Pflugers Arch - Eur J Physiol (2010) 459:333343

DOI 10.1007/s00424-009-0722-7

INTEGRATIVE PHYSIOLOGY

Klotho
Makoto Kuro-o

Received: 23 July 2009 / Accepted: 26 August 2009 / Published online: 4 September 2009
# Springer-Verlag 2009

Abstract The klotho gene was identified as an agingsuppressor gene in mice that accelerates aging when
disrupted and extends life span when overexpressed. It
encodes a single-pass transmembrane protein and is
expressed primarily in renal tubules. The extracellular
domain of Klotho protein is secreted into blood and urine
by ectodomain shedding. The two forms of Klotho protein,
membrane Klotho and secreted Klotho, exert distinct
functions. Membrane Klotho forms a complex with
fibroblast growth factor (FGF) receptors and functions as
an obligate co-receptor for FGF23, a bone-derived hormone
that induces phosphate excretion into urine. Mice lacking
Klotho or FGF23 not only exhibit phosphate retention but
also display a premature-aging syndrome, revealing an
unexpected link between phosphate metabolism and aging.
Secreted Klotho functions as a humoral factor that regulates
activity of multiple glycoproteins on the cell surface,
including ion channels and growth factor receptors such
as insulin/insulin-like growth factor-1 receptors. Potential
contribution of these multiple activities of Klotho protein to
aging processes is discussed.
Keywords Klotho . FGF23 . Phosphate .
Vitamin D . Klotho

M. Kuro-o (*)
Department of Pathology,
The University of Texas Southwestern Medical Center at Dallas,
6000 Harry Hines Blvd.,
Dallas, TX 75390-9072, USA
e-mail: makoto.kuro-o@utsouthwestern.edu

Introduction
The klotho gene was originally identified as a gene
disrupted in a mouse strain that inherited a syndrome
resembling human aging in an autosomal recessive
manner [48]. This strain, named after a Greek goddess
Klotho who spins the thread of life, was serendipitously
generated during an attempt to make transgenic mice by
conventional pronuclear microinjection of a transgene
[47]. Unfortunately, the transgene was not expressed in
the klotho mouse. However, integration of the transgene
into the mouse genome disrupted a promoter region of a
certain gene and shuts down its expression, which was
later identified as the klotho gene.
A defect in klotho gene expression in mice causes no
visible phenotypes until 34 weeks of age, but thereafter
leads to multiple aging-like phenotypes [48], including
growth retardation, hypogonadotropic hypogonadism, rapid
thymic involution [62], skin atrophy, sarcopenia, vascular
calcification, osteopenia [38], pulmonary emphysema [33,
81, 92], cognition impairment [67], hearing disturbance
[37], and motor neuron degeneration [1], and die prematurely around 2 months of age. In contrast, transgenic mice
that overexpress the klotho gene live longer than wild-type
mice [54]. Thus, the klotho gene may function as an agingsuppressor gene that extends life span when overexpressed
and accelerates aging when disrupted [46].
The klotho gene is composed of five exons [59, 90] and
encodes a type I single-pass transmembrane protein of
1,014 amino acid long. The intracellular domain is very
short (10 amino acid long) and has no known functional
domains. The extracellular domain is composed of two
internal repeats with weak homology to family 1 glycosidases that hydrolyze -glucosidic linkage in saccharides,
glycoproteins, and glycolipids [48, 61].

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The klotho gene is expressed predominantly in distal

convoluted tubules in the kidney and choroid plexus in the
brain [48]. It is also expressed in several endocrine organs,
including pituitary, parathyroid [5], pancreas, ovary, testis,
and placenta [48]. Despite the tissue-specific expression of
the klotho gene, a defect in klotho expression causes
systemic phenotypes, suggesting that Klotho may be
involved in the regulation of an endocrine system(s).
Recent studies have revealed multiple functions of
Klotho protein in endocrine regulation of mineral metabolism and growth factor signaling. The purpose of this
review was to summarize Klotho protein function and to
discuss its potential effects on aging processes in mammals.

