Renal and Extrarenal Actions of Klotho

Ming Chang Hu, MD, PhD,*,†,‡ Makoto Kuro-o, MD, PhD,*,§ and Orson W. Moe, MD*,†,储

Summary: Klotho is a single-pass transmembrane protein highly expressed in the kidney. Membrane Klotho
protein acts as a co-receptor for fibroblast growth factor-23. Its extracellular domain is shed from the cell surface
and functions as an endocrine substance that exerts multiple renal and extrarenal functions. An exhaustive review
is beyond the scope and length of this article; thus, only effects with pertinence to mineral metabolism and
renoprotection are highlighted here. Klotho participates in mineral homeostasis via interplay with other calcio-
phosphoregulatory hormones (parathyroid hormone, fibroblast growth factor-23, and 1,25-[OH]2 vitamin D3) in
kidney, bone, intestine, and parathyroid gland. Klotho also may be involved in acute and chronic kidney disease
development and progression. Acute kidney injury is a temporary and reversible state of Klotho deficiency and
chronic kidney disease is a sustained state of systemic Klotho deficiency. Klotho deficiency renders the kidney
more susceptible to acute insults, delays kidney regeneration, and promotes renal fibrosis. In addition to direct
renal effects, Klotho deficiency also triggers and aggravates deranged mineral metabolism, secondary hyperpara-
thyroidism, vascular calcification, and cardiac hypertrophy and fibrosis. Although studies examining the therapeu-
tic effect of Klotho replacement were performed in animal models, it is quite conceivable that supplementation of
exogenous Klotho and/or up-regulation of endogenous Klotho production may be a viable therapeutic strategy for
patients with acute or chronic kidney diseases.
Semin Nephrol 33:118-129 © 2013 Elsevier Inc. All rights reserved.
Keywords: Acute kidney injury, cardiac hypertrophy, chronic kidney disease, hyperparathyroidism, Klotho

T he Klotho gene was identified in 1997 when its

disruption in mice caused a phenotype of prema-
ture multiorgan failure including shortened life
span, growth retardation, accelerated thymic involution,
pulmonary emphysema, cognition impairment, skin atro-
phy, osteopenia, ectopic soft-tissue calcification, hyper-
phosphatemia, and high plasma fibroblast growth factor
(FGF)-23 levels.1 Most of the features observed in
Klotho hypomorph or knock-out mice could be rescued
by expressing Klotho,1,2 indicating that Klotho is anti-
aging gene.
*Charles and Jane Pak Center for Mineral Metabolism and Clinical
Research, University of Texas Southwestern Medical Center, Dallas,
TX.
†Department of Internal Medicine, University of Texas Southwestern
Medical Center, Dallas, TX.
‡Department of Pediatrics, University of Texas Southwestern Medical
Center, Dallas, TX.
§Department of Pathology, University of Texas Southwestern Medical
Center, Dallas, TX.
储Department of Physiology, University of Texas Southwestern Medical
Center, Dallas, TX.
Financial support: supported in part by the National Institutes of Health
(R01-DK091392, R01-DK092461), the George M. O’Brien Kidney Re-
search Center at UT Southwestern Medical Center (P30-DK-07938),
the American Heart Association (0865235F), the Simmons Family
Foundation, and the Charles and Jane Pak Foundation.
Conflict of interest statement: none.
Address reprint requests to Ming Chang Hu, MD, PhD, Charles and
Jane Pak Center of Mineral Metabolism and Clinical Research,
University of Texas Southwestern Medical Center at Dallas, 5323
Harry Hines Blvd, Dallas, TX 75390-8885. E-mail:
ming.chang.hu@utsouthwestern.edu
0270-9295/ - see front matter
© 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.semnephrol.2012.12.013
The Klotho gene encodes a single-pass transmembrane
protein and is expressed in multiple tissues, but in particu-
larly high levels in the kidney.1,3 In the mammalian kidney
including mouse, rat, and human, Klotho is expressed prom-
inently in distal convoluted tubules,1,3,4 but also unequivo-
cally is found in the proximal convoluted tubule4 and also in
the inner-medullary collecting duct– derived cell lines.5,6 In
addition to membrane-anchored Klotho, a secreted form of
Klotho protein is generated from the Klotho gene through
alternative splicing. Secreted Klotho is released di-
rectly into the extracellular compartment and is pres-
ent in body fluid. Another important form of soluble
Klotho in body fluid is ectodomain shedding from
membrane Klotho on cell surface by proteases acro-
nym for a desintegrin and metalloproteinase 10/17
(Fig. 1).7 Soluble Klotho protein is present in cerebro-
spinal fluid,8 blood,8,9 and urine of mammals.4,9
Membrane-anchored and soluble Klotho proteins
seem to have distinct functions (Table 1). Membrane
Klotho forms a tetrameric complex with FGF receptors
(FGFRs) and functions as a co-receptor for FGF23,10-12 a
bone-derived phosphatonin that induces negative phos-
phate balance through promotion of renal phosphate ex-
cretion (Fig. 1). Soluble Klotho is a pleiotropic protein
functioning as an endocrine factor with a multitude of
renal and extrarenal effects (Table 1). Recently, several
studies showed that nuclear Klotho13 and cytoplasm
Klotho14 are also bioactive molecules to protect cells
from senescence and apoptosis (Table 1).
PHYSIOLOGICAL ROLE OF KLOTHO IN THE KIDNEY
The kidney is not a mere excretory organ but also a hor-
monal source producing several active molecules such as

118 Seminars in Nephrology, Vol 33, No 2, March 2013, pp 118-129

Renal and extrarenal klotho 119

Figure 1. Physiologic roles of Klotho on solute channels and transporters and vitamin D metabolism in the kidney. Klotho is expressed prominently
in distal convoluted tubules, and less in proximal convoluted tubules.4 In proximal convoluted tubules, membrane Klotho at the basolateral side4
functions as a co-receptor of FGFRs and drives FGF23 signal transduction to inhibit NaPi cotransporters (NaPi: 2a/c and Pit2) and to suppress
cyp27␤1 encoding for 1-hydroxylase, and to stimulate cyp24␣1 encoding for 24-hydroxylase. The role of Klotho in the cytoplasm of renal tubules is
unclear. Whether membrane Klotho at the luminal side4 inhibits NaPi cotransporters (2a/c and Pit2) in an autocrine mode is not known (dashed line).
In distal convoluted tubules, membrane Klotho at the basolateral side4 functions as a co-receptor of FGFRs to induce FGF23 signal transduction.
What intermediate(s) are released from distal convoluted tubules and how intermediate(s) affect proximal convoluted tubules in a paracrine mode is
not known. One possible candidate is Klotho release from the distal convoluted tubules to act on the PCT. Whether membrane Klotho at the luminal
side directly regulates TRPV5 in an autocrine mode is unknown. Soluble Klotho in luminal urine derived from either blood or urine exerts regulatory
action on NaPi cotransporters in proximal convoluted tubules, and on TRPV5 in DCT. Dashed line, suspected action.

