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αKlotho Regulates Age-Associated Vascular Calcification and Lifespan in Zebrafish


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The hormone αKlotho regulates lifespan in mice, as knockouts die early of what appears to be accelerated aging due to hyperphosphatemia and soft tissue calcification. In contrast, the overexpression of αKlotho increases lifespan. Given the severe mouse phenotype, we generated zebrafish mutants for αklotho as well as its binding partner fibroblast growth factor-23 (fgf23). Both mutations cause shortened lifespan in zebrafish, with abrupt onset of behavioral and degenerative physical changes at around 5 months of age. There is a calcification of vessels throughout the body, most dramatically in the outflow tract of the heart, the bulbus arteriosus (BA). This calcification is associated with an ectopic activation of osteoclast differentiation pathways. These findings suggest that the gradual loss of αKlotho found in normal aging might give rise to ectopic calcification.
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aKlotho Regulates Age-Associated Vascular
Calcification and Lifespan in Zebrafish
Graphical Abstract
dZebrafish aklotho mutants display reduced lifespans
dThe aklotho phenotype occurs later in zebrafish than in mice
dZebrafish aklotho mutants display adult-onset vascular
dCalcification coincides with an increase in osteoclast
differentiation pathways
Ajeet Pratap Singh, Maria X. Sosa,
Jian Fang, ..., Samuel M. Cadena,
Mark C. Fishman, David J. Glass
In Brief
aKlotho regulates mineral homeostasis
and affects lifespans in mammals. Singh
et al. show that a loss of aklotho in
zebrafish results in reduced lifespans and
vascular calcification in the outflow tract
of the heart. Vascular calcification is
associated with an upregulation of bone
remodeling pathways and osteoclast
Singh et al., 2019, Cell Reports 28, 2767–2776
September 10, 2019 ª2019 Novartis Institutes for Biomedical Research.
Cell Reports
aKlotho Regulates Age-Associated
Vascular Calcification and Lifespan
in Zebrafish
Ajeet Pratap Singh,
Maria X. Sosa,
Jian Fang,
Shiva Kumar Shanmukhappa,
Alexis Hubaud,
Caroline H. Fawcett,
Gregory J. Molind,
Tingwei Tsai,
Paola Capodieci,
Kristie Wetzel,
Ellen Sanchez,
Guangliang Wang,
Matthew Coble,
Wenlong Tang,
Samuel M. Cadena,
Mark C. Fishman,
and David J. Glass
Zebrafish Group, Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, 181 Massachusetts Avenue, Cambridge,
MA 02139, USA
Preclinical Safety, Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA
DAx/Discovery and Translational Pharmacology, Novartis Institutes for Biomedical Research, 181 Massachusetts Avenue, Cambridge,
MA 02139, USA
Harvard Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Ave, Cambridge, MA 02138, USA
Age-Related Disorders Group, Chemical Biology and Therapeutics, Novartis Institutes for Biomedical Research, 181 Massachusetts
Avenue, Cambridge, MA 02139, USA
Lead Contact
The hormone aKlotho regulates lifespan in mice, as
knockouts die early of what appears to be acceler-
ated aging due to hyperphosphatemia and soft tis-
sue calcification. In contrast, the overexpression of
aKlotho increases lifespan. Given the severe mouse
phenotype, we generated zebrafish mutants for
aklotho as well as its binding partner fibroblast
growth factor-23 (fgf23). Both mutations cause
shortened lifespan in zebrafish, with abrupt onset
of behavioral and degenerative physical changes at
around 5 months of age. There is a calcification of
vessels throughout the body, most dramatically in
the outflow tract of the heart, the bulbus arteriosus
(BA). This calcification is associated with an ectopic
activation of osteoclast differentiation pathways.
These findings suggest that the gradual loss of
aKlotho found in normal aging might give rise to
ectopic calcification.
Systemic factors that regulate aging are of interest due to their
potential as novel drug targets in preventing or slowing down
age-related decline in animal health. aKlotho, a molecular scaf-
fold protein, is considered an anti-aging hormone that regulates
mineral homeostasis in mammals (Chen et al., 2018; Kuro-o,
2013; Kuro-o et al., 1997; Kurosu et al., 2006, 2005; Lindberg
et al., 2014; Shimada et al., 2004). It is one of the few systemic
secreted factors whose loss is sufficient to induce premature
morbidity and mortality that resembles accelerated aging
(Kuro-o et al., 1997), and its overexpression extends lifespans
(Kurosu et al., 2005). It is therefore of interest to understand
the cellular and molecular mechanisms by which aKlotho regu-
lates the aging process.
The mouse knockout models of aklotho are difficult to study;
animals die by 812 weeks of age and are difficult to maintain
´ndez et al., 2018; Kuro-o et al., 1997; unpublished data).
aklotho loss-of-function mice develop normally until about 3 or
4 weeks of age and then begin to display age-related conditions,
including ectopic calcification, arteriosclerosis, osteoporosis,
and reduced lifespans (Kuro-o et al., 1997). It was suggested
that the extended lifespan in mice overexpressing aklotho is
due to a suppression of insulin and the insulin-like growth fac-
tor-1 signaling (Kurosu et al., 2005), although it is likely that other
pathways are involved. Recently, it was shown that an increase
in autophagy levels could delay or prevent early mortality in
aklotho mutant mice (Ferna
´ndez et al., 2018). aKlotho acts as a
co-receptor for fibroblast growth factor-23 (FGF23) (Urakawa
et al., 2006). Knockouts of fgf23 have a similar accelerated aging
phenotype to that of aklotho and show a perturbation in vitamin
D metabolism (Shimada et al., 2004). aKlotho can also be
released from the cell surface. In such settings, it can still heter-
odimerize with FGF23, functioning as a co-ligand in forming a
high-affinity activator of FGF receptor signaling (Erben, 2018).
