commentary

effects that stromal mutations, including Dicer1 loss, have on kidney

development. It is striking that the
phenotypes in Dicer1 knockouts in the
ureteric bud and nephrogenic lineages
are by and large restricted to the mutant
lineage or cells, whereas the stromal
Dicer1 knockout seems to affect all
lineages. Just as the stromal cells above
the cap mesenchyme control the
nephron progenitors that lie beneath
them, at other sites in the kidney
stromal cells could influence other cell
types through close or direct contact
and modulating signals. The epithelial
stromal cross-talk proposed to be
responsible for the Wnt7b phenotype10
would be another example of this.
There is no reason why miRNAs would
not be involved in the control of these
modulating signals. In fact, through
their very nature miRNAs could target
multiple proteins or even multiple
signals in parallel, making them ideal
for such a task. As such, miRNAs might
be an ideal genetic entry point to
identify these signals.
There is clearly scope for more
detailed analyses of specific miRNAs
in the developing kidney. In some of
the renal Dicer1 knockouts, specific
miRNAs have been identified as important players in the observed phenotypes. In the case of the stromal Dicer1
knockout, miR-214, miR-199a-3p, and
miR-199a-5p are clearly implicated in
different aspects of the phenotype in
these mice; other miRNAs were identified in other renal Dicer1 mutants.
Transgenic mouse lines with reporter
and conditional knockout construct
alleles for miRNAs and the availability
of Cre drivers for most stages of kidney
development will undoubtedly lead to
an increased appreciation of the role of
specific miRNAs in kidney development
and disease.
DISCLOSURE

The authors declared no competing interests.

ACKNOWLEDGMENTS

The Roslin Institute receives Institute

Strategic Program Grant funding from the
Biotechnology and Biological Sciences
Research Council.
Kidney International (2015) 87

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2.

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4.

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Nakagawa N, Xin C, Roach AM et al. Dicer1

activity in the stromal compartment regulates
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Kobayashi A, Mugford JW, Krautzberger AM
et al. Identification of a multipotent selfrenewing stromal progenitor population
during mammalian kidney organogenesis.
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Levinson RS, Batourina E, Choi C et al. Foxd1dependent signals control cellularity in the
renal capsule, a structure required for normal
renal development. Development 2005; 132:
529539.
Nagalakshmi VK, Ren Q, Pugh MM et al. Dicer
regulates the development of nephrogenic
and ureteric compartments in the
mammalian kidney. Kidney Int 2011; 79:
317330.
Chu JY, Sims-Lucas S, Bushnell DS et al. Dicer
function is required in the metanephric
mesenchyme for early kidney development.

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Harvey SJ, Jarad G, Cunningham J et al.
Podocyte-specific deletion of dicer alters
cytoskeletal dynamics and causes glomerular
disease. J Am Soc Nephrol 2008; 19:
21502158.
Das A, Tanigawa S, Karner CM et al.
Stromal-epithelial crosstalk regulates kidney
progenitor cell differentiation. Nat Cell Biol
2013; 15: 10351044.
Hohenstein P, Pritchard-Jones K, Charlton J.
The yin and yang of kidney development and
Wilm's tumors. Genes Dev 2015; 29:
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Yu J, Carroll TJ, Rajagopal J et al. A Wnt7bdependent pathway regulates the orientation
of epithelial cell division and establishes the
cortico-medullary axis of the mammalian
kidney. Development 2009; 136: 161171.

see basic research on page 1141

The vascular secret of Klotho

Ewa Lewin1,2 and Klaus Olgaard2
Klotho is an evolutionarily highly conserved protein related to
longevity. Increasing evidence of a vascular protecting effect of the
Klotho protein has emerged and might be important for future
treatments of uremic vascular calcification. It is still disputed whether
Klotho is locally expressed in the vasculature or whether its vascular
effects arise uniquely from its presence in the circulation.
Kidney International (2015) 87, 10891091. doi:10.1038/ki.2015.80

