University of Groningen
Klotho in vascular biology
Mencke, Rik
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Mencke, R. (2018). Klotho in vascular biology. Rijksuniversiteit Groningen.
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Chapter 7
Imaging of incipient vascular calcification in
Klotho deficiency
R. Mencke J.W.A. Sijbesma J. Doorduin J.G. Hoenderop A. Pasch R.H.J.A. Slart J.L. Hillebrands Manuscript in preparation234
Abstract
Chronic kidney disease (CKD) patients suffer from vascular calcification and are generally deficient in the renal protein Klotho. In mice, Klotho deficiency causes the development of vascular calcification. Current imaging modalities can visualize the result, but not the process of calcification and therefore do not facilitate early detection. Furthermore, it is increasingly recognised that calciprotein particles (CPPs) are causally involved in CKD in the active process of smooth muscle cell (SMC)-mediated calcification, but it is unknown how CPPs relate to Klotho deficiency.
To study whether 18F-NaF, a bone tracer, can be used to investigate the calcification
propensity and development of incipient vascular calcification in Klotho-/- mice, we used 18
F-NaF as a tracer for small animal PET-CT scanning and autoradiography in a comparison to
histochemistry on Klotho-/- mouse aortas. We measured the T50 in Klotho-/- serum and we
explored the protective effects of Klotho on CPP-induced calcification in vitro.
While we did not detect any 18F-NaF signal on small animal PET-CT in the aortas of 7-week-old
or 10-week-old Klotho-/- mice, we did detect F-18 decay using autoradiography on aortas ex
vivo, indicative of active crystallisation. Interestingly, the aortic arch appeared to be affected
first (at 7 weeks of age), while the whole aorta was involved at 10 weeks of age. We detected almost no calcification using histochemistry, except for sporadically in the aortic arch. We
found that Klotho-/- mice have a significantly expedited CPP maturation time (T50), compared
to Klotho+/- and WT mice, indicative of an impaired serum calcification buffering capacity.
Finally, we found that recombinant Klotho inhibits the development of CPP-induced SMC calcification in vitro.
To conclude, we identified Klotho deficiency-associated calcification predilection spots similar
to the pattern in patients. 18F-NaF has the potential to be used for the detection of vascular
microcalcifications, but current small animal PET technology lacks the sensitivity for application in animal studies. Mechanistically, we find that CPPs are likely implicated in this process, while Klotho protects SMCs from CPP-induced calcification.
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Introduction
Chronic kidney disease (CKD) patients suffer from vascular calcification and an increased
cardiovascular mortality risk (1). CKD patients are also particularly Klotho-deficient (2-4).
Klotho is a transmembrane protein predominantly expressed in the kidney, from where soluble Klotho derives, which is found in the systemic circulation and in the urine. In mice, Klotho deficiency causes a premature ageing-like phenotype that includes the development
of severe vascular calcification (5), which raises the possibility that Klotho deficiency is causally
involved in the development of vascular calcification in CKD patients.
The medial smooth muscle cells (SMCs) in the vascular wall in Klotho-deficient mice generally start to calcify from around 4 weeks of age onwards in response to high phosphate and
calcium levels (6, 7), in combination with SMC senescence (8-11) and in spite of up-regulated
anti-calcification mechanisms. These include up-regulation of matrix Gla protein (12),
osteopontin (13, 14), osteoprotegerin (13), the pyrophosphate system (15), and higher plasma
fetuin A levels (13).
While it is generally accepted that vascular calcification is an active, cellular process involving SMC de-differentiation and subsequent activation of osteochondrogenic signalling pathways
(16), the possibly causal role of mature (secondary) calciprotein particles (CPPs) in the
induction of SMC calcification has recently come into focus (17, 18). These calciprotein
particles form when fetuin A binds to amorphous calcium phosphate crystals (primary CPPs),
thereby delaying their maturation to a crystalline state (secondary CPPs) (19-22). The CPP
maturation rate or serum calcification propensity in CKD patients is greatly increased and
predictive of mortality (23). However, a potential link between CPPs in the development of
vascular calcification and Klotho is currently unexplored.
While vascular calcification can be visualized very well using computer tomography and can be used as a prediction parameter, there are no imaging modalities that are currently routinely used in clinical practice to visualize the process of calcification, rather than the result. As a
bone tracer, 18F-NaF is currently being explored as a potential tracer for in vivo imaging of
vascular calcification. Early detection could enable us to facilitate early treatment of vascular calcification, in which a role can be envisioned for the maintenance of Klotho levels.
To further study the development of vascular calcification in Klotho deficiency, we evaluated a number of imaging techniques, including small animal PET-CT scanning and
autoradiography, using the positron emitter 18F-NaF as a tracer, which is suitable for the
evaluation of microcalcification (24). Using various imaging techniques in vivo and SMCs in
vitro, this study provides a number of novel insights to our understanding of the development
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Methods
Animals
Klotho+/- mice were kindly provided by Joost Hoenderop (Radboud University Medical Center,
Nijmegen, The Netherlands) (25) and were housed and bred at the University Medical Center
Groningen, generating Klotho-/-, Klotho+/-, and WT mice. The experimental protocol was
approved by the institutional University of Groningen ethical board and was in accordance
with the NIH Guide for the Use and Care for Laboratory Animals. Klotho-/- mice were housed
with 12-hour light/dark cycles in individually ventilated cages, with free access to drinking water and chow (containing 0.82% phosphate, 1.15% calcium, 0.29% magnesium, and 2900 IU/kg vitamin D), as well as ample nesting material and cage enrichment. Knockout and WT
mice were housed in pairs to counteract hypothermia in Klotho-/- mice.
Small animal PET-CT scans
At the age of 10 weeks, we scanned 4 Klotho-/- mice and 4 WT mice, with an additional 5
Klotho-/- mice undergoing scans at 7 weeks of age. Briefly, mice were anaesthetized with
isoflurane and injected intravenously with 18F-NaF in an average concentration of 8.0 MBq.
The tracer was allowed to circulate for 1 hour after which the animals were terminated using an intraperitoneal injection of pentobarbital (Euthasol, ASTFarma, The Netherlands). The scanning protocol for small animal PET consisted of 60 minutes, followed by 15 minutes of CT scanning, on an Inveon Hybrid small animal PET-CT Scanner (Siemens, Germany). Image reconstruction was performed using PMOD version 3.802 (PMOD Technologies, Switzerland).