Function of membrane Klotho protein

Klotho as a co-receptor for FGF23
Klotho protein function was not clear until it was realized
that phenotypes of Klotho-deficient mice were almost
identical with those of mice lacking fibroblast growth
factor-23 (FGF23). FGF23 had been identified as a gene
mutated in patients with autosomal dominant hypophosphatemic rickets (ADHR) [107]. ADHR is one of the
hereditary disorders that exhibit a phosphate wasting into
urine, indicating the involvement of FGF23 in phosphate
handling in the kidney.
Recent studies have identified FGF23 as a bone-derived
hormone that acts on kidney to promote phosphate
excretion into urine (phosphaturia) [75]. The amount of
phosphate excreted into urine is primarily determined by
the amount of phosphate reabsorbed at renal proximal
tubules. Phosphate in the luminal fluid in the proximal
tubules is taken up mainly through sodiumphosphate cotransporter type-2a (NaPi-2a) expressed on the apical brush
border membrane of proximal tubules [83, 84, 89]. FGF23
exerts its phosphaturic activity by suppressing NaPi-2a [57,
83]. Administration of recombinant FGF23 protein to
rodents reduces the number of NaPi-2a inserted in the
apical brush border membrane of proximal tubules and
induces phosphaturia within hours, although the precise
signaling pathway through which FGF23 regulates NaPi-2a
trafficking and/or expression remains to be determined.
ADHR patients carry missense mutations in the FGF23
gene that confer resistance to proteolytic inactivation on the
mutant FGF23 protein, resulting in high serum FGF23
levels and phosphate-wasting phenotypes including hypophosphatemia and defects in bone mineralization (rickets)
[88]. Furthermore, several mutations that affect expression
and/or proteolytic degeneration of FGF23 have been
identified in mice and humans, in which phosphatewasting phenotypes are associated with increased serum

Pflugers Arch - Eur J Physiol (2010) 459:333343

FGF23 levels [51, 75]. These observations have established

that FGF23 functions as a phosphaturic hormone.
In contrast, mice lacking FGF23 (Fgf23/ mice)
develop phosphate-retention phenotypes characterized by
extensive soft tissue calcification and hyperphosphatemia
[87]. In addition to these predictable phenotypes, Fgf23/
mice exhibit unexpected phenotypes, including growth
retardation, hypogonadism, premature thymic involution,
sarcopenia, osteopenia, skin atrophy, and pulmonary emphysema, which are reminiscent of the premature-aging
syndrome in Klotho-deficient mice. Likewise, Klothodeficient mice not only exhibit premature aging but also
suffer extensive soft tissue calcification and hyperphosphatemia, which are reminiscent of the phosphate-retention
phenotype in Fgf23/ mice. These observations revealed
an unexpected link between FGF23 and Klotho, suggesting that FGF23 and Klotho might function in a common
endocrine system that regulates phosphate metabolism. In
support of this notion, Klotho-deficient mice have
extremely high serum FGF23 levels, indicating that loss
of Klotho induces resistance to FGF23 [103].
Consistent with these genetic studies, biochemical and
cell biological studies have demonstrated that Klotho
protein functions as an obligate co-receptor for FGF23
[44, 53, 103]. FGF23 has very low affinity to FGF
receptors (FGFRs) and cannot activate its cognate receptors
without Klotho under physiological concentration [112].
Klotho protein forms a binary complex with several FGF
receptor isoforms (FGFR1c, 3c, and 4) and significantly
increases their affinity to FGF23 [53]. The fact that FGF23
requires Klotho for activating FGF receptors explains why
FGF23-deficient mice and Klotho-deficient mice develop
identical phenotypes and why Klotho-deficient mice are
resistant to FGF23. Also, kidney-specific expression of
Klotho explains why FGF23 can identify kidney as its
target organ among many other tissues that express multiple
FGF receptor isoforms. Thus, Klotho and FGF23 have
emerged as essential components of this newly identified
bonekidney endocrine axis that maintains phosphate
homeostasis [45].
Another important function of this endocrine axis is to
regulate serum levels of vitamin D. Vitamin D has an
activity that promotes absorption of dietary phosphate from
intestine [16]. The active form of vitamin D (1,25dihydroxyvitamin D3) is synthesized in the kidney from
its inactive precursor (25-hydroxyvitamin D 3 ) with
1-hydroxylase encoded by the Cyp27b1 gene and is
inactivated with 24-hydroxylase encoded by the Cyp24
gene. FGF23 suppresses Cyp27b1 gene expression and
increases Cyp24 gene expression, leading to reduction of
serum 1,25-dihydroxyvitamin D3 levels [86]. Thus, in
addition to functioning as a phosphaturic hormone, FGF23
functions as a counter-regulatory hormone for vitamin D [58].