-Klotho (Klotho) gene and protein

were discovered in 1997 by Kuro-o and
co-workers. The name Klotho refers to
Greek mythology, where Klotho is the
one of the Fates, who is spinning the
thread of life. Klotho-deficient mice
manifest a phenotype resembling accelerated human ageing. Klotho-deficient
mice have a short lifespan, and overexpression of Klotho in mice extends
1
Nephrological Department B, Herlev Hospital,
University of Copenhagen, Copenhagen, Denmark
and 2Nephrological Department P, Rigshospitalet,
University of Copenhagen, Copenhagen, Denmark
Correspondence: Ewa Lewin, Nephrological
Department B, Herlev Hospital, University of
Copenhagen, DK 2730 Copenhagen, Denmark.
E-mail: ewa.lewin@regionh.dk and
lewin@dadlnet.dk

lifespan significantly in comparison

with normal mice, which is taken as
proof of the concept that Klotho is
associated with longevity. In a human
population study, Klotho gene variations
were found to be associated with life
extension. Of particular interest was the
finding that Klotho deficiency in mice
was associated with a severe vascular
phenotype of arteriosclerosis, impaired
endothelial function, and impaired angiogenesis.
Klotho protein in mammals is present
in different isoforms, as a membranebound protein and as a soluble form.
Soluble Klotho can be generated by
shedding of the extracellular domain of
membrane Klotho, containing two internal repeats, KL1 and KL2. Membrane
1089

commentary

THE VASCULAR SECRETS OF KLOTHO

Klotho?
Adventitia?

Membrane Klotho?
Coreceptor for FGF23
Source of soluble Klotho

Soluble Klotho?
Maintenance of
endothelial integrity
and function

Soluble Klotho?
Protection against ageing
Protection of VSMC
against osteogenic
conversion by Wnt
antagonism

Soluble Klotho?
Resistance to oxidative
stress via insulin/IGF
signaling
Soluble Klotho?
Protection/inhibition
of VSMC calcification

Regulation of Ca2+
transport into VSMC
via TRPV5 or
Na+/K+-ATPase

1-hydroxylase
regulation in
vascular wall

Suppression of P
transport into VSMC
via Pit-1/2 regulation

Figure 1 | The vascular secrets of Klotho. Shown here are the three layers of aorta: the
intima, the media, and the adventitia. The different effects of Klotho, either membrane bound
or soluble, are indicated, but still marked with question marks, as the final settlement of the
presence and potential effects of Klotho is still a matter of discussion. The reference to Klotho
expression in the adventitia layer is based on the present report by Ritter et al.9 IGF, insulin-like
growth factor; VSMC, vascular smooth muscle cell.

Klotho can form a complex with fibroblast growth factor (FGF) receptors 1c,
3c, and 4, and thereby create high-affinity
binding sites for FGF23. FGF23 is an
osteocyte-derived hormone that acts on
the kidney, promoting phosphate excretion and inhibiting calcitriol synthesis. Membrane Klotho confers tissue
target specificity for FGF23. The function of Klotho as an obligatory coreceptor for FGF23 explains the nearly
identical phenotypes that are observed
in knockout mice lacking either Klotho
or FGF23. Soluble Klotho has, however,
been attributed to pleiotropic functions
that are independent of FGF23. Soluble
Klotho was subsequently shown to
possess glycosidase activity, playing a
role in the activation of the calcium
channels TRPV5 and TRPV6 and the
potassium channel ROMK1, and in
the suppression of the activities of the
sodium-dependent phosphate cotransporters Npt2a, Pit-1, and Pit-2. Recently, however, in an interesting paper,
it has been challenged that Klotho is an
activator of TRPV5, as Klotho was not
directly colocalized with TRPV5, but
1090

rather functioned as a coreceptor for

FGF23.1 Soluble Klotho has furthermore been shown to be involved in
regulation of signaling of the cytokines
and growth factors insulin-like growth
factor-1, Wnt, and transforming
growth factor-. Many of these proteins
and signaling pathways are important
for vascular function (Figure 1). The
Klotho gene is mainly expressed
in kidney, brain, and parathyroids.2
Soluble Klotho is secreted into serum,
urine, and cerebrospinal fluid. Serum
Klotho or Klotho fragments can have
humoral actions on tissues far distant
from the site of biosynthesis. This is
based on the observation of generalized
involvement of tissues and organs
outside the Klotho expression tissues
in the prematurely ageing phenotype of
Klotho-deficient mice and on the
finding that administration of soluble
Klotho ameliorated the premature
ageing-related features, such as growth
retardation, organ atrophy, and vascular
calcification.3
Uremia is potentially a state of
Klotho deficiency based on decreased