Autoradiography
Aortas were dissected after completion of the scanning protocol. Autoradiography was performed using a Cyclone phosphor system (Perkin Elmer, USA) and an exposure time of 1 minutes. Images were analysed using Optiquant software.
Histology
After aorta segments were formalin-fixed and paraffin-embedded, 4 µm sections were cut, de-paraffinized, and re-hydrated in demineralized water. Von Kossa staining was performed by incubating section for 1 hour in a 1% silver nitrate solution while exposed to sunlight, followed by incubation in 3% sodium thiosulfate for 5 minutes and counterstaining with nuclear fast red. Alizarin Red staining was performed by incubating sections in 2% Alizarin Red
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S (Sigma-Aldrich, USA) for 5 minutes (pH = 4.2), followed by passing them through acetone, xylene/acetone and xylene rinses.
Immunohistochemistry
FFPE kidney sections from Klotho-/- and WT mice were de-paraffinized and re-hydrated.
Heat-induced antigen retrieval was performed in 0.1 M Tris/HCl (pH = 9.0) buffer for 15 minutes
and endogenous peroxidase was inactivated in 0.3% H2O2/PBS for 30 minutes. Sections were
incubated with AF1819 (1:50; R&D Systems, USA) in 1% BSA/PBS for 1 hour at room temperature, followed by polyclonal rabbit anti-goat-HRP (1:100; P0449, Dako, USA) and polyclonal goat anti-rabbit-HRP (1:100; P0448, Dako) in 1% AB serum/1% BSA/PBS, both for 30 minutes at room temperature. Finally, the chromogenic reaction was performed using
0.03% H2O2 in 5% 3-amino-3-ethylcarbazole (AEC)/acetic acid for 15 minutes, followed by
hematoxylin counterstaining.
RNA in situ hybridization
FFPE kidney sections from Klotho-/- and WT mice were mounted on SuperFrost Plus glass slides
(Thermo Scientific, USA), baked for 1 hour at 60 °C, followed by antigen retrieval and RNA-ISH according to manufacturer instructions (ACDBio, Italy). We used a probe against the mouse
Klotho gene (422081, ACDBio) in conjunction with the RNAscope 2.5 HD Reagent kit (ACDBio).
Briefly, this approach hybridizes amplifier sequences to multiple double-Z probes, culminating in an HRP reaction with 3,3’-diaminobenzidine (DAB) as a chromogen and hematoxylin counterstaining.
PCR
RNA was isolated from Klotho-/- and WT kidneys using TRIzol (Ambion, USA), chloroform
2-propanol, and ethanol (Merck, USA). Transcription of cDNA was performed using random hexamer primers and SuperScript II (Thermo Fisher, USA). The program for qRT-PCR consisted of 2 minutes at 50 °C, 10 minutes at 95 °C, and 40 cycles of 15 seconds at 95 °C and 60 seconds at 60 °C, using Taqman assays for mouse Klotho (Mm00502002_m1) and Ywhaz (Mm01722325_m1) (ThermoFisher, USA) as a housekeeping gene. DNA was isolated with a high-salt method. Genotyping PCR was performed for the WT allele (using 5’-GATGGGGTCGACGTCA-3’ (forward) and 5’-TAAAGGAGGAAAGCCATTGTC-3’ (backward)) and the mutant allele (using 5’-GCAGCGCATCGCCTTCTATC-3’ (forward) and ATGCTCCAGACATTCTCAGC-3’ (backward)) and the following program: 15 seconds at 94 °C, 30 seconds at 65 °C, and 40 seconds at 72 °C for 10 cycles with the annealing temperature being lowered 1 °C per cycle, followed by 30 more cycles with the annealing temperature at 55 °C.
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PCR products were imaged on 2% agarose gels using ethidium bromide after gel electrophoresis.
T50 measurement
Sera were thawed for 48 hours at 4 °C before vortexing and centrifugation. Sera (40 µl) were admixed with supersaturated calcium (35 µl) and phosphate (25 µl) stock solutions in 384-wells plates. Nephelometry was performed for 600 minutes in a Nephelostar nephelometer (BMG Labtech, Germany) and non-linear regression analysis was used to determine the half-maximal precipitation time.
Cell culture
Human aortic smooth muscle cells (HASMCs, ScienCell, USA) were cultured in Smooth Muscle Cell Medium with 2% Fetal Calf Serum, 1% Smooth Muscle Growth Supplement, and 1%
Penicillin/Streptomycin (ScienCell) at 37 °C and 5% CO2. HASMCs were growth to confluence
in 96-wells plates. CPPs were generated by incubating DMEM with 10% FCS, 1% Penicillin/Streptomycin (all from Lonza, USA), supplemented with 3.5 mM phosphate and 1 mM calcium, at 37 °C for 7 days, followed by centrifugation, and calcium concentration measurement using the Calcium Colorimetric Assay (Sigma) per manufacturer instructions. CPPs were incubated with HASMCs at a final concentration equivalent to 100 µg/mL calcium, with and without 0.2 nM or 0.4 nM recombinant human Klotho (R&D Systems, USA), for 24 hours (8 wells per condition). Cells were then rinsed, fixed for 10 minutes in 2% paraformaldehyde, and stained with 2% Alizarin Red for 5 minutes, followed by three rinses in PBS.
Statistical analysis
Normally distributed variables are presented as mean ± standard deviation (SD) and non-normally distributed variables are presented as median [interquartile range]. Normality was tested using the Kolmogorov-Smirnov test. Differences between groups were tested using one-way ANOVA with the post-hoc Bonferroni correction for multiple-group comparisons or Student’s t test for two-group comparisons, in the case of a normal distribution, or with the Kruskal-Wallis test followed by Dunn’s post-hoc correction for multiple-group comparisons or Mann-Whitney U test for two-group comparisons, in the case of a non-normal distribution. Statistical analyses were performed using GraphPad Prism software version 5.0 (GraphPad Software, USA) and a p value < 0.05 was considered significant.