Fish diverged from tetrapods approximately 400 million years
ago (Daeschler et al., 2006; Romer, 1967). There are funda-
mental differences in physiology between fish and terrestrial ver-
tebrates owing to unique demands of aquatic versus terrestrial
environments. In terms of renal function and mineral and fluid ho-
meostasis, they have evolved different physiologies. For
example, renal function in freshwater fish serves partly to prevent
overhydration, whereas in mammals, it is designed to prevent
dehydration. We therefore examined if the roles of aKlotho, a
renal hormone, would be conserved in aging and mineral ho-
meostasis. The zebrafish also provide a tractable system for
measuring certain behaviors, including physical activity. The
zebrafish genome encodes one aklotho and one fgf23 (Mangos
et al., 2012; Sugano and Lardelli, 2011). Consistent with
Cell Reports 28, 2767–2776, September 10, 2019 ª2019 Novartis Institutes for Biomedical Research. 2767
This is an open access article under the CC BY-NC-ND license (
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2768 Cell Reports 28, 2767–2776, September 10, 2019
mammalian studies, zebrafish aklotho expression is detected in
multiple organs, including adult kidneys (Mangos et al., 2012).
Zebrafish fgf23 is expressed in corpuscles of Stannius, a
teleost-specific, kidney-associated endocrine gland involved in
mineral homeostasis (Elizondo et al., 2010; Mangos et al.,
2012). We generated knockouts of aklotho and fgf23 in zebrafish
in order to understand the mechanistic link between aging and
the aKlotho/FGF23 pathway.
Zebrafish Mutants in Both aklotho and fgf23 Have Early-
Onset Mortality
We targeted the aklotho gene using the CRISPR/Cas9 method
(Irion et al., 2014), generating mutations in two background ze-
brafish strains, T
u and AB, and identified aklotho alleles carrying
frameshift mutations and early stop codons (Table S1). We also
targeted fgf23 because the function of aKlotho in mammals de-
pends in large part upon its binding to FGF receptors and recruit-
ing FGF23 to activate FGF signaling (Chen et al., 2018; Kurosu
et al., 2006).
We find that aklotho
and fgf23
mutant zebrafish display
essentially indistinguishable phenotypes (Figure 1). As adults of
about 5 months of age, they develop emaciated bodies, tattered
fins, and an opaque overgrowth on the eyes (aklotho
Figures 1A, 1B, 1D, and 1E; fgf23
mutant; Figures 1C, 1F, and
S1A–S1F). In addition, female aklotho
and fgf23
displayed protruding eyes (Figures S1G–S1L).
The onset of mortality in mutant colonies began around
45 months post-fertilization (mpf; survival curves in Figures
1G–1J; p < 0.001), compared to wild-type strains of zebrafish,
which live for 35 years (Carneiro et al., 2016; Gerhard et al.,
2002). Both aklotho
and fgf23
mutant fish appear morpho-
logically comparable to wild-type siblings at 23 mpf (Figures
S1M–S1P) and are fertile as young adults, allowing us to breed
homozygotes. Among adult progeny (3 mpf) obtained by
inbreeding aklotho heterozygotes, we recovered homozygous
mutants in ratios consistent with Mendelian inheritance. Among
281 siblings raised together until adulthood, we obtained 70
wild-type siblings (25%), 130 heterozygous siblings (46%), and
79 homozygous aklotho mutants (28%). Among 241 adult zebra-
fish obtained from breeding parents fgf23 heterozygotes, we ob-
tained 66 wild-type siblings (27%), 128 heterozygous siblings
(52%), and 50 homozygous mutants (20%). This indicates that
and fgf23
mutants have no survival disadvantage
until adulthood, even when raised with wild-type siblings.
In order to probe the timing of more subtle aspects of physical
decline, we analyzed the behavior of the aklotho
and fgf23
zebrafish in two settings: a circular arena (Figure 2K) and an
arena resembling their home tank (Figure 2L). Although sponta-
neous behavior in zebrafish is intrinsically variable, aklotho
and fgf23
mutants demonstrated reduced activity in both
behavioral paradigms at 5 and 6 mpf, corroborating the physical
evidence of decline at this time (Figures 1M–1P).
We conclude from these data that there is an adult-onset, age-
related decline in the body condition in both aklotho
mutants. Although it is difficult to align developmental
frameworks between species, the adult-onset decline in both
and fgf23
mutant zebrafish appears to be propor-
tionally later than described for the mouse aklotho mutants
(Kuro-o et al., 1997; unpublished data).
Vascular Calcification and Inflammation across Organs
in aklotho
Mutant Zebrafish
In order to understand the phenotype at the cell and tissue level,
we performed comprehensive histopathological analysis by H&E
staining on sections of 5-month-old wild-type and aklotho
mutant fish (Figure 2; N = 3 males each). In aklotho
fish, there
was widespread calcification and inflammation. Within the integ-
ument of aklotho
fish, there was a reduction in the number of
mucosal cells and necrosis in areas of the epidermis and dermis,
with a mineralization of the dermal vasculature (Figure 2A).
Furthermore, in aklotho
mutants, there was calcification of
medium- to small-size blood vessels in the skeletal muscles (ar-
row in Figure 2B), accompanied by a degeneration and fibrosis of
adjacent skeletal muscles with immune cell infiltration (Fig-
ure 2B). Mineralization was often in a concentric pattern in the
affected areas. The gill arch demonstrated bone overgrowth (hy-
perostosis) with chondrodysplasia of the gill arch (Figure 2C) and
a loss of normal architecture of filaments and lamellae due to
blunting, fusion, and necrosis of the lamellae epithelium, along
with immune cell infiltration.