concentrations both at the tissue level

and in the circulation, although determinations of serum Klotho levels are
problematic as stated below. It is of high
priority to better understand whether
deficiency of Klotho contributes to the
reduced longevity and other many severe
complications in patients with chronic
kidney disease, which are accompanied
by a dramatic increase in the rate of
cardiovascular morbidity and death.4
In different experimental models
soluble Klotho has been shown to
protect against acute kidney injury,
renal fibrosis, uremic cardiomyopathy,
vascular calcification, and endothelial
dysfunction. These observations are
very important not only to understand
the pathogenetic mechanisms involved,
but also with respect to the development of future and more effective
treatments and prophylactic measures,
including replacement/substitution of
the potentially missing hormone. In
this context it is essential to know
whether the uremic state is associated
with reduced circulating levels of
Klotho, whether local vascular production is reduced, and whether vascular
tissue is an additional source of
humoral Klotho. It is still an open
question whether Klotho is present in
the vasculature under physiological
conditions, and, if so, whether it is
changed in the uremic state. Measurement of Klotho at the protein level, in
the circulation as well as in tissues, is
still accompanied by difficulty related to
problems of producing good antibodies.5 Recently these problems have
been explained by Klotho being an
evolutionarily highly conserved protein,
against which the organism is protected
to generate autoantibodies, and that this
might contribute to the difficulty in
developing antibodies by animal immunization.5 Indeed homologs of the
Klotho gene were detected and characterized in the nematode Caenorhabditis elegans, uncoding a predicted
protein with similarity to the KL1
domain of mammalian Klotho and
with functional relevance for the lifespan of the worm. Even though there
are many commercial antibodies available at present, most of them lack
Kidney International (2015) 87

commentary

sufficient information from the producers, and many producers do not

describe what part of the epitope of
the Klotho protein the antibody is
directed against.
The methods used to characterize
Klotho protein expression in tissues
usually include western blotting and
immunochemistry, both of which need
a specific antibody. Western blotting
uses the molecular weight of a protein
and provides information on the relative amount of protein present in the
different samples. The proteins are
separated by size by gel electrophoresis
and blotted, and then an antibody is
able to bind to its specific protein. A
secondary antibody is usually used for
visualization. In the case of Klotho, the
size of interest is 130 kDa. However,
nonspecific bands are observed, even
with the use of the best-characterized
antibody, KM2076. Theoretically, this
could represent degradation products of
Klotho; these bands were, however, also
observed in samples derived from
Klotho / mice, indicating a potentially
nonspecific signal.5 Such a non-Klothospecific signal might be detected by
immunohistochemistry as well, and
explain some of the very different and
even contradictory results in many
studies on vascular expression of Klotho
and Klotho protein in general. When
reverse transcription PCR is used for
detection of Klotho gene expression in
the vasculature, the method might in
some cases even be too sensitive,
providing irrelevant results.6 Often a
very high number of cycles are performed in the detection of Klotho in the
vasculatureeven 30 or morewhich
might not necessarily generate a
biologically relevant signal.7
Experimental studies have shown
that soluble Klotho, when delivered
as a humoral factor, can protect
the vasculature. Klotho gene delivery
by adenoviral vector increased endothelium-dependent nitric oxide synthesis and prevented adverse vascular
remodeling in an arteriosclerotic, obese
rat model. Results from several in vitro
models point toward a direct effect of
soluble Klotho on vascular tissue.
Recently, soluble Klotho has been
Kidney International (2015) 87