Pflugers Arch - Eur J Physiol (2010) 459:333343

These two distinct activities of FGF23 collectively induce a

negative phosphate balance in a Klotho-dependent manner.
The bonekidney endocrine axis mediated by Klotho
and FGF23
Vitamin D positively regulates expression of the FGF23
gene in the bone. Injection of 1,25-dihydroxyvitamin D3
increases serum FGF23 levels within hours in rodents [86].
Binding of 1,25-dihydroxyvitamin D3 to nuclear vitamin D
receptor (VDR) induces heterodimerization with another
nuclear receptor RXR. The VDR-RXR heterodimer in turn
binds to the promoter region of the FGF23 gene and
transactivates its expression [45]. FGF23 secreted from
osteocytes reaches the KlothoFGFR complex in the
kidney and transmits signal to suppress synthesis and
promote inactivation of 1,25-dihydroxyvitamin D3, thereby
closing a negative feedback loop (Fig. 1). This negative
feedback loop is essential for vitamin D homeostasis
because defects in either FGF23 or Klotho result in elevated
serum 1,25-dihydroxyvitamin D3 levels (hypervitaminosis
D) in mice and humans.
Phosphate also positively regulates FGF23 expression,
although the mechanism behind this regulation remains to

Fig. 1 The bonekidney endocrine axis that regulates phosphate and

vitamin D homeostasis. High serum phosphate increases FGF23
expression in the bone. FGF23 secreted from the bone binds to the
KlothoFGFR complex expressed on the kidney and suppresses
phosphate reabsorption by inhibiting NaPi-2a (a negative feedback
loop for phosphate homeostasis). High serum vitamin D increases
FGF23 expression. Active vitamin D (1,25-dihydroxyvitamin D3)

335

be determined. Phosphate overload by high phosphate diet

increases serum FGF23 levels in mice and humans [73, 79,
95]. Increased FGF23 in turn induces phosphaturia to
excrete excess phosphate into urine, thereby closing another
negative feedback loop for phosphate homeostasis (Fig. 1).
Suppression of serum vitamin D levels by increased FGF23
also contributes to prevention of phosphate retention
through reducing phosphate absorption from intestine.
However, these two negative feedback loops for phosphate
and vitamin D homeostasis can function independently.
Although VDR-deficient mice (VDR/ mice) have low
serum levels of phosphate and FGF23, the rescue diet
(rich in calcium and phosphate) restores serum FGF23
levels in VDR/ mice, indicating that phosphate can
increase FGF23 independently of vitamin D [113]. Also,
administration of low-dose vitamin D increases serum
FGF23 levels without significant increase in serum phosphate levels in mice, indicating that vitamin D can increase
FGF23 independently of phosphate [86].
Disruption of these negative feedback loops by ablating
either FGF23 or Klotho leads to hyperphosphatemia and
hypervitaminosis D. Hypercalcemia is also observed because vitamin D promotes both phosphate and calcium
absorption from intestine. The question is whether this

binds to vitamin D receptor (VDR) in osteocytes, which forms a

heterodimer with a nuclear receptor RXR and directly binds to a
promoter region of the FGF23 gene to transactivate its expression.
FGF23 acts on the KlothoFGFR complex in the kidney and
suppresses expression of Cyp27b1 gene that encodes 1-hydroxylase
(a negative feedback loop for vitamin D homeostasis)