In the aklotho
zebrafish, calcification was particularly strik-
ing within the walls of the bulbus arteriosus (BA) (the outflow tract
of the heart) (Figures 2D and 2E). The BA is composed of smooth
muscles and is lined by the endothelium, and its elasticity is
believed to buffer pulsatile bloodflow to the thin-walled capillaries
of the gills (Farrell, 1979; Grimes and Kirby, 2009). To validate
calcification in the BA, we used alizarinred, a stain for calcification
(Walker and Kimmel, 2007). The BA in aklotho
mutants is
prominently stained with alizarin red in contrast to wild-type ani-
mals (Figure 2D; insets), confirming calcification. Calcification
was also observed in the bile duct of the livers of aklotho
(arrow in Figure 2F). The kidneys of aklotho
mutants appeared
comparable to the wild-type controls (Figure 2G). Within the skel-
etal system, there were multifocal areas of hyperostosis and
Figure 1. Zebrafish aklotho and fgf23 Mutants
(A–F) Body condition of aklotho and fgf23 mutant males at 5 mpf: (A) T
u wild-type strain, (B) aklotho (kl
), and (C) fgf23 (fgf23
) mutant in T
u background; (D) AB
wild-type strain, (E) aklotho (kl
), and (F) fgf23 (fgf23
) mutant in AB background.
(G–J) Survival curves for (G) aklotho (n = 36 background controls, 32 mutants; p < 0.0001) and (H) fgf23 mutants (n = 14 wild-type siblings, 14 mutants; p < 0.0001)
in T
u background and for (I) aklotho (n = 24 wild-type siblings, 21 mutants; p = 0.0001) and (J) fgf23 mutants (n = 60 background contro ls, 68 mutants; p < 0.0001)
in AB background. Log-rank (Mantel-Cox) test for statistical analysis on survival curves in GraphP ad Prism.
(K and L) Analysis of speed (cm) in the (K) circular arena and (L) home-tank arena. Age (m; mpf) on x axis; n = number of fish.
(M and N) aklotho mutants and wild-type controls in (M) circular and (N) home-tank arena.
(O and P) fgf23 mutants and wild-type controls in (O) circular and (P) home-tank arena. Statistical analysis using unpaired t test in GraphPad Prism.
See also Figure S1 and Table S1.
Cell Reports 28, 2767–2776, September 10, 2019 2769
chondrodysplasia. These changes were prominent in the caudal
region (Figure S2A). Frequently, regions of bone overgrowth
were accompanied by areas of dystrophic calcification, connec-
tive tissue proliferation, and immune cell infiltration (Figure S2A).
Vascular calcification was the most prominent phenotype. In
fact, unprocessed and unstained BAs appear opaque white in
(Figure 2H), indicating severe calcification. fgf23
mutants phenocopy aklotho
mutants—the BAs in fgf23
mutants are prominently stained with alizarin red, in contrast to
wild-type animals (Figure S2B). It has been shown that calcifica-
tion in zebrafish is accompanied by an increase in osteoclast ac-
tivity (Apschner et al., 2014). Consistent with this, we observe
strong Tartrate-resistant acid phosphatase (TRAP) staining in
the outflow tract of the aklotho
mutant hearts (Figures 2I
and 2J), indicating the presence of osteoclasts in the BA.
Regulation of Osteogenesis in BAs of aklotho
In order to understand the molecular mechanisms underlying
ectopic calcification in aklotho
mutants, we performed an
Figure 2. Vascular Calcification and Inflam-
mation in aklotho Mutants
H&E staining on paraffin sections from 5-month-old
wild-type control (T
u) and aklotho (kl
) males.
Shown are (A) skin (arrows indicate mucous cells in
wild type); (B) muscle (arrows indicate vascular
calcification); (C) gills; (D) heart (arrow indicates
calcification in the BA); (D0and D00) alizarin red-
stained whole-mount hearts (arrows indicate the
BA); (E) BA (arrow indicates calcification); (F) liver
(arrows indicate bile-duct); and (G) kidney (arrows
indicate glomeruli). (H) Bright-field images of 5-mpf
wild-type (left) and kl
(right) hearts. TRAP staining
on (I) whole mount and (J) cryosection of 5-mpf
See also Figure S2.
RNA sequencing (RNA-seq) analysis of the
kidney, heart (including BA), and gills at
3 mpf, when mutantsappeared comparable
phenotypically to wild-type siblings, and at
mutants became
phenotypically distinct from wild-type sib-
lings (n = 8 animalsper genotype and condi-
tion; 4 malesand 4 females). In the wild-type
siblings, aklotho expression is highest in the
kidneys (Figure 3A[3]). As expected, a sig-
nificant downregulation of aklotho expres-
sion is observed in aklotho
mutants at
both 3 and 5 mpf (Figure 3A[1]). In wild-
type fish, fgf23 expression is detected in
the kidneys and, surprisingly, in the gills
(Figure 3B[2]). In aklotho
mutant gills,
fgf23 is the most significantly upregulated
gene at 3 mpf, indicating a dysregulation
of the aKlotho/FGF23 axis (Figure 3B[2]).
At 3 mpf, there were only modest
changes from the wild-type siblings in
the aklotho
patterns of gene expression in the kidney, heart,
and gills (Figures 3C–3G; Table S2) and no statistically significant
change at the pathway level (Figures 4A and S3A; Tables S3 and
S4). At 5 mpf, in the kidneys, which were histologically indistin-
guishable from the wild type (Figure 2G), the main noticeable
change was an upregulation of genes involved in the metabolism
of the heme (Figure 4A). In the gills, at 5 mpf, a time of wide-
spread disorganization based on histopathology, there was an
increase in the expression of genes of the extracellular matrix
(ECM) organization pathway (Figure 4A). There is minimal over-
lap between genes differentially regulated at 3 and 5 mpf in an
organ (Figures S3B and S3C).
Next, we analyzed tissue samples for pathway level changes
in the transcriptome by a hypergeometric test (Figure 4A; Table
S3) and gene set enrichment analysis by a weighted Kolmo-
gorov-Smirnov test (Figure S3A; Table S4). Both tests revealed
that at 5 mpf, multiple pathways were dysregulated in
mutant tissues. In heart samples collected for this
analysis, two out of eight hearts showed visual signs of calcifica-
tion in the BA at 3 mpf. All eight hearts displayed calcification in
2770 Cell Reports 28, 2767–2776, September 10, 2019
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Cell Reports 28, 2767–2776, September 10, 2019 2771
the BA at 5 mpf, indicating an adult-onset progressive vascular
calcification. At 3 mpf, we did not observe statistically significant
changes in the pathway level analysis (Figure 4A). However, at
the individual gene level, aklotho mutant hearts displayed an up-
regulation in genes involved in bone formation and remodeling,
such as matrix metallopeptidase-9 (mmp9), mmp13, and
osteopontin (secreted phosphoprotein 1/spp1)(Figures 4B, 4C,
4E, and S4A) (Page-McCaw et al., 2007; Standal et al., 2004).