1,25-(OH)2-vitamin D3 (1,25 VD3), renin, erythropoietin,
and Klotho. Klotho exerts multiple actions on the kidney but
only selected functions are highlighted in this article. This
includes regulation of 1,25 VD3 production and modulation
of urinary phosphate (Pi), Ca, and K excretion.
Interaction With Renal 1,25-(OH)2-Vitamin D3 Production
Klotho and the vitamin D system reciprocally regulate
each other. In homozygous Klotho-deficient (Kl⫺/⫺)
mice, extremely high plasma 1,25 VD3 levels were noted
as well as up-regulation of 1␣-hydroxylase and down-

Table 1. Biological Functions of Klotho Protein

Form of Klotho Nuclear Intracellular Membrane Extracellular

Locale Nucleus Cytoplasm Cell surface Blood, urine, cerebrospinal fluid

Domain N/A Kl1 or full length Full length Ectodomain containing Kl1 and Kl2
Biologic function Anti-age 2 cytokine production (1) FGF23-dependent: (FGF23 (1) FGF23-dependent: same as
Anti-senescence co-receptor for FGF23 transmembrane but in
Anti-age signal) endocrine or paracrine mode
Modulation of Anti-age (2) FGF23-independent:
Na/K-ATPase Anti-IGF Anti-oxidation
PTH release Calciophospho-regulatory Modulation of renal ion channels
Ca homeostasis hormone Anti-Wnt signal
Suppress PTH, 1,25 VD3 Anti-apoptosis
(2) FGF23-independent: Antisenescence
release soluble Klotho Anti-RAA
CSF, cerebrospinal fluid; IGF, insulin-like growth factor; N/A, no information; RAA, rennin-angiotensin-aldosterone.
120 M.C. Hu, M. Kuro-o, and O.W. Moe

regulation of 24-hydrolase,15 which provides in vivo ge-
netic, but indirect, evidence that the high circulating 1,25
VD3 levels are the result of overproduction and low
degradation of 1,25 VD3 (Fig. 1).15 In addition, Yoshida
et al15 found that normal genetic responses to vitamin D
supplementation, including down-regulation of 1␣-hy-
droxylase transcripts and up-regulation of 24-hydroxy-
lase and vitamin D receptor (VDR) transcripts, were
impaired in Kl⫺/⫺ mice, suggesting that normal expres-
sion of renal Klotho is required for normal vitamin D
homeostasis. On the other hand, the dysregulation of the
vitamin D system may be associated with high mortality
in both Klotho-deficient and FGF23-deficient mice be-
cause diminution of vitamin D activity by a vitamin
D– deficient diet16 or genetically deleting the 1␣-hydrox-
ylase gene17 successfully normalizes plasma 1,25 VD3
levels and rescues a significant fraction of the phenotypes
including renal Pi retention, vascular calcification, and
short life span in Kl⫺/⫺ mice. However, the mechanism
whereby inactivation of vitamin D activity rescues most
phenotypes in Kl⫺/⫺ mice is unknown. Recently, Ohnishi
and Razzaque18 proposed the role of phosphate toxicity
in mammalian aging because reducing blood Pi levels by
genetic deletion of Na-Pi cotransporter-2a (NaPi-2a) res-
cues most phenotypes in Kl⫺/⫺ mice. These beneficial
effects disappear and premature aging-like features reap-
pear when hyperphosphatemia is induced in mice with
double deletion of NaPi-2a and Klotho by feeding with a
high-phosphate diet.18
1,25 VD3 administration up-regulates renal Klotho
expression in normal animals.16 An in vitro study per-
formed by Forster et al5 showed that there are vitamin
D–responsive elements in the vicinity of the Klotho gene
promoter, and that 1,25 VD3 induces Klotho transcripts
in cultured mouse and human kidney cell lines. Thus 1,25
VD3 and Klotho forms a negative feedback control loop
similar to that of parathyroid hormone (PTH) and vitamin
D. A primary increase in 1,25 VD3 up-regulates Klotho
expression, which in turn suppresses 1,25 VD3 produc-
tion and likewise an increase in Klotho will suppress 1,25
VD3 to remove a major stimulator of Klotho production
(Fig. 2, left panel). Novel insights into Klotho–vitamin D
interaction will be valuable in understanding 1,25 VD3
therapy in chronic kidney disease (CKD) patients.
Modulation of Renal Ion Channel and Transporters
Klotho protein was considered one of the novel phospha-
tonins soon after its discovery because the Klotho-deficient
mouse has severe hyperphosphatemia and the transgenic
mouse overexpressing Klotho has hypophosphatemia. Sub-
sequent animal and cell culture studies revealed that Klotho