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metabolic state (hyperphosphatemia, hypercalcemia, and/or

hypervitaminosis D) is primarily responsible for the
premature-aging syndrome. Several studies have addressed
this question. First, Klotho- and FGF23-deficient mice,
when placed on vitamin D-deficient diet, exhibited normal
serum phosphate and calcium levels and no longer
developed aging-like phenotypes [91, 100]. Second, ablation of vitamin D action in Klotho-deficient mice and
FGF23-deficient mice by disrupting the Cyp27b1 gene [69,
77] or vitamin D receptor gene [25] rescued hyperphosphatemia and hypercalcemia, as well as the premature aging
syndrome. Lastly, low phosphate diet rescued shortened life
span and vascular calcification in FGF23- and Klothodeficient mice [64, 91]. These studies have clearly
demonstrated that the premature aging syndrome caused
by defects in the Klotho-FGF23 endocrine axis is attributed
to retention of phosphate, calcium, and/or vitamin D. It
should be noted that low phosphate diet significantly
increases serum levels of calcium and vitamin D [91]. This
is considered as an adaptation for limited phosphate
availability to maximize phosphate absorption from intestine by increasing serum vitamin D levels. However,
vitamin D promotes calcium absorption at the same time
and results in hypercalcemia. In fact, low phosphate diet
further increased already high serum calcium and vitamin D
levels in FGF23-deficeint mice but still rescued multiple
aging-like phenotypoes [91]. These findings suggest that
phosphate, but not calcium or vitamin D, is primarily
responsible for the aging-like phenotypes. It is likely that
low vitamin D diet and ablation of vitamin D activity
rescued accelerated aging through reducing serum phosphate levels, although it is possible that high serum vitamin
D and/or calcium levels may be a prerequisite for phosphate
to induce the premature aging syndrome.

Phosphate and aging

Phosphate and insulin sensitivity
Inorganic phosphate is one of the essential nutrients and a
major structural component of DNA, cell membrane
(phospholipids), and bone. In addition, phosphate participates in a myriad of biological processes including energy
metabolism (ATP production) and signal transduction
mediated by kinases. It is also involved in pathophysiology
of various common disorders such as bone diseases
(osteoporosis and osteomalasia), vascular calcification,
and chronic kidney disease among others. Although direct
effects of phosphate on aging have not been explored
extensively, several studies have demonstrated that inorganic phosphate affects glucose metabolism and oxidative
stress, which potentially modifies aging processes as well.