In mammals, mmp9 and mmp13 are required for the transition
from cartilage into bone (Page-McCaw et al., 2007; Stickens
et al., 2004). SPP1 is known to be an inhibitor of calcification
(Standal et al., 2004). At 5 mpf, multiple pathways involved in
bone formation, bone remodeling, osteoclast activity, ECM re-
modeling, and inflammation are upregulated in aklotho
mutant hearts (Figure 4). spp1 is the most significantly upregu-
lated gene in aklotho
mutant hearts at 5 mpf (Figure 4E).
We validated spp1 expression using qPCR on dissected BAs
and observed an 600-fold enrichment in spp1 transcript in
mutants at 5 mpf (Figure S4C).
entpd5a (ectonucleoside triphosphate diphosphohydrolase
5a), an osteoblast marker in zebrafish (Huitema et al., 2012), is
also upregulated in aklotho
mutant hearts at this stage (Fig-
ure 4D). However, we do not observe an upregulation of the con-
ventional markers of the osteoblast lineage in aklotho
hearts, including runx2,sp7,col10a1, and col1a2 (Huitema et al.,
2012; Vijayakumar et al., 2013; Yang et al., 2011). The qPCR
analysis for runx2a and runx2b on dissected BAs showed a
modest increase in runx2b levels and no change in runx2a levels
at 5 mpf (Figure S4C). Recent studies have identified a role for
osteolectin/clec11a and integrin-a11/itga11 signaling in osteo-
blast differentiation and the maintenance of adult skeletal bone
mass (Shen et al., 2019; Yue et al., 2016); both clec11a and
itga11a are upregulated in aklotho
mutant hearts at 5 mpf
(Figure S4B). Thus, our analysis reveals an upregulation of genes
involved in ECM remodeling and bone formation that could
explain the observed ectopic vascular calcification.
Calcification is remodeled and counter-regulated by the activ-
ity of hematopoietic stem cell-derived osteoclasts. The RANK/
RANKL/OPG pathway is required for osteoclast differentiation
from hematopoietic lineage (Edwards and Mundy, 2011; Novack
and Teitelbaum, 2008; Teitelbaum and Ross, 2003). In aklotho
mutant hearts, key members of this pathway (Figure S4D) and
osteoclast-enriched enzymes such as ctsk (encoding Cathepsin
K) and acp5 (encoding TRAP) are upregulated at 5 mpf (Figures
4F and 4G), suggesting an increase in osteoclast activity in the
ectopically mineralized region. In order to localize the ongoing
transcriptional activity in the heart, we performed RNAscope
analysis using spp1 probe; this analysis revealed a highly local-
ized spp1 expression in the BAs of aklotho
(Figure 4H).
The aKlotho/FGF23 pathway appears to play a role in the ag-
ing of zebrafish. Both aklotho
and fgf23
display early-onset morbidity, beginning at about 4 or 5 months
of age, accompanied by spinal deformities, loss of fin integrity,
and widespread ectopic calcification, especially of the outflow
tract of the heart. Soft-tissue calcification increases with
advancing age in humans, and vascular calcification is associ-
ated with an increase in atherosclerosis and cardiovascular
mortality (Leopold, 2013; McClelland et al., 2006; Shaw
et al., 2015; Thompson et al., 2013). The mechanisms that
lead to soft-tissue calcification remain poorly understood. It
has been suggested (Hortells et al., 2017, 2018; Persy and
D’Haese, 2009; Pillai et al., 2017), but debated (O’Neill and
Adams, 2014), that cardiovascular calcification actually re-
flects the osteogenic cell fate change of vascular smooth mus-
cle. Here, we find that in the absence of aKlotho/FGF23,
vascular tissue in the BA changes its pattern of gene expres-
sion to resemble that of bone: a pro-osteogenic reorganization
of the ECM may promote the observed calcification phenotype
in smooth muscle cells of the BA. This leads to a surge in anti-
osteogenic mechanisms, including a local differentiation of os-
teoclasts. The effect of aKlotho/FGF23 signaling on vascular
calcification is likely non-cell autonomous. aklotho is primarily
expressed in kidneys, whereas fgf23 is expressed in kidneys
and gills. Interestingly, a homozygous missense mutation of
aKlotho was reported in a human; this mutation resulted in se-
vere tumoral calcinosis including ectopic calcifications, indi-
cating the zebrafish model is predictive of the human condition
(Ichikawa et al., 2017).
The BA is an elastic, valveless cardiac outflow tract in tele-
osts that is believed to act as a windkessel to protect the deli-
cate gill vasculature from large variations of pressure generated
by the ventricle (Farrell, 1979; Grimes and Kirby, 2009; Maldanis
et al., 2016). The calcification of the BA may compromise its
elasticity, leading to large fluctuations in blood pressure in the
gill vasculature and a consequent loss of the gill architecture.
Thus, vascular calcification may be the primary cause of the
onset of morbidity in aklotho
and fgf23
zebrafish. This
has striking parallels with chronic kidney disease in humans:
vascular calcification is considered to contribute to mortality
in patients with chronic kidney disease (Go et al., 2004; Mizobu-
chi et al., 2009), and aKlotho treatment has been shown to be
helpful for the treatment of kidney disease in preclinical models
(Doi et al., 2011; Hum et al., 2017; Shi et al., 2016). Taken
together, the data suggest that one potential consequence of
age-related decline in aKlotho, as has been reported in humans,
could be inappropriate osteogenesis often observed in older
vascular smooth muscles, along with the decline in cardiac
Figure 3. RNA-Seq Analysis of Kidney, Heart, and Gills in aklotho Mutants
(A) aklotho expression in (A1) kidney, (A2) heart, and (A3) gills of wild-type siblings (red) and aklotho mutants (blue); 4 males and 4 females for each set.