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Results
Klotho knockout validation
To validate that the Klotho-/- mice we used in this study were an appropriate model for Klotho
deficiency, we ascertained that the mice carry the mutant allele (Figure 1A), that there was virtually no renal Klotho mRNA expression using qRT-PCR (p < 0.05, Figure 1B) and RNA in situ hybridisation (Figure 1C), that there was no renal Klotho protein expression (Figure 1D), and that the mice displayed the expected gross phenotype, including growth
Figure 1. Validation of knockout of the Klotho gene in Klotho-/- mice. (A) Validation in genomic DNA by
genotyping PCR, amplifying the mutant allele in Klotho-/- and Klotho+/- mice, and the WT allele in Klotho+/- and WT mice. (B) Confirmation of Klotho knockout using qRT-PCR on kidneys from Klotho-/- and WT mice. (C) Confirmation of Klotho knockout using RNA in situ hybridization in kidney sections from Klotho-/- and WT mice, depicting Klotho mRNA in predominantly distal tubules and not in the knockout kidney. (D) Confirmation of Klotho knockout using
immunohistochemistry, showing Klotho protein in WT mouse kidney in the distal tubules, but no Klotho protein in knockout mouse kidney. (E) Validation of the expected Klotho-deficient gross phenotype in a Klotho-/- mouse of 10 weeks of age, compared to its WT littermate (both post termination). The Klotho-/- mouse displays the expected stunted growth and kyphosis known to develop in Klotho deficiency. * p < 0.05.
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retardation and kyphosis (Figure 1E), all compared to wild-type littermates.
Small animal PET-CT imaging
To assess whether 18F-NaF as tracer is a feasible method of assessing vascular calcification in
mice, we performed small animal PET-CT scans of mice 1 hour after intravenous injection of
18F-NaF. We detected a clear signal from bones in both WT and Klotho-/- mice, indicating
successful delivery and circulation of 18F-NaF (Figure 2). However, we could not detect a signal
from the aorta in either Klotho-/- mice (neither at 7 weeks (Supplemental Figure 3), nor at 10
weeks of age (Supplemental Figure 2)) or in WT mice (at 10 weeks of age). There were also no vascular calcifications visible on small animal CT scans (Figure 2, Supplemental Figure 1).
Autoradiography
To assess whether 18F-NaF allows for visualisation of microcalcifications, we performed
autoradiography on Klotho-/- and WT littermates (at the age of 10 weeks) following the small
animal PET-CT scan. Radioactivity from 18F-NaF was detected in almost the entire Klotho
-/-aorta at 10 weeks of age, with particular concentrations in the aortic arch extending to the thoracic aorta, and in the abdominal aorta (Figure 3A). WT mouse aorta, however, only produced a low background signal corresponding to contamination with blood (Figure 3A).
The percentage of radioactivity compared to the injected dose was higher in Klotho-/- aortic
arch, thoracic aorta, and abdominal aorta, compared to homologous WT regions (Figure 3B). To assess the development of vascular calcification in Klotho deficiency, we included 7-week-old mice, in which the signal was more restricted and limited to the aortic arch and the abdominal aorta (Figure 3C).
Histology
Assessing the aortic arch, thoracic aorta, abdominal aorta, and aorta/iliac artery around the
level of the bifurcation in Klotho-/- and WT mice, we detected virtually no vascular calcification
using Von Kossa and Alizarin Red stainings (Figure 4), except sporadically in the aortic arch
from 10-week-old Klotho-/- mice (Figure 4A, B). This corroborates the autoradiography findings
that indicate that the aortic arch is the first and most severely affected segment of the aorta.
Furthermore, using 18F-NaF in combination with autoradiography is a more sensitive
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Figure 2. Small animal PET-CT scans of 10-week-old Klotho-/- and WT mice after circulation of 18F-NaF. (A)
Median sagittal cross-section of a 10-week-old Klotho-/- mouse, showing tracer uptake in skeletal structures and tracer
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Figure 3. Autoradiography on Klotho-/- and WT aorta after circulation of 18F-NaF. (A) At 10 weeks of age, Klotho -/- aortas (N = 4) exhibit a pattern of vascular calcification most prominent in the aortic arch, thoracic aorta, and in the abdominal aorta, whereas WT aortas (N = 4) may only display a little background signal from blood. (B)
Quantification of the signal in the aortic arch, thoracic aorta, and abdominal aorta (N = 3 mice per genotype) indicates reveals higher tracer uptake in the Klotho-/- aortas, compared to the injected dose and compared to a calibration curve. (C) Autoradiography on aortas from Klotho-/- mice at 7 weeks of age reveals that the pattern develops in the aortic arch and in the abdominal aorta. Patchy involvement of the carotid artery, renal artery, and bifurcation can also be observed.
(Figure 2. Cont’d.) presence in the bladder, but no discernible signal from the aorta. Note the pronounced
kyphosis. (B) Median sagittal cross-section of a 10-week-old WT mouse, showing a pattern similar to (A). (C)
Prevertebral coronal cross-section (A), showing tracer uptake in skeletal structures and tracer presence in the bladder, but no discernible signal from the aorta. (D) Prevertebral coronal cross-section of (B), showing tracer
uptake in skeletal structures and tracer presence in a kidney and in the bladder, but no discernible signal from the aorta. (E) Transverse section at the thoracic level of (A), with a similar pattern. (F) Transverse
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Serum calcification propensity
To investigate the mechanism of incipient vascular calcification and the role of calciprotein particles (CPPs), we focused on the anti-calcific serum buffer capacity using the previously
described T50 test (26, 27) that measures the maturation rate for CPPs from inert primary CPPs
to noxious secondary CPPs. To address whether an increased serum calcification propensity
could be involved in instigating vascular calcification in Klotho deficiency, we determined T50
in serum from Klotho-/- (N = 9), Klotho+/- (N = 14), and WT (N = 11) mice. We found that T50
Figure 4. Histochemical analysis for vascular calcification of 10-week-old Klotho-/- and WT mouse aortas. (A)
Von Kossa staining on Klotho-/- aortic arch, including a calcified area. (B) Alizarin Red staining on Klotho-/- aortic arch, including a calcified area. (C) Von Kossa staining on WT aortic arch. (D) Alizarin Red staining on WT aortic
arch. (E) Von Kossa staining on Klotho-/- thoracic aorta. (F) Alizarin Red staining on Klotho-/- thoracic aorta. (G) Von Kossa staining on WT thoracic aorta. (H) Alizarin Red staining on WT thoracic aorta. (I) Von Kossa staining on
Klotho-/- abdominal aorta. (J) Alizarin Red staining on Klotho-/- abdominal aorta. (K) Von Kossa staining on WT abdominal aorta. (L) Alizarin Red staining on WT abdominal aorta. (M) Von Kossa staining on Klotho-/- aortic bifurcation. (N) Alizarin Red staining on Klotho-/- aortic bifurcation. (O) Von Kossa staining on WT aortic bifurcation. (P) Alizarin Red staining on WT aortic bifurcation. Original magnifications are 400× and arrows
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was significantly decreased (meaning that serum calcification propensity was increased) in
Klotho-/- mouse serum (252 ± 82 min), compared to serum from both Klotho+/- (456 ± 67 min)
and WT mice (462 [432-477]) (Figure 5).