Figure 2. Proposed physiological role of Klotho in mineral metabolism and pathophysiological consequences of Klotho deficiency in CKD. Left
panel: in the setting of normal kidney function with normal Klotho levels, Klotho may suppress FGF23 production and release from the bone.
But there are no data to date to prove a direct effect of Klotho on FGF23 production in the bone. Klotho functions as a co-receptor of FGFR
to allow FGF23 to suppress PTH production and release from the parathyroid. PTH stimulates and increases plasma levels of FGF23 and 1,25
VD3. Increased 1,25 VD3 further stimulates FGF23, and directly and indirectly suppresses PTH levels. Increased 1,25 VD3 also stimulates
Klotho production in the kidney. Taken together, through several negative or positive feedback loops, Klotho functions as both a phosphate and
calcium regulatory hormone to directly or indirectly suppress PTH, 1,25 VD3, and FGF23 production and release. The final outcome of Klotho’s
action on the kidney is to prevent renal Pi retention and to prevent renal Ca loss. Right panel: in CDK and end-stage renal disease, the network
is deranged (red arrows). Renal Klotho is decreased followed by a decrease in plasma Klotho. The down-regulation of Klotho increases FGF23
production via unknown mechanisms, which in turn suppresses 1,25 VD3 production in the kidney. Whether low plasma Klotho renders the
parathyroid gland resistant to the suppressive effect of FGF23 on PTH production is not proven. However, decreased FGFR1/3 and Klotho
expression in the uremic parathyroid gland could make the gland resistant to FGF23, and triggers and/or promotes secondary hyperparathy-
roidism. Low plasma Ca levels also participate in SHPT development. Hyperphosphatemia amplifies the high FGF23 and PTH, and low Klotho
in the blood. The high plasma PTH, Pi, and FGF23, and low plasma 1,25 VD3 and Klotho, in concert, contribute to the development of
complications such as metabolic bone disease, secondary hyperparathyroidism, cardiomyopathy, and vascular calcification. Dashed line:
unproven putative roles of Klotho.
Renal and extrarenal klotho
not only controls phosphate homeostasis through modulat-
ing Na-dependent phosphate cotransporters (NaPi-2a and
2c),4,19,20 but also regulates calcium homeostasis by modu-
lating the renal calcium channel, transient receptor potential
ion channel (TRPV5),21,22 and potassium homeostasis by
regulation of the renal outer medullary K channel 1
(ROMK1).23 One unique aspect is that as a regulator of ion
transport, Klotho functions as an enzyme. The two Kl1 and
Kl2 repeats in the extracellular domain of membrane Klotho
share 20% to 40% amino acid sequence homology to family
1 glycosidase.1,24 But two important and highly preserved
glutamate residues critical for the glycosidase activity are
replaced by an asparagine within the Kl1 domain and an
alanine or a serine within the Kl2 domain, rendering the
glycosidase activity null.1,24 However, in vitro studies
showed that Klotho possesses ␤-glucuronidase4,22,24 or siali-
dase activity21,23 through which Klotho exerts regulation of
several renal ion channels and transporters. Therefore,
Klotho is emerging as a principal calciophosphoregulatory
hormone.
Inhibition of NaPi-2a activity
Three NaPi cotransporters, NaPi-2a, NaPi-2c, and Pit-2,
with different expression in the kidney proximal tubules,
participate in renal regulation of phosphate homeostasis
with different isoforms possessing different substrate
specificities, pH sensitivities, and kinetics of response in
regulation.25
Two lines of independent studies showed that hyper-
phosphatemic Kl⫺/⫺ mice display an increase in activity
of NaPi cotransport in the kidney,4 and in NaPi-2a and
NaPi-2c protein expression compared with wild-type
(WT) mice,4,19 suggesting that hyperphosphatemia at least
in part is of renal origin. Most importantly, phosphate
restriction26,27 or induction of renal phosphate leak by
deletion of NaPi-2a,18 successfully rescues the multior-
gan failure in the Klotho-deficient mice, indicating that
phosphotoxicity is a principal pathogenic factor in these
animals. Transgenic Klotho-overexpressing mice have
lower plasma Pi, whereas renal fractional excretion of Pi
is increased,4 indicating a renal leak of Pi. Soluble Klotho
increases renal fractional excretion of Pi and decreases
plasma Pi in the normal rat and in FGF23 knock-out
mice,4 indicating that the Klotho-induced phosphaturia is
FGF23-dependent as well as FGF23-independent.
The direct suppression of NaPi-2a by soluble Klotho
protein in a FGF23-independent fashion is mediated by
direct inhibition of NaPi cotransport activity without
change in protein abundance on the cell surface, which is
a novel mode of regulation for this class of transporters.4
This can be mimicked by recombinant ␤-glucuronidase
and blocked by glucuronidase inhibitor, but not affected
by sialidase.4 The model of a direct effect of Klotho on
NaPi-2a protein is proposed as follows: glucuronate on
an as yet unknown substrate is removed by Klotho,
leading to suppression of NaPi cotransport activity,
which subsequently renders NaPi-2a protein more sus-
121
ceptible to proteases residing in brush-border membrane.
Although protease inhibitors abolish the proteolysis, they
do not reverse the Klotho-induced inhibition of trans-
port,4 supporting the theory that Klotho-induced degly-
cosylation is sufficient to suppress NaPi cotransport ac-
tivity and that subsequent proteolysis is not required to
suppress NaPi transport during the acute phase (Fig. 1).
Regulation of TRPV5
In addition to being a potent phosphaturic hormone,
Klotho also maintains calcium homeostasis.15 Through
its suppression of PTH28 and 1,25 VD3,15 Klotho indi-
rectly decreases intestinal calcium absorption. Suppres-
sion of PTH also is expected to promote calciuria, but the
potent action of Klotho on distal calcium reabsorption
partially counteracts the reduction in gut absorption. The
net effect at the whole organism level is maintenance of
balance but with lower turnover. This is supported by the
fact that Klotho gene deletion or overexpression has
rather mild effects on plasma calcium as opposed to the
severe hyperphosphatemia and hypophosphatemia in
Klotho-deficient and Klotho-excess animals, respec-
tively.1,4 We focus on the most novel aspect, which is the