(B) fgf23 expression in (B1) kidney and (B2) gills of wild-type siblings (red) and aklotho mutants (blue); no expression detected in heart.
(C–E1) Volcano plots showing differentially expressed genes at 3 and 5 mpf in (C and C1) kidney, (D and D1) heart, and (E and E1) gills.
(F and G) Euler diagram, obtained by R package eulerr, showing the number of (F) upregulated and (G) downregulated genes by aklotho mutation in the kidney,
heart, gills, and their overlaps at 3 and 5 mpf. The area of each disjointed shape is proportional to the number of its elements as marked.
See also Figures S3 and S4 and Table S2.
2772 Cell Reports 28, 2767–2776, September 10, 2019
elasticity and function (de Carvalho Filho et al., 1996; Fleg and
Strait, 2012; Hamczyk et al., 2018; McClelland et al., 2006;
Semba et al., 2011a, 2011b; Shaw et al., 2015). Such findings
could suggest particular therapeutic readouts of aKlotho sup-
plementation in aged humans where aKlotho levels are low.
In vertebrates, calcium phosphate must be carefully regulated
to avoid ectopic precipitation of calcium-phosphate crystals. It is
suggested that a shift from a calcium carbonate-based skeleton
in invertebrates to a calcium phosphate-based skeleton in verte-
brates necessitated the evolution of mechanisms to regulate
Figure 4. Pathway Enrichment Analysis of Differentially Upregulated Genes
(A) Gene set enrichment analysis comparing upregulated pathways by aklotho mutation in the kidney, heart, and gills. Each row is a pathway, annotated on the
right-hand side, and each column corresponds to the significance of the enrichment analysis for each tissue and adult stage (m3, 3 mpf; m5, 5 mpf). The colors
from white to red represent the negative log
adjusted p value from low to high. Only pathways that were enriched significantly (adjusted p value < 0.05) in at least
one tissue were included.
(B–G) Box plots showing select examples of genes upregulated at 5 mpf in aklotho mutant hearts (blue, kl) compared to wild-type (WT) siblings (red). Shown are
(B) mmp9 (matrix metallopeptidase 9), (C) mmp13a (matrix metallopeptidase 13a), (D) entpd5a (ectonucleoside triphosphate diphosphohydrolase 5a), (E) spp1
(secreted phosphoprotein 1), (F) ctsk (cathepsin K), and (G) acp5a (acid phosphatase 5a, tartrate resistant). Age: 3 and 5 mpf.
(H) RNAscope for spp1: (H1 and H2) WT control; (H3 and H4) aklotho (kl
) mutant hearts stained with DAPI (blue), and spp1 RNAscope probe (red). White lines
outline the BA (arrow) and the blood vessel leading to gills.
See also Figure S4 and Tables S3 and S4.
Cell Reports 28, 2767–2776, September 10, 2019 2773
calcium phosphate homeostasis (Kuro-o and Moe, 2017). In
mammals, aKlotho is highly expressed in the kidneys, whereas
bones are a primary source of FGF23 (Kuro-o et al., 1997; Rimi-
nucci et al., 2003). Interestingly, although aklotho is expressed in
zebrafish kidneys, fgf23 is expressed in corpuscles of Stannius,
a teleost-specific, kidney-associated gland involved in mineral
homeostasis (Elizondo et al., 2010; Mangos et al., 2012), and in
the gills of adults (this study). The gills play a major role in mineral
homeostasis—these are the primary site of calcium uptake in
adult fish (Evans et al., 2005; Flik et al., 1985, 1995; Liao et al.,
2007). In terrestrial vertebrates, the main source of calcium is
food. It is absorbed primarily in the intestines and kidneys, and
the bones serve as the major reservoir of the calcium. Taken
together, this suggests that although the focus organs may
have shifted during the evolutionary transition from aquatic to
terrestrial life, the function of the aKlotho/FGF23 pathway in
maintaining mineral homeostasis has been conserved.
Detailed methods are provided in the online version of this paper
and include the following:
BLifespan Analysis
BBehavioral Analysis
BRNA-Seq Sample Preparation
BRNA-Seq Data Analysis
Supplemental Information can be found online at
The study was funded by Novartis. We thank the Zebrafish Group, the Age-
Related Disorders Group, and the Chemical Biology and Therapeutics groups
for their enthusiastic support; T. Shavlakadze and A. Jaffe for discussions; T.
Scott and P. Stolyar for comments; O. Iartchouk and ASI for help with RNA-
seq; U. Plikat and NIBR Informatics for the RNA-seq analysis pipeline; G.
Zhang, Z. Li, C. Russ, and the NGS facility for NGS; N. Kirkpatrick and the mi-
croscopy facility; and K. Maloney, F. Vetrano-Olsen, H. Clark, M.-K. Paulina, L.
Ponczek, J. Tobin, V. Afere, A. Elliott, R. Brown, N. Jones-Bolduc, J. Fremont-
Rahl, E. Theve, and the laboratory animal services for zebrafish care.
Experiment Design, A.P.S., M.X.S., S.M.C., M.C.F., and D.J.G.; Experiment
Execution, A.P.S., M.X.S., A.H., C.H.F., G.J.M., E.S., G.W., and M.C.; Gener-
ation and Characterization of the Knockouts, A.P.S. and M.X.S.; Histology,
C.H.F., K.W., and P.C.; Analysis of Histopathology Slides, S.K.S. and A.P.S.;
Sample Collection and RNA Preparation, M.X.S., A.P.S., and E.S.; RNA-seq
Analysis, M.X.S. and J.F.; qPCR, A.H.; RNAscope, C.H.F.; Behavioral Data
Collection, G.J.M. and A.P.S.; Behavioral Data Analysis, T.T. and W.T.; Manu-
script Preparation, A.P.S. and D.J.G., with inputs from all authors.