In vitro calcification of SMCs
It was recently shown that SMCs calcify within 24 hours of incubation with secondary (but not
primary) CPPs(17, 18). To examine whether recombinant Klotho is capable of directly
preventing CPP-induced SMC calcification, we stimulated SMCs with secondary CPPs with and without 0.2 or 0.4 nM recombinant human Klotho. Alizarin Red staining revealed that Klotho inhibited the development of CPP-induced calcification (Figure 6).
Figure 5. Serum calcification propensity in Klotho-/-, Klotho+/-, and WT mice. T50 was significantly lower in Klotho -/- mouse sera, indicative of a higher serum calcification propensity. ** p < 0.01, *** p < 0.001.
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Discussion
This study focused on the development of incipient vascular calcification in Klotho deficiency. The autoradiography data indicate that the pattern in which vascular calcification develops in Klotho deficiency is very similar to the pattern that is commonly found both in ageing or CKD patients and experimentally, in that it affects the aortic arch and the abdominal aorta first (28, 29). Furthermore, this study cements a role for CPPs in the development of Klotho deficiency-induced vascular calcification, which is in line with emerging paradigms of how vascular calcification develops.
18F-NaF is currently being re-explored in humans as a possible PET tracer not only for bone,
but for vascular calcification as well (24, 29-41). One of our aims was therefore to test the
feasibility of 18F-NaF as a small animal PET tracer for vascular calcification in animal studies
and we found that 18F-NaF in combination with autoradiography can be used to visualize
microcalcifications that are not yet manifest in small animal CT imaging. However, the inability of current small animal PET technology to resolve microcalcifications limits the potential
practical applications of 18F-NaF. The detection of a vascular signal using autoradiography
corroborates data from Irkle et al., who show that 18F-NaF detects both established
Figure 6. Recombinant Klotho inhibits secondary calciprotein particle (CPP)-induced calcification of human aortic smooth muscle cells (HASMCs). (A) Alizarin Red staining of HASMCs incubated with PBS. (B) Alizarin Red
staining of HASMCs incubated with secondary CPPs for 24 hours. (C) Alizarin Red staining of HASMCs incubated
with secondary CPPs and 0.2 nM recombinant Klotho for 24 hours. (D) Alizarin Red staining of HASMCs incubated
with secondary CPPs and 0.4 nM recombinant Klotho for 24 hours. (E) Representative quantification for one
experiment with 8 replicates per condition. Experiments were performed three times independently with similar results. Original magnification is 40×.
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macrocalcifications and actively developing microcalcifications (24), with CT imaging being
used in their study to discriminate between the two. Our data indicate that 18F-NaF indeed
detects active microcalcifications and confirm that it can do so in vivo (as opposed to ex vivo incubation in tracer solution) in an animal model. Finally, building upon the data from Irkle et
al., the calcification pattern as detected by autoradiography includes primarily
microcalcifications that are also undetectable upon histological examination, attesting to the
sensitivity of 18F-NaF as a suitable tracer, with the caveat that an accordingly sensitive detector
be used.
The developing calcification pattern in Klotho deficiency as seen in Figure 2 matches indications found in previous studies. Notably, Bai et al. in 2009 used (contact) radiography on
aortas from Klotho-deficient mice in which the arch was calcified (42). More recently, Alesutan
et al. detected a similar pattern with predominant calcification in the aortic arch and
abdominal aorta, using Alizarin Red staining on whole kl/kl aorta (43). Hum et al. and
Nakamura et al. also detected a similar pattern of aortic arch calcification on small animal CT at 4 weeks of age in kl/kl mice, which progressed to encompass the rest of the aorta at 8 weeks
of age (44, 45). The fact that sporadic calcifications, when detected by using histochemistry in
our study, were present in the aortic arch further confirms these studies and is in line with our own autoradiography data.
While the general lack of development of overt vascular calcifications did allow us to focus on the early changes and development of incipient microcalcifications, it was slightly surprising
that the Klotho-/- mice we studied did not develop marked vascular calcifications. Many
different Klotho-deficient mice have been generated over the years, including the original kl/kl
mice (with a disrupted Klotho promoter) (5), Klotho-/- mice (with constitutive deletion of an
exon) (46), various Klothoflox/flox mice, producing deletion of an exon upon Cre recombinase
expression under various promoters, including β-actin (47) and Six2 (48), and
N-ethyl-N-nitrosourea-induced mutations affecting Klotho expression (49). All of these mice develop
spontaneous vascular calcification, although there does appear to be some variation. To an extent, this variation is known to be a function of the level of any potentially residual Klotho expression, however, we hypothesize that the genetic background of the strain, and
environmental factors like diet (6, 25, 46, 50) and housing conditions (51) can greatly influence
the rate at which the Klotho deficiency phenotype develops. The validation of our model
(Figure 1) and the finding that microcalcifications are detectable using 18F-NaF and
autoradiography in combination with an increased serum calcification propensity indicate that the development of vascular calcification, as expected in Klotho deficiency, is progressing but likely at a lower rate than in other strains in other labs.
This is the first study in which serum calcification propensity is investigated in Klotho-/- mice.