Pflugers Arch - Eur J Physiol (2010) 459:333343

Animals placed on low phosphate diet were reported to

exhibit increased gluconeogenesis and decreased glycolysis
[109, 110]. This metabolic state is reminiscent of that
induced by diet restriction, which delays aging and extends
life span in diverse organisms. To adapt reduced food
availability, animals under diet restriction reduces blood
insulin levels and attenuates overall activity of intracellular
insulin signaling in tissues, which triggers changes in the
expression of insulin-responsive genes and leads to global
changes in glucose metabolism, including increased gluconeogenesis and decreased glycolysis [9, 39, 55, 106].
Although phosphate restriction does not induce hypoinsulinemia like diet restriction, low phosphate diet induces
moderate insulin resistance by unknown mechanisms [23,
72]. The moderate insulin resistance induced by phosphate
restriction also attenuates overall activity of intracellular
insulin signaling in tissues, leading to changes in expression of insulin-responsive genes and metabolic state similar
to those induced by hypoinsulinemia upon diet restriction.
Conversely, phosphate retention is associated with increased insulin sensitivity and accelerated aging as observed in Klotho-deficient mice and FGF23-deficient mice
[77, 87, 104]. They exhibit hypoglycemia, hypoinsulinemia, and extremely increased insulin sensitivity, which can
be rescued by resolving hyperphosphatemia [25]. Thus,
serum phosphate levels positively correlate with insulin
sensitivity.
Increased insulin resistance does not necessarily mean
diabetes and short life span. Rather, it has become
increasingly clear that partial or tissue-specific inhibition
of insulin-like signaling pathway is a mechanism for antiaging and life span extension evolutionarily conserved from
worms to mammals. Hypomorphic alleles of the genes
encoding orthologs of insulin receptor, insulin receptor
substrates (IRS), and PI3 kinase have been associated with
extended life span in Caenorhabditis elegans and Drosophila [14, 40, 41, 65, 96]. In rodents, extended longevity was
reported in mice lacking insulin receptor in adipose tissues
[6], mice heterozygous for a null allele of the insulin-like
growth factor-1 (IGF-1) receptor gene [28], mice lacking
IRS-1 [85], mice lacking IRS-2 in the brain [94], and dwarf
mice with impaired somatotrophic endocrine axis (the
growth hormoneIGF-1 endocrine axis) [4, 8, 19]. In
humans, some centenarians carry loss-of-function mutations in the IGF-1 receptor gene and show resistance to
IGF-1, short statue, and high serum IGF-1 levels [93]. In
addition, some long-lived mice exhibit insulin resistance
[54, 85], indicating that increased insulin sensitivity is not a
prerequisite for long life span and anti-aging. Although life
span extension by diet restriction is associated with
increased insulin sensitivity, it is associated with hypoinsulinemia and the metabolic state compatible with attenuated
insulin signaling activity in tissues.

Pflugers Arch - Eur J Physiol (2010) 459:333343

Phosphate and oxidative stress

In addition to its involvement in glucose metabolism and
insulin sensitivity, inorganic phosphate increases oxidative
stress both in vivo and in vitro. Phosphate retention caused
by Klotho deficiency in mice is associated with cognition
impairment due to increased oxidative damage to hippocampus neurons, which can be rescued by administration of
an antioxidant [67]. Furthermore, human vascular endothelial cells exposed to high phosphate medium (2.5 mM) have
higher levels of cellular reactive oxygen species (ROS) than
those cultured in normal phosphate medium (1.0 mM) [15].
These observations have raised the possibility that hyperphosphatemia by itself may be a cause of endothelial
dysfunction and cardiovascular disease in chronic kidney
disease (CKD) patients because blood phosphate levels
higher than 2.5 mM are often observed in these patients.
Indeed, hyperphosphatemia has been identified as a potent
risk of death in CKD patients [21, 99]. Of note, the
National Kidney Foundation task force has indicated that
the cardiovascular mortality of a 35-year-old patient on
dialysis is equivalent to that of an 80-year-old healthy
individual, rendering CKD to be a premature vascular aging
[60]. Also, the American Heart Association identified CKD
patients as the highest-risk group for cardiovascular disease
[80]. Importantly, Klotho expression is significantly reduced in CKD patients [43]. Thus, CKD may be viewed as
a segmental progeroid syndrome associated with Klotho
deficiency and phosphate retention.
Extracellular phosphate enters into the cell primarily
through sodium-phosphate co-transporter type-3 (NaPi-3,
also known as Pit-1/2) [105]. Cytoplasmic phosphate is
transported into mitochondria to be utilized for oxidative
phosphorylation. It has been known that extramitochondrial phosphate concentration (or cytoplasmic
phosphate concentration) positively correlates with mitochondrial membrane potential () [7], and positively
correlates with ROS production in mitochondria [70]. In
addition, phosphate enhances delivery of reducing equivalent to cytochrome c in complex III in the electron transport
chain [7]. These effects of phosphate on mitochondrial
function potentially increase ROS production in the cell and
may affect aging processes.
Phosphate and longevity
The fact that the premature aging syndrome in Klothodeficient mice and FGF23-deficient mice is rescued by
resolving hyperphosphatemia has raised the possibility
that aging process may be accelerated by inorganic
phosphate that plays multiple roles in glucose metabolism, insulin sensitivity, and oxidative stress in health
and disease. Several lines of circumstantial evidence