This study was funded by Novartis AG. All authors, except for M.C.F., were
employees of Novartis at the time the study was conducted. Some authors,
including D.J.G., own Novartis stock. M.C.F. is on the BOD of Semma Thera-
peutics and Beam Therapeutics, the SAB of Tenaya Therapeutics, and serves
as advisor to MPM and Burrage Capital.
Received: March 28, 2019
Revised: July 2, 2019
Accepted: July 31, 2019
Published: September 10, 2019
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2776 Cell Reports 28, 2767–2776, September 10, 2019
Biological Samples
Zebrafish Kidney This study N/A
Zebrafish Gills This study N/A
Zebrafish Heart This study N/A
Chemicals, Peptides, and Recombinant Proteins
Alizarin Red-S Sigma-Aldrich Cat No. A5533-25G
Tartrate-resistant acid phosphatase Sigma-Aldrich Cat No. 387A-1KT
Modified Davidson’s fixative Fisher Scientific Cat No. 50-292-28
Hematoxylin (Gill’s Hematoxylin III) Poly Scientific R&D Corp Cat No. s211-32oz
Eosin Y Alcoholic Working Solution Poly Scientific R&D Corp Cat No. s2186-32oz
Critical Commercial Assays
RNAscopeProbe- Dr-spp1 Advanced Cell Diagnostics Cat No. 409501
RNALater Stabilization Solution ThermoFisher Cat No. AM7021
RNeasy Fibrous Tissue Mini kit QIAGEN Cat No. 74704
Ambion MEGAshortscript T7 Kit ThermoFisher Cat no. AM1354
RNAeasy kit QIAGEN Cat No. 74104
Cas9 Protein PNA Bio Cat No. CP01
Deposited Data
RNaseq data This study Sequence Read Archive, NCBI. BioProject
Accession: PRJNA556842
Experimental Models: Organisms/Strains
Zebrafish (Danio rerio), T
u strain N
usslein-Volhard lab RRID:ZIRC_ZL57
Zebrafish (Danio rerio), AB strain ZIRC RRID:ZIRC_ZL1
u strain) This study N/A
(AB strain) This study N/A
u strain) This study N/A
(AB strain) This study N/A
T7 universal primer for the DNA template for
Integrated DNA Technologies,
Inc., USA
klotho-specific primer for the DNA template for
Integrated DNA Technologies,
Inc., USA
Forward primer for genotyping aklotho mutant:
Integrated DNA Technologies,
Inc., USA
Reverse primer for genotyping aklotho mutant:
Integrated DNA Technologies,
Inc., USA
fgf23-specific primer for the DNA template for
Integrated DNA Technologies,
Inc., USA
Forward primer for genotyping fgf23 mutant:
Integrated DNA Technologies,
Inc., USA
(Continued on next page)
Cell Reports 28, 2767–2776.e1–e5, September 10, 2019 e1
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, David J.
Glass ( Zebrafish mutants generated in this study are available upon request, under a Materials Transfer
Agreement, to be negotiated with Novartis.
All animals were maintained and used for scientific research in accordance with the guidelines of The Institutional Animal Care and
Use Committee (IACUC) of the Novartis Institutes for BioMedical Research, Cambridge, USA. Zebrafish were housed in 3L tanks in a
recirculating Aquatic Habitats facility (Pentair, USA) on a 14:10 hour light:dark cycle at 28C. Larvae were fed Zeigler Larval Diet
AP100 Z3 to M3 (< 100 microns; Zeigler Bros., Inc, USA) from 5 day post-fertilization to 20 days post-fertilization. Juvenile fish
were fed Brine shrimps hatched from Premium Grade Brine Shrimp Eggs (Brine Shrimp Direct, USA) and TetraMin Tropical Flake
(Tetra, Germany) twice per day. Adults fish were fed a diet of GEMMA Micro 300 (Skretting France) once per day. Zebrafish were
anesthetized using 0.0168% buffered Tricaine-S (MS-222, Syndel).
u and AB strains of zebrafish were used as wild-type for the study.
Generation of Zebrafish Knockouts
Knockouts for klotho and fgf23 were generated by CRISPR/Cas9 method.
DNA template for in vitro transcription of CRISPR sgRNA was prepared by PCR using gene-specific primer and T7 universal primer:
klotho-specific primer:
fgf23-specific primer:
For sgRNA generation, PCR product was purified and in vitro transcription was performed using the Ambion MEGAshortscript T7
Kit (cat no. AM1354). sgRNA was purified using RNAeasy kit (QIAGEN; cat no. 74104), and diluted to 500 ng/ml. Equal volume of pu-
rified sgRNA and Cas9 protein (500 ng/ml; PNA Bio – CP01) was co-injected into single-cell stage zebrafish embryos. Injected fertil-
ized embryos were raised to adulthood (F
). F
adults were crossed to wild-type zebrafish to identify F
generation by NGS. Animals
carrying frameshift mutation were identified and propagated for the purpose of this study. For genotyping, DNA was isolated from fin-
clips (Meeker et al., 2007) for PCR and next-generation sequencing (NGS).
sgRNA target for klotho: GGCTGGAGTAATTCGGTTATGG; primers for NGS (forward: CGGCACCGCTGCATATTCAGTGG; and
Reverse primer for genotyping fgf23 mutant:
Integrated DNA Technologies,
Inc., USA
Software and Algorithms
Exon Quantification Pipeline Schuierer and Roma, 2016 N/A
MultiQC Ewels et. al, 2016
DESeq2 Love et al., 2014
apeglm Zhu et al., 2019
msigdbr msigdbr package
ClusterProfiler Yu et al., 2012
e2 Cell Reports 28, 2767–2776.e1–e5, September 10, 2019
Lifespan Analysis
In order to analyze the lifespan of control and mutant lines, survival analysis based on humane end-points was performed using
GraphPad Prism 7. Parameters for humane end-points were determined in consultation with institutional veterinarian services. An-
imals displaying the following signs were euthanized: Signs of tissue degeneration and tumors, inability or unwillingness to swim,
inability to maintain balance, abnormal swelling or tumors, severe eye protrusion, gross abnormalities in body shape, posture or spi-
nal deformities that affect animal’s ability to swim or eat.