The finding that serum calcification propensity is markedly increased at a time when histological methods are only beginning to sporadically identify a nodus of calcification fits with the experimental evidence and emerging paradigm that CPPs are causally involved in the
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pathophysiology of vascular calcification. The substantial decrease in T50 is similar to the
decrease observed in fetuin A-/- mice, compared to WT mice, or in hemodialysis patients,
compared to healthy controls (27). This illustrates the importance of Klotho in preventing
vascular calcification. It has long been known that Klotho counteracts vascular calcification via various indirect mechanisms, including preventing hyperphosphatemia by promoting
NaPi2a-mediated phosphaturia directly (52-54) and indirectly, serving as a co-receptor with fibroblast
growth factor (FGF) receptor 1 for FGF23 (55-57), with a clear role for the soluble Klotho
protein (44, 53). In addition to these indirect mechanisms, there are also indications that
Klotho inhibits vascular calcification directly. Hu et al. find that in a CKD mouse model, there is a beneficial effect of Klotho overexpression even after correction for plasma creatinine and
plasma phosphate (3). Recently, it was shown that in mice with a loss-of-function mutation in
Enpp1, fed a high-phosphate diet resulting in ectopic calcification, overexpression of Klotho
also inhibited the development of vascular calcification (58), outside the context of CKD. Furthermore, Hum et al. found that vascular calcification in Klotho-deficient mice is markedly reduced by overexpression of circulating Klotho protein(44). Moreover, recombinant Klotho
has been shown to inhibit the uptake of phosphate by SMCs and SMC calcification in vitro (3,
59, 60). These in vitro studies, however, were performed by increasing the phosphate concentration in the medium and are likely dependent on the slow formation and ripening of CPPs in vitro. Our study confirms that Klotho inhibits SMC calcification also when directly induced by secondary CPPs.
To conclude, in investigating the mechanism of incipient vascular calcification in Klotho
deficiency, we found, using 18F-NaF as a tracer in Klotho-/- mice, that vascular calcification
develops in a specific pattern in the aorta, and that microcalcifications are already detectable
with 18F-NaF before they are generally detectable with histochemical stainings or computer
tomography. 18F-NaF may therefore be a suitable tracer for the detection of
microcalcifications, which could ultimately be used in the early detection and treatment of
patients at risk for developing vascular calcification. Our data further indicate that Klotho
-/-mice have a markedly increased serum calcification propensity, which further implicates CPPs in the pathophysiology of vascular calcification and which is further corroborated by the rapid induction of SMC calcification in vitro, which could be inhibited by soluble Klotho. These findings provide new insights in the development of vascular calcification in Klotho deficiency and in the anti-calcification effects of Klotho.
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Acknowledgments
We are grateful to Inês Farinha Antunes, Beatrix Blanchard, Mattias Bachtler, Parisa Aghagolzadeh, and Isabel Gsponer for their technical assistance. This study was funded by the
Dutch Kidney Foundation consortium program [CP10.11] (NIGRAM consortium; PIs: P.M. ter
Wee and M.G. Vervloet, VU University Medical Center, Amsterdam, The Netherlands; J.G. Hoenderop and R.J. Bindels, Radboud University Medical Center, Nijmegen, The Netherlands; G.J. Navis, M.H. de Borst, and J.L. Hillebrands, University Medical Center Groningen, Groningen, The Netherlands).
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References
1. A. S. Go, G. M. Chertow, D. Fan, C. E. McCulloch, C. Y. Hsu, Chronic kidney disease and the risks of death, cardiovascular events, and hospitalization. N. Engl. J. Med. 351, 1296-1305 (2004).
2. N. Koh, T. Fujimori, S. Nishiguchi, A. Tamori, S. Shiomi, T. Nakatani, K. Sugimura, T. Kishimoto, S. Kinoshita, T. Kuroki, Y. Nabeshima, Severely reduced production of klotho in human chronic renal failure kidney. Biochem.
Biophys. Res. Commun. 280, 1015-1020 (2001).
3. M. C. Hu, M. Shi, J. Zhang, H. Quinones, C. Griffith, M. Kuro-o, O. W. Moe, Klotho deficiency causes vascular calcification in chronic kidney disease. J. Am. Soc. Nephrol. 22, 124-136 (2011).
4. S. L. Barker, J. Pastor, D. Carranza, H. Quinones, C. Griffith, R. Goetz, M. Mohammadi, J. Ye, J. Zhang, M. C. Hu, M. Kuro-o, O. W. Moe, S. S. Sidhu, The demonstration of alphaKlotho deficiency in human chronic kidney disease with a novel synthetic antibody. Nephrol. Dial. Transplant. 30, 223-233 (2015).
5. M. Kuro-o, Y. Matsumura, H. Aizawa, H. Kawaguchi, T. Suga, T. Utsugi, Y. Ohyama, M. Kurabayashi, T. Kaname, E. Kume, H. Iwasaki, A. Iida, T. Shiraki-Iida, S. Nishikawa, R. Nagai, Y. I. Nabeshima, Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature. 390, 45-51 (1997).
6. M. Ohnishi, M. S. Razzaque, Dietary and genetic evidence for phosphate toxicity accelerating mammalian aging. FASEB J. 24, 3562-3571 (2010).
7. T. Shiraki-Iida, A. Iida, Y. Nabeshima, H. Anazawa, S. Nishikawa, M. Noda, M. Kuro-o, Y. Nabeshima, Improvement of multiple pathophysiological phenotypes of klotho (kl/kl) mice by adenovirus-mediated expression of the klotho gene. J. Gene Med. 2, 233-242 (2000).
8. M. Eren, A. E. Boe, S. B. Murphy, A. T. Place, V. Nagpal, L. Morales-Nebreda, D. Urich, S. E. Quaggin, G. R. Budinger, G. M. Mutlu, T. Miyata, D. E. Vaughan, PAI-1-regulated extracellular proteolysis governs senescence and survival in Klotho mice. Proc. Natl. Acad. Sci. U. S. A. 111, 7090-7095 (2014).
9. Y. Nabeshima, M. Washida, M. Tamura, A. Maeno, M. Ohnishi, T. Shiroishi, A. Imura, M. S. Razzaque, Y. Nabeshima, Calpain 1 inhibitor BDA-410 ameliorates alpha-klotho-deficiency phenotypes resembling human aging-related syndromes. Sci. Rep. 4, 5847 (2014).
10. S. Sato, Y. Kawamata, A. Takahashi, Y. Imai, A. Hanyu, A. Okuma, M. Takasugi, K. Yamakoshi, H. Sorimachi, H. Kanda, Y. Ishikawa, S. Sone, Y. Nishioka, N. Ohtani, E. Hara, Ablation of the p16(INK4a) tumour suppressor reverses ageing phenotypes of klotho mice. Nat. Commun. 6, 7035 (2015).
11. R. Mencke, J. L. Hillebrands, NIGRAM consortium, The role of the anti-ageing protein Klotho in vascular physiology and pathophysiology. Ageing Res. Rev. 35, 124-146 (2016).