direct effect of Klotho on renal calcium reabsorption
(Fig. 1).
Chang et al22 showed that soluble Klotho stimulates
TRPV5, one of the key regulators for urinary calcium
excretion, by stabilizing TRPV5 on the cell surface and
proposed that Klotho functions as a glucuronidase.
Glucuronic acid is a very uncommon moiety of N-glycan
chains on a membrane channel such as TRPV5. A second
study by Cha et al21 supported an alternative mode of
Klotho effect on TRPV5. Klotho functions as a sialidase,
which removes ␣2,6-sialic acids from the N-glycan
chains on TRPV5 and exposes underlying N-acetyl-D-
lactosamine for binding to galectin-1.21 This association
prevents TRPV5 from internalization via a dynamin-
dependent process, leading to stabilization of more
TRPV5 on the cell surface. These results clearly support
the concept that Klotho is a calciotropic protein that
prevents renal calcium loss.
Regulation of ROMK1
ROMK1 is one of the major mediators of urinary K
reabsorption. Cha et al23 found that acute infusion of
Klotho leads to antikaliuresis mediated by increased api-
cal membrane abundance of ROMK1. They proposed a
mechanism of Klotho action similar to that of TRPV5.23
Klotho removes terminal sialic acids from N-glycan of
ROMK1, and exposes underlying disaccharide galactose-
N-acetylglucosamine, a ligand for a ubiquitous galec-
tin-1. Binding to galectin-1 at the cell surface prevents
clathrin-mediated endocytosis and causes accumulation
of functional ROMK1 on the plasma membrane.23 Pres-
ently, the role of Klotho as a potassium homeostatic
hormone is unclear because there does not appear to be
disturbances in plasma potassium concentrations in either
122
the Klotho-deficient or Klotho-overexpressing mice. If
Klotho has significant antikaliuretic effects, one should
not anticipate undesirable effects in Klotho-deficient
states in acute kidney injury (AKI) and CKD but theo-
retically should be more concerned with therapeutic ad-
ministration of Klotho leading to hyperkalemia. These
theoretical effects remain to be explored.
PHYSIOLOGICAL ROLE OF
KLOTHO IN EXTRARENAL ORGANS
Membrane Klotho and soluble Klotho protein exert dis-
tinct but possibly overlapping actions. FGFRs are ex-
pressed ubiquitously, but FGF23 signal transduction is
controlled by co-expression with membrane Klotho.12
The restricted expression of Klotho protein in very few
organs (kidney, heart, brain, and parathyroid gland) in
concert with multiple functions in multiple tissues (Table
1) suggests that soluble Klotho may function indepen-
dently of FGFRs as an endocrine hormone and an en-
zyme to directly modulate target proteins.
Modulation of PTH Production
Klotho modulates PTH directly and indirectly. Klotho
indirectly regulates PTH production through modulation
of plasma levels of 1,25 VD3, Pi, and FGF23. In addition,
Klotho may exert a direct effect on PTH production and
release (Fig. 2, left panel).
In genetically manipulated mice whose Klotho gene is
replaced by a LacZ reporter, ␤-X-gal staining is clearly
positive in the kidney, the sinoatrial node region of the
heart, choroid plexus of brain, and the parathyroid
gland,29 which is compatible with earlier findings by
reverse-transcription polymerase chain reaction.1 Immu-
nohistochemistry further confirmed the localization of
Klotho in the parathyroid and but not the surrounding
thyroid tissue,28 thus excluding the possibility that the
positive reverse-transcription polymerase chain reaction
results are caused by contamination. Ben-Dov et al28
found FGFR1 and FGFR3 in the parathyroid tissue, sug-
gesting that FGFRs may form a complex with Klotho and
FGF23 in the parathyroid gland. FGF23 binds to Klotho/
FGFR and activates the mitogen-activated protein kinase
cascade signal pathway in cultured cells transfected with
Klotho,12 parathyroid cells,28 as well as in the kidney,4,10
indicating that the parathyroid gland is a likely target
organ of FGF23, and Klotho is a prerequisite for FGF23
to modulate PTH production.
In vivo animal and in vitro cell culture experiments
performed in independent laboratories revealed that
FGF23 decreases PTH production, increases expression
of both the parathyroid calcium-sensing receptor and the
vitamin D receptor (both of which contributes to sup-
pression of PTH), and decreases cell proliferation28,30
when normal Klotho and FGFR are expressed in para-
thyroid glands. Interestingly, FGF23 seems to increase
Klotho in the parathyroid gland,28 which may posi-
M.C. Hu, M. Kuro-o, and O.W. Moe
tively facilitate the action of FGF23 on suppression of
PTH production. The expression of Pit-1, one of the
NaPi-3 isoforms, was found in the parathyroid gland
and was up-regulated by a 1,25 VD3 and low Pi diet,
and down-regulated by vitamin D– deficient diet.31 Hu
et al9 showed that Klotho suppresses the activity and
expression of Pit-1 in the rat vascular smooth muscle
cell line in vitro, but to date, there is no evidence for
a Klotho effect on Pit-1 in parathyroid.
Ionized calcium activity is a key modulator of PTH
synthesis and release from parathyroid. An intriguing
model was proposed as an alternative mode of Klotho
action on the parathyroid cells.32 In this model, intracel-
lular Klotho binds to Na/K-adenosine triphosphatase
(ATPase) to form a complex in response to low intracel-
lular [Ca2⫹] and bring Na/K-ATPase to the cell surface.32
The resultant change in electrochemical gradient was
proposed to trigger the release of PTH through an un-
identified signal pathway.32 Thus, Klotho expression in
the parathyroid may suppress PTH production by the
FGF23 signal pathway and increase PTH production by
the Na/K-ATPase signal pathway stimulated by low
blood Ca levels. However, how the complex of Na/K-
ATPase with Klotho is formed in response to low Ca2⫹
levels, how Na/K-ATPase activity is stimulated, and
what intracellular signal is required to couple Na/K-
ATPase to PTH release remain to be illustrated.
Inhibition of Intestinal Phosphate Absorption
In addition to increased renal reabsorption of Pi, in-
creased absorption of dietary Pi may exacerbate hyper-
phosphatemia in Kl⫺/⫺ mice19 because expression of the
intestinal phosphate transporter NaPi-2b is significantly
higher in Kl⫺/⫺ mice than in WT mice.19 In vitro studies
revealed that Klotho directly decreases Pi-induced cur-