Behavioral Analysis
For the behavioral assay performed in the circular arena, six fish of matching size and age were used in each assay. Controls and
mutants were assayed in parallel. Experiments were conducted at a similar time of day and monitored by video without human pres-
ence. Behavioral rooms had room temperature and light cycles consistent with the main facility. The circular arena consisted of the
following features: acrylic tank; outside diameter = 50.8cm; inside diameter = 48.9cm; tank height = 20.3cm height; open top (Plastic
Supply, Inc., USA). The tanks were filled to a depth of 4.4cm (9 L total volume) with zebrafish system water fed directly from the main
zebrafish housing unit to ensure all water parameters were identical to the housing conditions. The circular arenas were coated on the
outside (I00810, Frosted Glass Finish, Krylon, USA) to prevent the fish from being able to see outside of the arenas without compro-
mising the transparency to infrared light. Underneath the tanks were adjustable infrared panels (940 nm IR LEDs, Shenzhen VICO).
Basler Ace 2040-90um Near Infrared (NIR) cameras (Order#-106541, Graftek Imaging, USA) were mounted 58.4cm above the arena
to collect a dorsal view of zebrafish. Infrared long-pass filters (Midopt LP780-62, Graftek Imaging, USA) were attached to the lens
(Schneider Cinegon 1.9, Graftek Imaging, USA) and were set to an aperture of six. Six fish were transferred directly from home tanks
to the behavioral arena by netting. All trials were recorded after 10 minutes of habituation to allow for recovery from any stress due to
netting from the home tank. Each trial was a recording of 30 min at 60 frames per second. Arenas were rinsed clean with system water
at the end of the day and put through a cabinet washer once a week on a hot water only cycle.
For behavioral analysis in the home-tank arena, five size and age-matched fish were placed in a 1.4 L zebrafish tank (Pentair, USA)
with 1 L of zebrafish system water. We used a side-mounted camera (acA2000-165u mNIR, Basler). To make the background uniform
for tracking, we placed a 25cmX25cm infrared illuminating board on the obverse side of the fish tank to illuminate the fish. An optical
filter (LP780-72 filter, MidOPT) was placed on a lens (LM8XC 1.3’’ (4/3’’) 8.5mm, F2.8, KOWA) to permit recording of infrared light.
Each trial was a recording of 30 minutes at 60 frames per second. Behavioral data were analyzed as described (Tang et al., 2018).
Adult zebrafish were euthanized by exposure to chilled water (0-4C). Samples were fixed in Modified Davidson’s fixative (Fisher Sci-
entific; catalog number 50-292-28) for up to 72 hours. At the time of fixation, abdominal cavity was cut open in order to expose in-
ternal organs for efficient fixation. The gills were flushed gently with the fixative, and an incision was made across the spinal cord
posterior to the brain for efficient fixation of the central nervous system. After fixation, Zebrafish were placed in Immuno Cal Decal-
cifier (Stat Lab-McKinney, TX) for a total of 48 hours with continuous agitation, fresh solution was added after 24 hours. The Zebrafish
were then rinsed in running tap water to remove any residual calcium salts, and processed through a graded series of alcohols and
xylene to be embedded in paraffin wax in a sagittal orientation. Paraffin embedded blocks were then serially sectioned at 5mmona
rotary microtome, and each individual section was placed on a charged glass slide. Every tenth slide was stained with a hematoxylin
and eosin (H&E; Gill’s Hematoxylin III (s211-32oz) and Eosin Y Alcoholic Working Solution (s2186-32oz), Poly Scientific R&D Corp,
Bay Shore NY) staining procedure to identify different tissue structures. Slides were then scanned into an Aperio slide Scanner (Leica
Biosystems). A board-certified histopathologist analyzed the H&E stained samples. Alizarin red (Alizarin Red-S, Sigma-Aldrich;
A5533-25G) staining was performed as described (Walker and Kimmel, 2007). Tartrate-resistant acid phosphatase staining was per-
formed as per the manufacturer’s instructions (Sigma-Aldrich; 387A-1KT).
RNA-Seq Sample Preparation
Tissue Collection and Dissection
Adult zebrafish were euthanized by exposure to chilled water (0-4C). Gills, heart and kidney were collected from 32 individual fish at
two time-points, namely at 3 and 5 mpf for aklotho mutants and wild-type sibling controls (AB background; 8 fish per genotype per
time-point - 4 males, 4 females) for a total of 96 samples. Tissues were dissected in cold PBS and immediately stored in RNALater
Stabilization Solution (ThermoFisher Cat# AM7021). RNALater was removed after an overnight incubation at 4C, and samples were
stored at 80C until processing.
RNA Extraction
All tissues were homogenized using the TissueLyser II (QIAGEN) plus Lysis buffer containing b-mercaptoethanol and stored at
80C. RNA extraction was performed using the automated protocol in the QIAcube workstation utilizing the RNeasy Fibrous Tissue
Cell Reports 28, 2767–2776.e1–e5, September 10, 2019 e3
Mini kit (QIAGEN Cat No./ID: 74704). RNA integrity and quality were assessed by Agilent TapeStation using High Sensitivity RNA
ScreenTapes. Samples were normalized and 300ng of RNA was used for library prep for each sample. ERCC RNA Spike-in mix
was added for quality control.
RNASeq Library Prep and Sequencing
RNASeq libraries were prepared using the Illumina TruSeq stranded mRNA HS sample preparation kit using an automated pipeline.