12. R. Nakano-Kurimoto, K. Ikeda, M. Uraoka, Y. Nakagawa, K. Yutaka, M. Koide, T. Takahashi, S. Matoba, H. Yamada, M. Okigaki, H. Matsubara, Replicative senescence of vascular smooth muscle cells enhances the calcification through initiating the osteoblastic transition. Am. J. Physiol. Heart Circ. Physiol. 297, H1673-84
250
13. C. B. Leibrock, I. Alesutan, J. Voelkl, D. Michael, T. Castor, U. Kohlhofer, L. Quintanilla-Martinez, L. Kubler, J. G. Mannheim, B. J. Pichler, K. P. Rosenblatt, M. Kuro-O, F. Lang, Acetazolamide sensitive tissue calcification and aging of klotho-hypomorphic mice. J. Mol. Med. (Berl). 94, 95-106 (2015).
14. Q. Yuan, T. Sato, M. Densmore, H. Saito, C. Schuler, R. G. Erben, B. Lanske, Deletion of PTH rescues skeletal abnormalities and high osteopontin levels in Klotho-/- mice. PLoS Genet. 8, e1002726 (2012).
15. T. Nakatani, M. Ohnishi, M. S. Razzaque, Inactivation of klotho function induces hyperphosphatemia even in presence of high serum fibroblast growth factor 23 levels in a genetically engineered hypophosphatemic (Hyp) mouse model. FASEB J. 23, 3702-3711 (2009).
16. C. M. Shanahan, N. R. Cary, J. R. Salisbury, D. Proudfoot, P. L. Weissberg, M. E. Edmonds, Medial localization of mineralization-regulating proteins in association with Monckeberg's sclerosis: evidence for smooth muscle cell-mediated vascular calcification. Circulation. 100, 2168-2176 (1999).
17. P. Aghagolzadeh, M. Bachtler, R. Bijarnia, C. Jackson, E. R. Smith, A. Odermatt, R. Radpour, A. Pasch, Calcification of vascular smooth muscle cells is induced by secondary calciprotein particles and enhanced by tumor necrosis factor-alpha. Atherosclerosis. 251, 404-414 (2016).
18. P. Aghagolzadeh, R. Radpour, M. Bachtler, H. van Goor, E. R. Smith, A. Lister, A. Odermatt, M. Feelisch, A. Pasch, Hydrogen sulfide attenuates calcification of vascular smooth muscle cells via KEAP1/NRF2/NQO1 activation. Atherosclerosis. 265, 78-86 (2017).
19. A. Heiss, V. Pipich, W. Jahnen-Dechent, D. Schwahn, Fetuin-A is a mineral carrier protein: small angle neutron scattering provides new insight on Fetuin-A controlled calcification inhibition. Biophys. J. 99, 3986-3995 (2010).
20. A. Heiss, W. Jahnen-Dechent, H. Endo, D. Schwahn, Structural dynamics of a colloidal protein-mineral complex bestowing on calcium phosphate a high solubility in biological fluids. Biointerphases. 2, 16-20 (2007).
21. A. Heiss, T. Eckert, A. Aretz, W. Richtering, W. van Dorp, C. Schafer, W. Jahnen-Dechent, Hierarchical role of fetuin-A and acidic serum proteins in the formation and stabilization of calcium phosphate particles. J. Biol. Chem.
283, 14815-14825 (2008).
22. A. Heiss, A. DuChesne, B. Denecke, J. Grotzinger, K. Yamamoto, T. Renne, W. Jahnen-Dechent, Structural basis of calcification inhibition by alpha 2-HS glycoprotein/fetuin-A. Formation of colloidal calciprotein particles. J. Biol.
Chem. 278, 13333-13341 (2003).
23. E. R. Smith, M. L. Ford, L. A. Tomlinson, E. Bodenham, L. P. McMahon, S. Farese, C. Rajkumar, S. G. Holt, A. Pasch, Serum calcification propensity predicts all-cause mortality in predialysis CKD. J. Am. Soc. Nephrol. 25,
339-348 (2014).
24. A. Irkle, A. T. Vesey, D. Y. Lewis, J. N. Skepper, J. L. Bird, M. R. Dweck, F. R. Joshi, F. A. Gallagher, E. A. Warburton, M. R. Bennett, K. M. Brindle, D. E. Newby, J. H. Rudd, A. P. Davenport, Identifying active vascular microcalcification by (18)F-sodium fluoride positron emission tomography. Nat. Commun. 6, 7495 (2015).
25. T. E. Woudenberg-Vrenken, B. C. van der Eerden, A. W. van der Kemp, J. P. van Leeuwen, R. J. Bindels, J. G. Hoenderop, Characterization of vitamin D-deficient klotho(-/-) mice: do increased levels of serum 1,25(OH)2D3 cause disturbed calcium and phosphate homeostasis in klotho(-/-) mice? Nephrol. Dial. Transplant. 27,
251
26. A. Pasch, Novel assessments of systemic calcification propensity. Curr. Opin. Nephrol. Hypertens. 25, 278-284
(2016).
27. A. Pasch, S. Farese, S. Graber, J. Wald, W. Richtering, J. Floege, W. Jahnen-Dechent, Nanoparticle-based test measures overall propensity for calcification in serum. J. Am. Soc. Nephrol. 23, 1744-1752 (2012).
28. T. Tani, H. Orimo, A. Shimizu, S. Tsuruoka, Development of a novel chronic kidney disease mouse model to evaluate the progression of hyperphosphatemia and associated mineral bone disease. Sci. Rep. 7,
2233-017-02351-6 (2017).
29. T. Derlin, Z. Toth, L. Papp, C. Wisotzki, I. Apostolova, C. R. Habermann, J. Mester, S. Klutmann, Correlation of inflammation assessed by 18F-FDG PET, active mineral deposition assessed by 18F-fluoride PET, and vascular calcification in atherosclerotic plaque: a dual-tracer PET/CT study. J. Nucl. Med. 52, 1020-1027 (2011).
30. B. A. Blomberg, A. Thomassen, R. A. Takx, M. H. Vilstrup, S. Hess, A. L. Nielsen, A. C. Diederichsen, H. Mickley, A. Alavi, P. F. Hoilund-Carlsen, Delayed sodium 18F-fluoride PET/CT imaging does not improve quantification of vascular calcification metabolism: results from the CAMONA study. J. Nucl. Cardiol. 21, 293-304 (2014).