337

support this notion. First, serum phosphate levels

positively correlate with all-cause mortality in humans.
An epidemiological study using patients whose serum
phosphate levels are within normal range demonstrated
that those with high serum phosphate levels (4.0 mg/
dL) exhibited ~70% higher mortality rate than those
with low serum phosphate levels (<2.5 mg/dL) [99].
Furthermore, the longevity of various species in mammals
inversely correlates with the serum phosphate level, but
not with the serum calcium level (Table 1, Fig. 2),
indicating an association between phosphate metabolism
and aging processes.

Function of secreted Klotho protein

Regulation of ion channel activity
In addition to functioning as a co-receptor for FGF23,
Klotho protein functions as a humoral factor. The extracellular domain of Klotho protein is clipped on the cell surface
(ectodomain shedding) by membrane-anchored proteases
ADAM10 and ADAM17 to generate a secreted form of
Klotho protein [13]. In fact, the entire extracellular domain
of ~120130 kDa (Klotho ectodomain) is detected in the
blood, urine, and cerebrospinal fluid (CSF) [29, 54]. Thus,
Klotho protein exists at least in two forms. One is
membrane Klotho expressed primarily in renal tubular
cells, and the other is secreted Klotho that exists in the
blood, urine, and CSF.
Although membrane Klotho functions as a coreceptor for FGF23, secreted Klotho cannot function
as a soluble receptor for FGF23 because it is the
KlothoFGFR complex that has high affinity to FGF23,
but not Klotho protein or FGFR alone [53]. These
findings suggest that secreted Klotho may have function
independent of FGF23. Recent studies have revealed
novel function of secreted Klotho distinct from that of
membrane Klotho, which includes regulation of multiple
ion channels and growth factor receptors on the cell
surface.
Transient receptor potential cation channel, subfamily
V, member 5 (TRPV5) is one of the ion channels
regulated by secreted Klotho protein [11, 12]. TRPV5 is
a calcium channel expressed on the apical side of the renal
tubular cells and is primarily responsible for the Ca2+
entry in transepithelial Ca2+ reabsorption in the kidney
[26]. Secreted Klotho protein inhibits internalization of
TRPV5 and increases the number of TRPV5 on the cell
surface, thereby increasing TRPV5-mediated Ca2+ influx
and renal Ca2+ reabsorption. In fact, Klotho-deficient mice
exhibit higher calcium excretion into urine than wild-type
mice [101]. The Klotho-induced retention of TRPV5 on

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Pflugers Arch - Eur J Physiol (2010) 459:333343

Table 1 Relation between longevity and serum phosphate level in mammals

Species
Mouse (Klotho/)
Mouse
Rat
Hamster
Gerbil
Nutria
Rabbit
Guinea pig
Sheep
Squirrel
Porcupine
Naked mole rat
Flying fox
Bear
Rhinoceros
Camel
Elephant
Human
Human (centenarian)
a

Longevity (year)

Serum phosphate (mg/dL)a

Serum calcium (mg/dL)a

0.15
3
3
4
6
9
10
12

14.0
7.8
9.0
6.5
5.4
7.5
7.4
5.3

10.3
9.2
11.8
9.7
4.8
9.6
14
10.2

[48,
[27,
[27,
[18,
[18,
[17,
[27,
[22,

5.5
5.4
4.4
4.6
4.4
4.9
3.7
4.6
4.3
3.6
3.1

8.4
7.5
9.6
9.6
9.4
9.6
12.4
9.3
9.5
9.5
8.6

[27, 74]
[2, 22]
[22, 63]
[22, 111]
[24, 27]
[27, 76]
[27, 66]
[27, 78]
[27, 102]
[27, 71]
[71]

21
24
27
28
30
50
50
50
70
75
100

Reference
84]
84]
97]
22, 97]
22]
22]
97]
97]