rent in NaPi-2b– expressing oocytes,20 which supports the
model that Klotho inhibits Pi absorption in the intestine.
In addition, a recent study showed that another
Na-coupled phosphate transporter Pit-1 protein also is
present in the apical membrane of enterocytes of rat
duodenum and jejunum, but not in the ileum.33 Unlike
NaPi-2b, PiT-1 protein in the brush-border membrane
and Pit-1 messenger RNA (mRNA) expression are not
changed in the duodenum or jejunum as a function of
dietary Pi.33 Therefore, the relative contribution of
PiT-1 to intestinal Pi absorption remains to be deter-
mined, and Klotho’s effect on intestinal Pit-1 remains
to be addressed.
Taken together, Klotho is a calciophosphoregulatory
protein. The direct effect of Klotho on the kidney is to
promote phosphaturia and to prevent renal calcium loss.
Klotho regulates Pi and Ca homeostasis directly and by
interplay with other calcium and phosphate regulatory
hormones: FGF23, PTH, and 1,25 VD3 (Figs. 1 and 2,
left panel).
Renal and extrarenal klotho
PATHOPHYSIOLOGICAL ROLE OF
KLOTHO DEFICIENCY IN KIDNEY DISEASE
Klotho Deficiency Renders the
Kidney More Susceptible to Injury
Liu et al34 showed increased senescence in progenitor
cells in many tissues of Kl⫺/⫺ mice. Knock-down of
endogenous Klotho promotes augmentation of senes-
cence in cultured cells.34 Administration of exogenous
Klotho significantly decreases senescence in endothelial
cells35 and fibroblasts.36 Furthermore, Klotho depletion-
induced cell senescence may be associated with up-reg-
ulation of Wnt signaling activity because administration
of exogenous Wnt accelerates cell senescence in vivo and
in vitro, and Wnt signaling is increased significantly in
Kl⫺/⫺ mice, and suppressed by genetic Klotho overex-
pression.34 Soluble Klotho can bind to various Wnt fam-
ily members and inhibit their biological activity.34 Re-
cently, intracellular Klotho was shown to be able to
suppress cell senescence by inhibiting retinoic-acid–in-
ducible gene-I–induced expression of interleukin-6 and
interleukin-8 both in vitro and in vivo.14 Klotho defi-
ciency from kidney diseases enhances cell senescence
induced by oxidative stress.37-39 Excessive senescence or
resultant apoptosis and stem cell deletion may decrease
the kidney’s ability to defend against renal insults and
impair regeneration.
Klotho is an anti-apoptotic protein. Klotho deficiency
significantly increases apoptosis in cultured kidney and
endothelial cells,35,40 and in the kidney.39 Oxidative stress
down-regulates Klotho expression and in turn increases
cell damage when exposed to H2O2.6 Increase of Klotho
by genetic manipulation or viral delivery decreases the
number of apoptotic cells and improves kidney function
and renal morphology after acute40 and chronic kidney
damage.39 These results form the basis for potential clin-
ical application of Klotho in the treatment of AKI and
CKD.
Klotho Deficiency Delays Kidney Recovery
Two independent studies showed that Kl⫺/⫺ mice have
stem cell depletion in several organs34 and progenitor cell
senescence in the kidney,39 which may delay tissue re-
covery after kidney injury. In addition, Kl⫺/⫺ mice have
severe abnormal endothelial function41 and integrity,42
and impairment of angiogenesis and vasculogenesis after
ischemic limbs.43 If those results were translated into
ischemic kidney damage, kidney regeneration may be
delayed after kidney injury. Increased Klotho by genetic
manipulation was shown to accelerate angiogenesis and
vasculogenesis, decrease limb loss, and promote limb
recovery after ischemic injury in Kl⫺/⫺ mice,44 which
suggest that Klotho replacement may promote kidney
recovery.
Klotho Deficiency Promotes Renal Fibrosis
Renal fibrosis is not only a histologic characteristic in
CKD, but also is pathogenic for chronic progression.
123
Transforming growth factor (TGF)-␤ is considered to
contribute to renal fibrosis.45 Kl⫺/⫺ mice have more glo-
merular and tubulointerstitial fibrin deposition and higher
active plasminogen activator inhibitor-1 antigen in
plasma and plasminogen activator inhibitor-1 mRNA ex-
pression in the kidney,46 and Kl-/⫹ mice have more fibro-
sis than WT mice do at baseline and after unilateral
ureteral ligation to induce unilateral ureteral obstruction
(UUO).47 Kl-/⫹ mice with UUO also have higher TGF-␤
levels, and lower Klotho mRNA and protein than WT
mice.47 The obstructed kidneys from Kl-/⫹ mice express
significantly higher levels of fibrosis markers such as
␣–smooth muscle actin, fibronectin, and TGF-␤ than
those from WT mice.47 In cultured rat kidney cell lines,
Klotho alleviates TGF-␤–induced epithelial-mesenchy-
mal transition or phenotype, suppresses TGF-␤–induced
target genes activation, and reduces TGF-␤1–induced
Smad2 phosphorylation,48 suggesting that Klotho protein
inhibits renal fibrosis primarily through inhibiting
TGF-␤1 signaling. More importantly and interestingly,
Doi et al48 recently showed that intraperitoneal injection
of soluble Klotho protein suppresses renal fibrosis in-
duced by UUO, suggesting that soluble Klotho protein
may be a novel therapeutic agent for renal fibrosis. In
addition to the effect of Klotho on TGF-␤1, suppressive
effects of Klotho on the insulin-like growth factor path-
way2 and on the Wnt signal pathway34 also may be
associated with Klotho’s inhibitory action on renal fibro-
sis, but to date there is no direct evidence to confirm this
concept.
We propose that Klotho deficiency renders the kidney
more susceptible to injury, accelerates renal fibrogenesis,
retards renal tissue regeneration, and eventually promotes
chronic progression. Supplementation of Klotho may
provide beneficial impact in preventing and slowing
down CKD progression.
PATHOPHYSIOLOGICAL ROLE OF
KLOTHO IN THE METABOLIC SYNDROME
The metabolic syndrome (MS) is characterized by obe-
sity, serum lipid profile alterations, hypertension, and
fasting hyperglycemia, and is a risk factor for the devel-
opment of diabetes and cardiovascular disease.49,50 Re-
cent studies have indicated that the MS also is associated
independently with an increased risk for incident CKD in
nondiabetic adults51 because MS patients are at signifi-
cantly higher risk for microalbuminuria and/or CKD, and
the level of risk is related to the number of components
of the syndrome. Any component of MS may indepen-
dently favor the development of renal abnormalities and