31. T. Derlin, U. Richter, P. Bannas, P. Begemann, R. Buchert, J. Mester, S. Klutmann, Feasibility of 18F-sodium fluoride PET/CT for imaging of atherosclerotic plaque. J. Nucl. Med. 51, 862-865 (2010).
32. B. A. Blomberg, A. Thomassen, P. A. de Jong, M. G. E. Lam, A. C. P. Diederichsen, M. H. Olsen, H. Mickley, W. P. T. M. Mali, A. Alavi, P. F. Hoilund-Carlsen, Coronary fluorine-18-sodium fluoride uptake is increased in healthy adults with an unfavorable cardiovascular risk profile: results from the CAMONA study. Nucl. Med. Commun. 38,
1007-1014 (2017).
33. Y. Ishiwata, T. Kaneta, S. Nawata, A. Hino-Shishikura, K. Yoshida, T. Inoue, Quantification of temporal changes in calcium score in active atherosclerotic plaque in major vessels by (18)F-sodium fluoride PET/CT. Eur. J. Nucl.
Med. Mol. Imaging. 44, 1529-1537 (2017).
34. T. Derlin, T. Janssen, J. Salamon, S. Veldhoen, J. D. Busch, G. Schon, J. Herrmann, F. O. Henes, P. Bannas, G. Adam, Age-related differences in the activity of arterial mineral deposition and regional bone metabolism: a 18F-sodium fluoride positron emission tomography study. Osteoporos. Int. 26, 199-207 (2015).
35. T. Kitagawa, H. Yamamoto, S. Toshimitsu, K. Sasaki, A. Senoo, Y. Kubo, F. Tatsugami, K. Awai, Y. Hirokawa, Y. Kihara, (18)F-sodium fluoride positron emission tomography for molecular imaging of coronary atherosclerosis based on computed tomography analysis. Atherosclerosis. 263, 385-392 (2017).
36. T. Derlin, C. Wisotzki, U. Richter, I. Apostolova, P. Bannas, C. Weber, J. Mester, S. Klutmann, In vivo imaging of mineral deposition in carotid plaque using 18F-sodium fluoride PET/CT: correlation with atherogenic risk factors. J. Nucl. Med. 52, 362-368 (2011).
37. J. M. Lee, J. I. Bang, B. K. Koo, D. Hwang, J. Park, J. Zhang, T. Yaliang, M. Suh, J. C. Paeng, Y. Shiono, T. Kubo, T. Akasaka, Clinical Relevance of (18)F-Sodium Fluoride Positron-Emission Tomography in Noninvasive Identification of High-Risk Plaque in Patients With Coronary Artery Disease. Circ. Cardiovasc. Imaging. 10,
10.1161/CIRCIMAGING.117.006704 (2017).
38. A. Rominger, T. Saam, S. Wolpers, C. C. Cyran, M. Schmidt, S. Foerster, K. Nikolaou, M. F. Reiser, P. Bartenstein, M. Hacker, 18F-FDG PET/CT identifies patients at risk for future vascular events in an otherwise asymptomatic cohort with neoplastic disease. J. Nucl. Med. 50, 1611-1620 (2009).
252
39. M. R. Dweck, M. W. Chow, N. V. Joshi, M. C. Williams, C. Jones, A. M. Fletcher, H. Richardson, A. White, G. McKillop, E. J. van Beek, N. A. Boon, J. H. Rudd, D. E. Newby, Coronary arterial 18F-sodium fluoride uptake: a novel marker of plaque biology. J. Am. Coll. Cardiol. 59, 1539-1548 (2012).
40. N. V. Joshi, A. T. Vesey, M. C. Williams, A. S. Shah, P. A. Calvert, F. H. Craighead, S. E. Yeoh, W. Wallace, D. Salter, A. M. Fletcher, E. J. van Beek, A. D. Flapan, N. G. Uren, M. W. Behan, N. L. Cruden, N. L. Mills, K. A. Fox, J. H. Rudd, M. R. Dweck, D. E. Newby, 18F-fluoride positron emission tomography for identification of ruptured and high-risk coronary atherosclerotic plaques: a prospective clinical trial. Lancet. 383, 705-713 (2014).
41. J. H. Rudd, E. A. Warburton, T. D. Fryer, H. A. Jones, J. C. Clark, N. Antoun, P. Johnstrom, A. P. Davenport, P. J. Kirkpatrick, B. N. Arch, J. D. Pickard, P. L. Weissberg, Imaging atherosclerotic plaque inflammation with [18F]-fluorodeoxyglucose positron emission tomography. Circulation. 105, 2708-2711 (2002).
42. X. Bai, Q. Dinghong, D. Miao, D. Goltzman, A. C. Karaplis, Klotho ablation converts the biochemical and skeletal alterations in FGF23 (R176Q) transgenic mice to a Klotho-deficient phenotype. Am. J. Physiol. Endocrinol. Metab.
296, E79-88 (2009).
43. I. Alesutan, M. Feger, R. Tuffaha, T. Castor, K. Musculus, S. S. Buehling, C. L. Heine, M. Kuro-O, B. Pieske, K. Schmidt, A. Tomaschitz, W. Maerz, S. Pilz, A. Meinitzer, J. Voelkl, F. Lang, Augmentation of phosphate-induced osteo-/chondrogenic transformation of vascular smooth muscle cells by homoarginine. Cardiovasc. Res. 110,
408-418 (2016).
44. J. M. Hum, L. M. O'Bryan, A. K. Tatiparthi, T. A. Cass, E. L. Clinkenbeard, M. S. Cramer, M. Bhaskaran, R. L. Johnson, J. M. Wilson, R. C. Smith, K. E. White, Chronic Hyperphosphatemia and Vascular Calcification Are Reduced by Stable Delivery of Soluble Klotho. J. Am. Soc. Nephrol. 28, 1162-1174 (2017).
45. K. Nakamura, D. Miura, Y. Saito, K. Yunoki, Y. Koyama, M. Satoh, M. Kondo, K. Osawa, O. F. Hatipoglu, T. Miyoshi, M. Yoshida, H. Morita, H. Ito, Eicosapentaenoic acid prevents arterial calcification in klotho mutant mice.
PLoS One. 12, e0181009 (2017).
46. H. Tsujikawa, Y. Kurotaki, T. Fujimori, K. Fukuda, Y. Nabeshima, Klotho, a gene related to a syndrome resembling human premature aging, functions in a negative regulatory circuit of vitamin D endocrine system.