Average or median values, whichever available in the literatures

the plasma membrane was associated with modification of

the glycans of TRPV5 [11, 12]. Consistent with the fact
that Klotho belongs to the family 1 glycosidases that
cleave glycosidic bonds, secreted Klotho was reported to
have weak -glucuronidase activity in vitro [98]. However, the -glucuronidase activity of Klotho may not be
involved in the modification of TRPV5 glycans because
typical N-linked glycans of mammalian glycoproteins do
not contain glucuronic acids. Instead, sialic acids cap the

Fig. 2 Relation between longevity and serum levels of phosphate

and calcium in mammals (based on Table 1). The longevity is
inversely correlated with serum phosphate levels (closed circles,
y
1:5183Lnx 10:015, R2 =0.8945). On the other hand, there
is no significant correlation between longevity and serum calcium
levels (open circles)

terminals of branched N-linked glycans. Secreted Klotho

removes these terminal sialic acids though its putative
sialidase activity [11]. Removal of sialic acids exposes
underlying galactose or disaccharide N-acetyllactosamine
in the glycans. Once exposed, these sugars can bind to
galectin-1, a lectin (sugar-binding protein) abundant in the
extracellular matrix. Thus, removal of terminal sialic acids
from the glycans of TRPV5 by secreted Klotho promotes
interaction between TRPV5 glycans and galectin-1, which
traps TRPV5 on the cell surface and prevents its
endocytosis, resulting in accumulation of TRPV5 on the
plasma membrane (Fig. 3) [11].
Furthermore, secreted Klotho regulates renal outer
medullary potassium channel (ROMK1) through the mechanism identical with that for TRPV5 regulation [10].
ROMK1 is a potassium channel expressed on the apical
side of renal tubular cells and is primarily responsible for
potassium secretion into urine. Secreted Klotho removes
terminal sialic acids from the glycans of ROMK1, which
triggers interaction between the ROMK1 glycans and
galectin-1, tethers ROMK1 to extracellular matrices, and
prevents its internalization. Thus, secreted Klotho increases
the number of ROMK1 on the plasma membrane and
increases potassium secretion into urine. Indeed, injection
of secreted Klotho into mice promotes potassium excretion
into urine [10]. This action of secreted Klotho represents a
novel mechanism for regulation of internalization of cellsurface glycoproteins.

Pflugers Arch - Eur J Physiol (2010) 459:333343

339

Fig. 3 Klotho inhibits internalization of cell-surface calcium channel

TRPV5. The number of TRPV5 on the plasma membrane is
determined by counterbalance between insertion by forward trafficking from Golgi and removal by endocytosis to endosomes. Terminals
of sugar chains of many cell-surface glycoproteins are capped with
sialic acids (red). Secreted Klotho protein removes these sialic acids

through its putative 26 sialidase activity and exposes underlying

galactose residues (green) in the glycans. The exposed galactose then
binds to galectin-1 (blue) in the extracellular matrix. Galectin-1 traps
TRPV5 on the cell surface and prevents its endocytosis, leading to
accumulation of TRPV5 on the plasma membrane and increase in
calcium influx

Regulation of growth factor signaling

action and may partly explain why Klotho-deficient mice

are extremely insulin sensitive.
It was reported that secreted Klotho protein bound to
several Wnt ligands and inhibited Wnt signaling through
preventing Wnt binding to its cognate cell-surface receptor
[56]. Although activation of Wnt signaling is essential for
stem cell proliferation and survival, continuous and
prolonged Wnt signaling activation can cause exhaustion
and depletion of stem cells [42, 82]. Since stem cell
dysfunction limits tissue regeneration and potentially
affects aging processes, the ability of secreted Klotho
protein to inhibit Wnt signaling may also contribute to
aging-like phenotypes in Klotho-deficient mice. The number of epidermal stem cells in hair follicles in Klothodeficient mice was much lower than that in wild-type mice.
Enhanced Wnt signaling activity and expression of SAgalactosidase, a marker for cell senescence, were observed
in epidermal stem cells in Klotho-deficient mice. These
findings can be explained by assuming that Klotho
deficiency caused continuous activation of Wnt signaling
and senescence of stem cells. The reduced number of
epidermal stem cells may also explain poor wound healing
observed in Klotho-deficient mice.