shown to regulate vascular tonus, and

a nitric oxide stimulatory ability was
confirmed in vitro.8 Soluble Klotho
suppressed the expression of vascular
cell adhesion molecule-1 and intercellular adhesion molecule-1 in endothelial cells in vitro, and inhibited highphosphate-induced vascular smooth
muscle cell calcification, through inhibition of Pit-1 and Pit-2.
Whether vascular smooth muscle
cells express endogenous Klotho is
currently a matter of debate.6 This
matter has now been addressed again
in an interesting article by Ritter et al.9
(this issue), and it remains an important topic for future investigations. In
this respect, we would like to underline
again that factors mainly related to the
specificity and sensitivity of the antibody used for detection of Klotho may
critically account for the discrepant
findings.
Ritter et al.9 examined in an elaborated experimental study the presence
of Klotho protein in aortas of normal
rats, uremic rats in the absence
of pharmacologic treatment, and uremic rats treated with paricalcitol. They
found expression of Klotho in vascular
smooth muscle cells of intima
and media layers of aorta in normal
tissue. This expression was completely
suppressed in uremic rats and was not
affected by treatment with paricalcitol.
Of particular interest was their
finding that Klotho immunostaining in
adventitia of aortas from uremic rats
was significantly increased and that
treatment with paricalcitol blocked
this increase. This observation was in
stark contrast to renal expression of the
Klotho protein, which was found to be
ubiquitously expressed in all tubular
segments including the collecting duct,
and was significantly decreased in the
uremic state. Notably, this decrease in
the uremic rats could be prevented by
paricalcitol treatment. On the basis of
these findings, the authors point toward
a differential regulation of the expression
of Klotho by uremia and paricalcitol
in different tissues. This concept was
further expanded by showing that the
expression of Klotho in human uremic
parathyroid glands was increased in

oxyphil cells, as compared with chief

cells.9
One still needs to be cautious in the
interpretation of Klotho immunoreactivity in the aorta, and one might still be
cautious when using antibodies that are
not sufficiently well characterized. This
is the case when dealing with the
presence of Klotho in the kidney, where
the present study finds expression of
Klotho evenly distributed in both distal
and proximal tubules and in the vessels,
where Klotho now has been found to be
present in the adventitia. These are very
interesting new findings in a study of
high scientific quality; however, like
most other investigations in this field,
they are limited by use of presently
available, unfortunately as-yet incompletely characterized antibodies. As
such, the field is still left open for
future new and fascinating studies.
DISCLOSURE

The authors declared no competing interests.

REFERENCES
1.

2.

3.

4.

5.

6.

7.

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Andrukhova O, Smorodchenko A, Egerbacher M

et al. FGF23 promotes renal calcium
reabsorption through the TRPV5 channel. EMBO
J 2014; 33: 229246.
Lindberg K, Amin R, Moe OW et al.
The kidney is the principal organ mediating
klotho effects. J Am Soc Nephrol 2014; 25:
21692175.
Chen T-H, Kuro-o M, Chen C-H et al. The
secreted Klotho protein restores phosphate
retention and suppresses accelerated aging
in Klotho mutant mice. Eur J Pharmacol 2013;
698: 6773.
Lewin E, Olgaard K. Klotho, an important new
factor for the activity of Ca2+ channels,
connecting calcium homeostasis, ageing and
uraemia. Nephrol Dial Transplant 2006; 21:
17701772.
Barker SL, Pastor J, Carranza D et al. The
demonstration of Klotho deficiency in human
chronic kidney disease with a novel synthetic
antibody. Nephrol Dial Transplant 2015; 30:
223233.
Lindberg K, Olauson H, Amin R et al. Arterial
klotho expression and FGF23 effects on
vascular calcification and function. PLoS One
[online] 2013; 8: e60658.
Donate-Correa J, Mora-Fernandez C,
Martinez-Sanz R et al. Expression of FGF23/
KLOTHO system in human vascular tissue.
Int J Cardiol 2013; 165: 179183.
Six I, Okazaki H, Gross P et al. Direct, acute
effects of Klotho and FGF23 on vascular
smooth muscle and endothelium. PLoS One
[online] 2014; 9: e93423.
Ritter C, Zhang S, Delmez J et al. Differential
expression and regulation of Klotho by
paricalcitol in the kidney, parathyroid, and
aorta of uremic rats. Kidney Int 2015; 87:
11411152.

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