potentially is considered a modifiable risk factor for
CKD. Thus, correction of any component should be a
rationale for intervention of MS and effectively can pre-
vent the development and progression of renal damage.
One cross-sectional study examining genetic variants
of the Klotho gene polymorphism with MS showed as-
sociation of the KL-VS variant (F352V and/or C370S
124
within exon 2, simply termed VS alleles) to high blood
glucose level, high blood pressure, insulin resistance, and
a trend toward its association with hypertriglyceridemia
in Asian Indians.52 Several animal models such as dia-
betes induced by streptozotocin,53 diabetes from leptin
deficiency,54 spontaneous non–insulin-dependent diabe-
tes (Otsuka Long-Evans Tokushima Fatty),55,56 and hy-
pertension (spontaneous hypertension and volume-de-
pendent hypertension)55,57 have disclosed a dramatic
reduction of renal Klotho mRNA and/or protein expres-
sion, suggesting that a decrease in Klotho may be part of
the MS. At present, we do not know whether Klotho is a
cause or a result of MS, or a parallel phenomenon.
The administration of thiazolidinedione, an agonist
of peroxisome proliferator-activated receptor-␥ in-
creases renal Klotho mRNA expression, attenuates ab-
normal lipid and glucose metabolism, and reduces
systolic blood pressure in Otsuka Long-Evans
Tokushima Fatty rats.56 More interestingly, adenovi-
rus-mediated Klotho gene delivery can repeat thiazo-
lidinedione’s action,58 indicating the therapeutic po-
tential of Klotho gene delivery in MS.
In rats with streptozotocin-induced diabetes,59
Klotho protein in the kidney is notably decreased
along with kidney destruction.59 Both insulin and phlo-
ridzin corrects hyperglycemia, reverses the reduced
renal Klotho expression, and improves kidney function
and histology of diabetic rats. Klotho protein in Ma-
din–Darby canine kidney cells is reduced by incuba-
tion in high glucose medium.7 Insulin has been shown
to stimulate shedding of the extracellular domain of
membrane Klotho protein,7 which may increase the
blood Klotho concentrations.
Similarly, in rats with spontaneous hypertension,
Klotho gene delivery via adeno-associated virus carrying
mouse Klotho full-length complementary DNA reverses
reduced Klotho expression in the kidney, controls blood
pressure, improves kidney function, and attenuates renal
fibrosis.57
Taken together, the MS appears to be a state of Klotho
deficiency before development of kidney injury. Klotho
deficiency may render the kidneys more susceptible to
acute and chronic renal insults, and kidney damage fur-
ther exacerbates Klotho deficiency. Improvement of glu-
cose metabolism or control of blood pressure could in-
crease Klotho expression in the kidney considerably. On
the other hand, Klotho possesses anti-insulin activity and
induces insulin resistance.2 Unger60 in 2006 proposed
that insulin resistance induced by Klotho may decrease
insulin-stimulated intracellular glucose availability and
may prevent intracellular caloric and lipid overload and
toxicity. A frequently used medication, peroxisome pro-
liferator-activated receptor-␥ agonist for type II diabetes
mellitus, has been confirmed to increase Klotho.61 Thus,
modulation of Klotho expression in the kidney is a po-
tential future treatment option for the MS.
M.C. Hu, M. Kuro-o, and O.W. Moe
PATHOPHYSIOLOGICAL ROLE OF KLOTHO DEFICIENCY
IN COMPLICATIONS OF CHRONIC KIDNEY DISEASE
Secondary Hyperparathyroidism
Secondary hyperparathyroidism (SHPT) is a common
and severe complication in CKD,62 contributing to renal
metabolic bone disease, cardiac disease, and anemia. In
addition, hyperphosphatemia, hypovitaminosis D, and
low expression of VDR are proposed to contribute to
maintaining increased PTH levels in CKD patients. But if
one analyzes the profile of changes of those parameters,
it is more likely that these abnormalities are involved in
the acceleration of SHPT in later stages rather than
triggering them early.62 Epidemiologic observation63 and
animal studies64 show that a plasma FGF23 increase is a
very early event. Plasma Klotho decreases may even be
an earlier event,65 which in turn can stimulate FGF23
overproduction. Thus, the conceptual mode of develop-
ment of SHPT is revised to include aberrant FGF23/
Klotho activity to play a more fundamental role in SHPT
development (Fig. 2, right panel).
CKD in both human beings and animals is a state of
high plasma levels of FGF2366 and low FGFR1 and
Klotho expression in the parathyroid gland,67-69 which
renders FGF23 to lose its inhibitory actions on PTH
production, and to fail to increase the calcium-sensing
receptor and VDR.30 Indeed, Galitzer et al70 confirmed
that FGF23 fails to decrease plasma PTH, suppress cell
proliferation, and activate the mitogen-activated protein
kinase pathway in parathyroid glands of rats with ad-
vanced CKD, indicating that the gland is resistant to
FGF23, possibly from low FGFR1 and Klotho (Fig. 2,
right panel). In addition, the aberrant Klotho-Na/K-
ATPase axis, an FGF23-independent mode, also may be
involved in SHPT development in CKD. Hofman-Bang
et al71 reported an unexpected finding of an increase in
Klotho, FGFR, and Na/K ATPase in the parathyroid
glands of early CKD rats, which is seemingly opposite of
findings by other laboratories,30,68-70 but may be owing to
different stages of the CKD model and different levels of
plasma calcium concentrations. These investigators fur-
ther reasoned that when plasma calcium levels are in the
normal range, the increased expression of Klotho in
parathyroid glands will be reduced,71 because Imura
et al32 proposed that low plasma calcium increases intra-
cellular Klotho. Thus far, a model can be constructed in
which PTH production in CKD may be controlled by two
Klotho-dependent signal pathways. In early CKD,
Klotho-Na/K-ATPase signaling is overactive owing to
higher expression of Klotho and FGFR in the parathyroid
glands and possibly regulated by plasma calcium con-
centration. Hypocalcemia promotes formation of the
Klotho/Na/K-ATPase complex and induces PTH synthe-
sis in the parathyroid. The increase of plasma FGF23
with a high expression of Klotho and FGFR in the
parathyroid glands should decrease PTH secretion to
maintain normal mineral metabolism. However, in ad-
Renal and extrarenal klotho 125