Mol. Endocrinol. 17, 2393-2403 (2003).
47. H. Olauson, K. Lindberg, R. Amin, T. Jia, A. Wernerson, G. Andersson, T. E. Larsson, Targeted deletion of klotho in kidney distal tubule disrupts mineral metabolism. J. Am. Soc. Nephrol. 23, 1641-1651 (2012).
48. K. Lindberg, R. Amin, O. W. Moe, M. C. Hu, R. G. Erben, A. Ostman Wernerson, B. Lanske, H. Olauson, T. E. Larsson, The kidney is the principal organ mediating klotho effects. J. Am. Soc. Nephrol. 25, 2169-2175 (2014).
49. C. T. Esapa, F. M. Hannan, V. N. Babinsky, P. Potter, G. P. Thomas, P. I. Croucher, M. A. Brown, S. D. Brown, R. D. Cox, R. V. Thakker, N-ethyl-N-Nitrosourea (ENU) Induced Mutations within the Klotho Gene Lead to Ectopic Calcification and Reduced Lifespan in Mouse Models. PLoS One. 10, e0122650 (2015).
50. K. Morishita, A. Shirai, M. Kubota, Y. Katakura, Y. Nabeshima, K. Takeshige, T. Kamiya, The progression of aging in klotho mutant mice can be modified by dietary phosphorus and zinc. J. Nutr. 131, 3182-3188 (2001).
51. M. Kawai, S. Kinoshita, K. Ozono, T. Michigami, Inorganic Phosphate Activates the AKT/mTORC1 Pathway and Shortens the Life Span of an alphaKlotho-Deficient Model. J. Am. Soc. Nephrol. 27, 2810-2824 (2016).
253
52. N. Ide, H. Olauson, T. Sato, M. J. Densmore, H. Wang, J. I. Hanai, T. E. Larsson, B. Lanske, In vivo evidence for a limited role of proximal tubular Klotho in renal phosphate handling. Kidney Int. 90, 348-362 (2016).
53. M. C. Hu, M. Shi, J. Zhang, T. Addo, H. J. Cho, S. L. Barker, P. Ravikumar, N. Gillings, A. Bian, S. S. Sidhu, M. Kuro-O, O. W. Moe, Renal Production, Uptake, and Handling of Circulating alphaKlotho. J. Am. Soc. Nephrol. 27,
79-90 (2016).
54. M. C. Hu, M. Shi, J. Zhang, J. Pastor, T. Nakatani, B. Lanske, M. S. Razzaque, K. P. Rosenblatt, M. G. Baum, M. Kuro-o, O. W. Moe, Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule. FASEB J. 24, 3438-3450 (2010).
55. H. Kurosu, Y. Ogawa, M. Miyoshi, M. Yamamoto, A. Nandi, K. P. Rosenblatt, M. G. Baum, S. Schiavi, M. C. Hu, O. W. Moe, M. Kuro-o, Regulation of fibroblast growth factor-23 signaling by klotho. J. Biol. Chem. 281,
6120-6123 (2006).
56. E. G. Farrow, S. I. Davis, L. J. Summers, K. E. White, Initial FGF23-mediated signaling occurs in the distal convoluted tubule. J. Am. Soc. Nephrol. 20, 955-960 (2009).
57. I. Urakawa, Y. Yamazaki, T. Shimada, K. Iijima, H. Hasegawa, K. Okawa, T. Fujita, S. Fukumoto, T. Yamashita, Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature. 444, 770-774 (2006).
58. R. Watanabe, N. Fujita, Y. Sato, T. Kobayashi, M. Morita, T. Oike, K. Miyamoto, M. Kuro-O, T. Michigami, S. Fukumoto, T. Tsuji, Y. Toyama, M. Nakamura, M. Matsumoto, T. Miyamoto, Enpp1 is an anti-aging factor that regulates Klotho under phosphate overload conditions. Sci. Rep. 7, 7786-017-07341-2 (2017).
59. T. Chen, H. Mao, C. Chen, L. Wu, N. Wang, X. Zhao, J. Qian, C. Xing, The Role and Mechanism of alpha-Klotho in the Calcification of Rat Aortic Vascular Smooth Muscle Cells. Biomed. Res. Int. 2015, 194362 (2015).
60. L. Cheng, L. Zhang, J. Yang, L. Hao, Activation of peroxisome proliferator-activated receptor gamma inhibits vascular calcification by upregulating Klotho. Exp. Ther. Med. 13, 467-474 (2017).
254
Supplemental Figure 1. Small animal CT scans of 10-week-old Klotho-/- and WT mice after circulation of 18F-NaF
as depicted in Figure 2. (A) Median sagittal cross-section of a 10-week-old Klotho-/- mouse, showing no densities in the aorta. Note the pronounced kyphosis. (B) Median sagittal cross-section of a 10-week-old WT mouse,
showing a pattern similar to (A). (C) Prevertebral coronal cross-section (A), showing no densities in the aorta. (D)
Prevertebral coronal cross-section of (B). (E) Transverse cross-section at the thoracic level of (A), with a similar
255
Supplemental Figure 2. Small animal PET scans of 10-week-old Klotho-/- and WT mice after circulation of 18
F-NaF as depicted in Figure 2. (A) Median sagittal cross-section of a 10-week-old Klotho-/- mouse, showing tracer uptake in skeletal structures and tracer presence in the bladder, but no discernible signal from the aorta. Note the pronounced kyphosis. (B) Median sagittal cross-section of a 10-week-old WT mouse, showing a pattern
similar to (A). (C) Prevertebral coronal cross-section (A), showing tracer uptake in skeletal structures and tracer
presence in the bladder, but no discernible signal from the aorta. (D) Prevertebral coronal cross-section of (B),
showing tracer uptake in skeletal structures and tracer presence in a kidney and in the bladder, but no discernible signal from the aorta. (E) Transverse cross-section at the thoracic level of (A), with a similar pattern. (F) Transverse
256
Supplemental Figure 3. Small animal PET-CT scans of 7-week-old Klotho-/- mouse after circulation of 18F-NaF.
(A) Transverse cross-section at the thoracic level of a 10-week-old Klotho-/- mouse, showing tracer uptake in skeletal structures, but no discernible signal from the aorta. (B) Median sagittal cross-section of (A). (C)
