whereas this is not the case in mice revealing a few hundred mg/g up to ∼2 g/g [40, 57] or comparable
increased urinary excretion of albumin only [31]. Further- to 24-h protein excretions [56, 59, 60]. The urinary
more, some endocytosis of VDBP is observed in the protein excretion is correlated with the excretion of

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 278
cubilin ligands, such as albumin, α1-microglobulin and
transferrin, whereas the correlation between the total
protein excretion and urinary β2-microglobulin, a ligand
for megalin, and immunoglobulin-G was less obvious
[57]. So far no consistent changes in the renal structure
have been reported in biopsies from cases of IGS. In one
patient, with concomitant haematuria, focal mesangial ex-
pansion with immunoglobulin-A deposits consistent with
coexisting IgA nephropathy was observed [40], while in
others no or non-specific glomerular changes have been
described [60–62]. Although cubilin has been identified
in the podocyte [34], no characteristic glomerular pathol-
ogy has been reported in animal models of cubilin
deficiency or dysfunction, and thus the specificity of the
glomerular changes observed in a few IGS patients
remains elusive, and may in fact represent bias in the se-
lection of patients for renal biopsy. In conclusion, current
evidence suggest that proteinuria observed in patients
with IGS is the result of tubular dysfunction; however,
further studies in humans are needed. The tubular origin
of the proteinuria in IGS is supported by the observation
that generally the proteinuria is stable and not associated
with progressive loss of renal function [52].
Urinary tract abnormalities have been reported in Nor-
wegian patients with IGS [5]; however, as these are
associated with mutations in AMN this may be caused by
other developmental functions of AMN and independent
of cubilin. Similarly, impaired transport of B12 into the
CNS was suggested in one patient with IGS associated
with a mutation in AMN [63], and despite the expression
of cubilin in other tissues than intestine and kidneys, no
consistent phenotype relating to these tissues have been
reported in IGS.
Cubilin, albuminuria, and chronic kidney disease
Albuminuria is one of the most sensitive and widely used
markers of kidney dysfunction and an important risk
marker for the progression of renal disease as well as for
mortality and death [64]. The importance of cubilin in
tubular albumin uptake suggests a crucial role in regulat-
ing urinary albumin excretion. As discussed, mutations in
cubilin and AMN often result in albuminuria; however,
it is not clear whether cubilin dysfunction is associated
with the progression of renal disease or with the increased
mortality associated with albuminuria in larger popu-
lations. A genome-wide association study has identified, a
single-nucleotide polymorphism (SNP) in CUBN as a
gene locus for albuminuria both in the general population
and in diabetes [65], supporting a role of cubilin in regu-
lating urinary albumin excretion. Whether this link is
casual and whether this is associated with a greater risk of
progressive renal disease remain to be established. As
also mentioned above, a single base pair deletion of
CUBN exon 53 (∼CUB domain 20) was recently found
by exome sequencing in two siblings with albuminuria
but no megaloblastic anaemia [58]. Recently, a different
SNP in cubilin was associated with a greater risk for end-
stage renal disease, as well as proteinuria and graft failure
in transplanted kidneys, with the latter being associated
with the donor rather than the recipient genotype [66].
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E.I. Christensen et al.
While any causal relationship is uncertain, this may
suggest that cubilin function or dysfunction may play a
role for the development or progression of CKD although,
as evident from patients with IGS, this is not obligatory.
Changes in cubilin function and/or expression have
been reported in animal models of acute and chronic renal
disease including lipopolysaccharide-induced endotoxe-
mia [67], ischaemia–reperfusion kidney injury [67, 68],
and chronic kidney disease using the remnant kidney
model of chronic kidney disease [69]. Increased urinary
excretion of cubilin, along with megalin, has been ident-
ified in models of Alport syndrome [70], and experimen-
tal as well as human diabetes [71, 72]. In cystic fibrosis
mouse models [73], an increased excretion of cubilin but
not of megalin was observed together with increased
LMW proteinuria of cubilin ligands. Whether these
changes are secondary or part of the primary events
leading to kidney dysfunction is not clear. Excessive
tubular uptake of filtered proteins, including albumin, has
been implicated in the progression of chronic kidney
disease [74] as also recently reviewed [75, 76]. Studies
have suggested a number of mechanisms by which
albumin and other filtered proteins may activate cellular
pathways in proximal tubule cells leading to apoptosis,
endoplasmic reticulum stress, interstitial inflammation and
fibrosis [74, 76, 77]. Antisense RNA delivered by adeno-
virus and leading to partial knockdown cubilin is shown
to inhibit adriamycin-induced glomerulosclerosis and tu-
bulointerstitial damage in rats despite higher level of albu-
minuria [78]; however, additional studies are needed to
establish the role of cubilin-mediated albumin uptake for
the progression of proteinuric kidney disease [76].
Cubilin in acute kidney injury
Cubilin has been shown to bind and mediate uptake
of several nephrotoxic proteins including light chains
[79–81], myoglobin [82] and haemoglobin [83], which
may cause acute kidney failure if filtered in excessive
amount. Since all these proteins also bind to megalin, the
specific role of cubilin in mediating nephrotoxicity is
unresolved.
Other diseases
In Dent’s disease, proximal tubular expression of cubilin
is significantly reduced [84] together with a decreased
expression of megalin [84, 85] probably as a consequence
of a disturbed intracellular trafficking of the receptors,
leading to LMW proteinuria. Association studies have
identified polymorphisms in CUBN associated with type
1 diabetes [86] and spina bifida [87], observations which
need confirmation.
Conclusion
The physiological importance of cubilin in both the intes-
tine and the kidneys is evident both from animal models
and humans. The interaction of cubilin with other mem-
brane proteins is also well established; however, further
studies are needed to identify the tissue specificity of
 Cubilin in bowel and kidneys
these interactions and to clarify the significance of these
in the individual, intracellular transport steps. The func-
tional role of cubilin expression in podocytes awaits
further clarification. Finally, while the phenotype of rare,
inherited diseases involving mutations and dysfunction of
cubilin or associated proteins is well described, the poss-
ible role of cubilin in the development of albuminuria and
CKD as suggested by gene association studies requires
further confirmation and clarification of the underlying
pathogenic mechanisms.
Acknowledgements. The work was supported in part by the Danish
Medical Research Council, Lundbeck Foundation, Novo Nordisk Foun-
dation, Aarhus University and the program of the European Community,
EUNEFRON (FP7, GA#201590).
Conflict of interest statement. None declared.
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Received for publication: 11.9.12; Accepted in revised form: 29.10.12