vanced CKD, a low expression of Klotho and FGFR in

the parathyroid glands blunts suppressive activity of Table 2. Comparison of Phenotypes Klotho Deficiency and
FGF23/Klotho signaling. The nonunanimous blood lev- CKD
els of PTH in CKD patients would be interpreted by the Chronic
Klotho Deficiency Kidney Disease
complicated mechanisms of PTH modulation. Thus,
Klotho restoration in the parathyroid with normalization Blood chemistry
of plasma Ca levels may be a novel approach to block Phosphate 1111 1 or 111*
Calcium 1 ↔ or 22
SHPT development.
Creatinine 1 111
1,25 VD3 111 222
Cardiovascular Calcification PTH ↔ or 2 11
FGF23 111 11
Cardiovascular disease is the leading cause of mortality Klotho 222 or disappear 22 at ESRD†
of CKD and cardiovascular calcification is prevalent in Gross phenotypes
CKD. In addition to the classic traditional factors, FGF23 Body weight 222 22
and Klotho are novel contributors to ectopic calcification Growth retardation 2222 22 in children
in soft tissues including the aorta (Fig. 2B).1,9,72,73 Thus Physical activity 222 2
far, there are no effective targeted therapies for cardio- Fertility 2222 22
Life span 2222 22
vascular disease in CKD other than manipulation of the
Cardiovascular
classic risk factors. disease
Tangri et al74 examined the association of the func- Cardiac 11 111
tional Kl-VS variant of Klotho with valvular and vascular hypertrophy
calcifications on 1389 cases and 2139 controls from the Cardiac fibrosis 11 111
Framingham Heart Study Offspring Cohort and did not Vascular 1111 11
calcification
observe any association of the Kl-VS variant of Klotho
Arthrosclerosis 11 111
valvular or vascular calcifications. However, experimen- Blood pressure 1 1111
tal animal studies documented that CKD is a state of Hematocrit levels 2 2222
systemic Klotho deficiency (Table 2). Kl⫺/⫺ mice have Bone disease 222 222
extensive ectopic calcification in soft tissues akin to that Abbreviation: ESRD, end-stage renal disease.
observed in CKD subjects, suggesting a pathogenetic *During early CKD the blood Pi is in the normal range.
association between Klotho deficiency and calcification. †Blood Klotho may be increased in early CKD.89
An increase in Klotho by genetic manipulation inhibits
vascular calcification in CKD animals.9 The suppressive
influence of Klotho on vascular calcification in CKD is
multifactorial, including the following: (1) decreasing human arterial organ cultures from CKD patients.77 Further-
plasma Pi by promoting a negative Pi balance4,72,73; (2) more, VDR activators exert their anticalcific effects by
inhibiting Pi-induced Pit-1 and Pit-2 activation in the restoration of Klotho and regaining FGF23 responsive-
vasculature9; (3) suppressing cell senescence, apoptosis, ness.77 This study provides some novel insights into vascu-
and death in vascular endothelial cells and smooth mus- lar calcification in uremia: both chronic metabolic and
cle cells induced by a variety of insults including mechanistic stress could induce vascular calcification by
Pi36,38,75; and (4) serving as an anti-inflammatory modu- dysregulation of Klotho/FGF23 signaling. Administration
lator.14,54 of VDR activators can restore Klotho expression and un-
Data from independent laboratories showed that Klotho mask the FGF23 anticalcific effect.77 This concept is similar
and FGFR1/3 protein and/or mRNA, but not FGF23 and to the mode of the effect of defect in FGF23/Klotho signal-
FGFR4, are expressed in human vasculature,76,77 and human ing in SHPT in CKD (Fig. 2, right panel).
aorta– derived smooth muscle cells.77 Interestingly, both Recently, one study examined the role of stanniocalcin
Klotho and FGFR1/3 expression are down-regulated in hu- (STC) 1 and 2, which are up-regulated in the kidney of
man arteries from CKD patients who have vascular calcifi- Kl⫺/⫺ mice,78 in ectopic calcification. STC2 protein is
cation.77 When human aorta-derived smooth muscle cells localized focally with the calcified lesions of renal arte-
are incubated with pooled uremic blood, high Pi, and Ca, rioles, renal tubular cells, heart, and aorta in Kl⫺/⫺ mice.
both FGFR1/3 and Klotho expressions are down-regulated High Pi medium increases STC2 mRNA levels as well as
and vascular smooth muscle cells undergoing “osteogenic- that of osteocalcin, osteopontin, and PiT-1 in rat aortic
chondrogenic” transdifferentiation are triggered. Vascular vascular smooth muscle. Interestingly, knockdown with a
Klotho deficiency by Klotho knockdown potentiates the small interfering RNA or the overexpression of STC2
development of calcification and decreases the ability of showed acceleration and inhibition of Pi-induced calci-
vascular tissue to respond to FGF23.77 Lim et al also fication, respectively, in this cell line.79 These results
showed that vascular Klotho deficiency driven by pro-cal- suggest that STC2 might represent a novel target for
cific stressors could be restored by VDR activators in an in ectopic calcification in the condition of Klotho defi-
vitro cell culture model and an ex vivo model by using ciency.
126
The prospect of Klotho restoration affecting the course
of CKD in terms of prevention, forestalling, slowing, or
reversal of vascular calcification is a high priority ques-
tion in terms of therapeutics and requires investigation.
Cardiac Remodeling
Cardiac remodeling, which often is used as a general
term of changes in cardiac structure and function with
implied negative connotations, is used here to describe
the cardiac hypertrophy and fibrosis in CKD; sometimes
referred to as uremic cardiomyopathy, which distin-
guishes it pathogenically from highly prevalent hyperten-
sive and ischemic cardiomyopathy. In addition to tradi-
tional cardiovascular risk factors, novel risk factors such
as vitamin D deficiency,80 high plasma FGF23,81,82 and
low plasma Klotho81 all can contribute to cardiac hyper-
trophy (Fig. 2, right panel).
In the heart, Klotho is expressed solely at the sinoatrial
node.29 Kl⫺/⫺ mice have sinoatrial conduction defects,29