Klotho-deficient mice are hypoglycemic, hypoinsulinemic,

and extremely sensitive to insulin [25, 104]. In contrast,
Klotho-overexpressing transgenic mice are resistant to
insulin and IGF-1, although they maintain normal fasting
blood glucose levels and are not diabetic [54]. These
observations suggest that Klotho may have an inhibitory
effect on insulin/IGF-1 activity. In fact, secreted Klotho has
an activity that inhibits insulin- and IGF-1-induced autophosphorylation of insulin receptor and IGF-1 receptor
when applied to cultured cells [54, 108]. Although the
mechanism by which secreted Klotho inhibits activity of
insulin/IGF-1 receptors remains to be determined, it is
possible that secreted Klotho may alter internalization and
cell surface abundance of insulin/IGF-1 receptors by
modifying their glycans. The ability of Klotho to inhibit
insulin/IGF-1 receptors may contribute to the anti-aging
properties of Klotho since numerous lines of genetic
evidence indicate that partial inhibition of insulin-like
signaling pathway is one of the evolutionarily conserved
mechanisms for suppressing aging as discussed above. The
fact that insulin promotes Klotho shedding [13] is of
potential importance in the regulation of insulin activity in
vivo. Insulin may increase serum levels of secreted Klotho
protein, which in turn inhibits insulin signaling in peripheral tissues and prevents prolonged insulin action. This may
represent a novel negative feedback mechanism of insulin
signaling by secreted Klotho through its endocrine mode of

Concluding remarks
The discovery of the klotho gene has led to identification of
a novel bonekidney endocrine axis that maintains phos-

340

phate and vitamin D homeostasis: FGF23 is secreted from

bone and acts on kidney where Klotho is expressed to
induce phosphaturia and to counteract vitamin D. Although
FGF23 belongs to the FGF ligand superfamily, amino acid
sequence analysis has segregated FGF23 and two additional
FGFs (FGF19 and FGF21) from the other classic FGF
ligands [36]. These atypical FGFs (FGF19, FGF21, and
FGF23) are collectively called endocrine FGFs [20]
because they function as hormones unlike the other classic
FGFs that primarily act as paracrine and/or autocrine
factors. FGF19 is secreted from intestine upon feeding
and acts on liver to suppress bile acid synthesis [30].
FGF21 is secreted from liver upon fasting and acts on white
adipose tissue to promote lipolysis and to mediate fasting
metabolic responses [3, 31]. Like FGF23 requires Klotho,
FGF19 and FGF21 require Klotho as an obligate coreceptor for high affinity binding to their cognate FGF
receptors [49, 68]. Klotho was identified based on its
sequence similarity to Klotho [35]. Unlike Klotho, Klotho
is expressed predominantly in the liver and white adipose
tissue [35, 49], the target organs of FGF19 and FGF21,
respectively. Consistent with the fact that Klotho is an
obligate co-receptor for FGF19, mice lacking either
Klotho or FGF15 (the mouse ortholog of human
FGF19) exhibited increased bile acid synthesis [30, 34].
Thus, Klotho gene family may have evolved in the
regulation of endocrine FGFs to confine their target organs
in the redundant receptor-ligand system [45, 5052].
Multiple novel endocrine axes mediated by endocrine
FGFs and Klothos have emerged that regulate various
metabolic processes. FGF23 and Klotho affect aging
processes through regulating phosphate metabolism.
Interestingly, transgenic mice that overexpress FGF21
exhibit short statue and resistance to growth hormone,
which is reminiscent of dwarf mice [32]. It remains to be
determined whether FGF19, FGF21, and Klotho may
also affect aging processes through regulating metabolic
responses to feeding and fasting.
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