which may be a cause of sudden death under restraint
stress. The absence of degenerative structural change in
the sinoatrial node of Kl⫺/⫺ mice29 suggests that haplo-
sufficient levels of Klotho might be sufficient to maintain
the function of sinoatrial node as a pacemaker.
One recent study showed that Klotho deficiency is
associated with cardiac hypertrophy.81 Klotho-deficient
mice have left ventricular hypertrophy, which is not
hypertension-dependent, but may be FGF23-dependent
because either intramyocardial or intravenous injection
of FGF23 results in cardiac hypertrophy in WT mice, or
an FGFR blocker attenuates cardiac hypertrophy in 5/6
nephrectomized CKD, but does not change blood pres-
sure.81 One interpretation is the importance of FGF23,
but the effect of low Klotho is equally likely owing to the
possible interplay between Klotho and FGF23 in the
pathogenesis of cardiac remodeling in CKD. It is clear
that uremic cardiomyopathy is a complex metabolic dis-
ease that is way beyond the classic cardiac risk factors. In
addition to FGF23 and Klotho, aberrant vitamin D and
phosphate metabolism and cardiac renin-angiotension-
aldosterone activation also may participate in cardiac
hypertrophy in CKD (Fig. 2, right panel).
KLOTHO AS A POTENTIAL THERAPEUTIC AGENT
Although there are no published clinical studies showing
the therapeutic efficacy of Klotho administration in either
acute or chronic kidney disease, animal experiments thus
far have shown unequivocal therapeutic effects of Klotho
gene delivery or direct administration of Klotho protein
on several models. Restoration of endogenous Klotho or
administration of exogenous Klotho potentially provides
novel treatment strategies for CKD patients. We high-
light the recent advances of Klotho administration in
animal models of kidney disease.
M.C. Hu, M. Kuro-o, and O.W. Moe
Administration of Exogenous Klotho
Animal studies have shown that delivery of Klotho gene
via viral carrier efficiently protects the kidney from acute
injury induced by ischemia reperfusion,83 and also affects
the course of disease progression of CKD.57,58,84 Com-
pared with Klotho gene delivery to animal subjects, ad-
ministration of recombinant Klotho protein might be a
safer and easier approach to correct endocrine Klotho
deficiency.
In vitro studies have shown that soluble Klotho, a full
length of the extracellular domain, is active in inhibition
of insulin-like growth factor signal transduction,2 sup-
pression of Wnt signal,34 modulation of several ion chan-
nels or transporters,4,21,23 or control of FGF23 signal
transduction.12 In vivo studies further showed its thera-
peutic potential in several kidney disease models. Klotho
administration has been proven successful in the protec-
tion of kidney function in AKI animals induced by isch-
emia-reperfusion injury (IRI).85 More recently, the same
Klotho preparation also was documented to be efficient
in the inhibition of renal fibrosis and in better preserva-
tion of renal function in the UUO model.48 Thus, exog-
enous Klotho protein supplementation may be a poten-
tially feasible way of replacement therapy in Klotho-
deficient states.
Up-Regulation of Endogenous Klotho Protein
Transgenic overexpression of Klotho to plasma levels
about twice that of normal2 is effective in preventing
kidney injury acutely from ischemia-reperfusion,85 im-
mune-mediated glomerulonephritis,39 and in a renal ab-
lation model of CKD.9 This provides the basis for devel-
oping ways to increase endogenous Klotho expression
via stimulation or removal of suppression of Klotho
when residual kidney function is somewhat preserved
and endogenous Klotho-producing cells in the kidney are
not destroyed but simply suppressed. Furthermore, strat-
egies to increase Klotho production in extrarenal tissues
might be of particular importance for end-stage renal
disease patients whose functional kidney tissue is totally
lost.
It has been shown that the peroxisome proliferator-
activated receptor-␥ agonist,86 and anti-oxidants6 in-
crease renal Klotho expression in AKI animals. Some
animal experiments and cell culture studies showed that
restricting Pi intake,26 active vitamin D,16 inhibition of
3-hydroxy-3-methylglutaryl CoA reductase,87 and inhibi-
tion of Ang II88 increase Klotho expression in the kidney
and/or in cultured cell lines. Therefore, these conven-
tional maneuvers potentially offer additional therapeutic
mechanisms for treating Klotho deficiency and their clin-
ical utility needs to be explored.
CONCLUSIONS AND PERSPECTIVES
In concert, animal data and the limited clinical observa-
tions to date are overwhelmingly strong to suggest that
Renal and extrarenal klotho
Klotho is a pleiotropic protein and plays multiple phys-
iological roles in modulation of kidney function and
pathophysiological roles in acute kidney damage, pro-
gression of CKD, and extrarenal complications in CKD.
Klotho is not merely an early biomarker for kidney
damage, but also has a pathogenic role for kidney dis-
ease. The understanding of the renal and extrarenal ac-
tions of Klotho will advance novel strategies for both
diagnosis and treatment of AKI and/or CKD.
The potential utility of Klotho in clinical practice is
anticipated to be at least two-fold. First, Klotho may
serve as an early and sensitive biomarker to kidney
diseases. But its specificity and its prognostic value and
differential diagnostic value in human beings remain to
be examined. Second, Klotho exogenous supplementa-
tion and/or up-regulation of endogenous Klotho produc-
tion may provide novel therapy for AKI patients to retard
or block its progression to CKD and for CKD by arrest-
ing or slowing progression as well as preventing and
reversing complications. Because we are moving to use
Klotho as a novel diagnostic, prognostic, and therapeutic
strategy for CKD patients, identification of proper indi-
cations including CKD stage, composition of abnormal
mineral metabolism, and state of complications is of the
highest priority.
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