Magnetic poly-T oligo beads were used to purify poly-A containing mRNA for cDNA synthesis. The library quality was assessed by
Agilent TapeStation using High Sensitivity DNA 1000 ScreenTapes and quantitated using Invitrogen Quant-iT PicoGreen dsDNA
assay kit. Samples were pooled in equal amount before checking them in an Illumina MiSeq flow cell for quality and to optimize clus-
tering. Four samples failed the library preparation step (two aklotho mutant females, one aklotho mutant male, one wild-type sibling
male) and could not be sequenced. Final sequencing was performed on a HiSeq 2500 instrument (76 base pair, paired-end).
qPCR for the Quantification of RNA Expression Levels
RNA levels were quantified and normalized using the Qubit RNA HS Assay kit (Thermo Fisher). Reverse transcription was performed
using the Superscript III First-Strand Synthesis System (Thermo Fisher) following the manufacturer’s instructions (with a 1:1 mix of
oligo-dT and random hexamers). qPCR was then performed in triplicates using the Power SYBR Green Mix (Thermo Fisher) on a
QuantStudio 7 Flex system following the manufacturer’s instructions. The primers were validated for specificity (melting-curve)
and efficiency (dilution curve). The following qPCR primer pairs were used (Vijayakumar et al., 2013; Yang et al., 2011):
runx2a (runt-related transcription factor 2): Forward primer: AGCCGACCCACGCCAGTTTGAG Reverse primer: TGGGGTGTAG
runx2b: Forward primer: ACGCAAACGGAGGACATACG
osteopontin/spp1: Forward primer: GAGCCTACACAGACCACGCCAACAG
tnf-a: Forward primer: GCGCTTTTCTGAATCCTACG
b-actin: Forward primer: CGAGCAGGAGATGGGAAC
values were automatically calculated by the QuantStudio 7 Flex system and outliers among technical triplicates were manually
eliminated. Data were last analyzed using the DDC
method: C
values were averaged, then subtracted to the average C
value of
b-actin (DC
), and last the fold change was determined using the formula 2–(
sample –
RNAscope probe targeting spp1 gene (ACDBio; Cat No. 409501) was used to visualize spp1 expression as described (Gross-Thebing
et al., 2014). Briefly, Adult zebrafish were euthanized in ice-cold water and decapitated. The hearts were dissected out and fixed in
4% PFA overnight at 4C. The hearts were washed and then dehydrated through serial methanol incubations and stored at 20C
overnight. Following rehydration, the samples were permeabilized and then incubated in the probe mixture overnight at 40C.
Following incubation, the embryos were washed and fluorescence detection steps were performed as described (Gross-Thebing
et al., 2014). Samples were then imaged using a Zeiss Lightsheet microscope (Lightsheet Z.1, Zeiss).
RNA-Seq Data Analysis
Alignment and quantification were performed with a Novartis internal pipeline, Exon Quantification Pipeline (EQP) (Schuierer and
Roma, 2016), using STAR and the Zebrafish Reference GRCz11. MultiQC package was used to do the pre-alignment QC check
(Ewels et al., 2016). Two samples failed the QC checked and were removed from further analysis (one kidney sample, one heart sam-
ple). The failed samples correlated with low RIN scores. The average total mapped reads per sample was 23.7 million PE reads.
Genes with read counts < 10 were filtered out before Differential gene expression analysis (DGE). DGE was performed with the
DESeq2 package (Love et al., 2014), comparing mutants to wild-type using sex as a covariate for each time-point with adjusted p
value cutoff = 0.05 and using ‘apeglm’ for LFC shrinkage (Zhu et al., 2019). Adjusted p values were calculated using the
Benjamini-Hochberg False Discovery Rate approach to correct for multiple testing.
Gene Set Enrichment Analysis
Gene set enrichment analysis was performed on the up- or downregulated genes in aklotho mutants (adjusted p value < 0.05 and log-
2-fold change > 1.5) from each tissue (kidney, heart, and gills) and adult stage (three and five mpf). The curated gene sets, including
canonical pathways and hallmark pathways were included in the analysis. The gene sets were downloaded from the Molecular Sig-
natures Database ( and were converted to zebrafish homologs using the R package
msigdbr. For each pathway, the corresponding gene set was compared with an up- or downregulated gene list. The overlapping
genes were counted, and a hypergeometric test was performed to measure the statistical significance, e.g., p value, on whether
the number of overlaps occurred by chance. Finally, adjusted p values were derived by the Benjamini-Hochberg (BH) procedure
e4 Cell Reports 28, 2767–2776.e1–e5, September 10, 2019
to control the False Discovery Rate (FDR). In a separate analysis, for each pathway and comparison, a weighted Kolmogorov-
Smirnov test was performed using the gsea function (with the number of permutations to be 1e8) from the R package ClusterProfiler
(Yu et al., 2012). Adjusted p values were derived by the BH procedure to control the FDR.
Sequencing data have been deposited to the Sequence Read Archive at NCBI under the BioProject Accession: PRJNA556842.
Cell Reports 28, 2767–2776.e1–e5, September 10, 2019 e5
... In zebrafish, αklotho expression is detected in the adult kidney and fgf23 is continuously expressed in the corpuscles of Stannius, an endocrine gland close to the nephron that contributes to calcium homeostasis, where Fgf23 is responsible for adjusting and regulating calcium metabolism [53,54]. As for the mouse mutants, zebrafish mutants for αklotho and fgf23 have a short lifespan [34]. However, the disease phenotype only occurs at approximately five months of age, later than in the mouse model (i.e., as soon as one month of age). ...
... Thus, zebrafish mutants can reach adulthood and reproduce, allowing the maintenance of a mutant brood stock, a major drawback of the mouse model. Both zebrafish mutants display ectopic calcification of the vessels throughout the body, especially in the outflow tract of the heart and the bulbus arteriosus, a pathological calcification likely associated with premature aging, ectopic activation of osteoclast differentiation, and ageassociated vascular calcification [34]. Indeed, in vivo studies using αklotho, fgf23, and ennp1 zebrafish mutants have consistently shown an increase in osteoclast activity around mineralized soft tissues, hinting at the existence of osteoclasts that develop as a response to ectopic calcifications [30,34,55,56]. ...
... Both zebrafish mutants display ectopic calcification of the vessels throughout the body, especially in the outflow tract of the heart and the bulbus arteriosus, a pathological calcification likely associated with premature aging, ectopic activation of osteoclast differentiation, and ageassociated vascular calcification [34]. Indeed, in vivo studies using αklotho, fgf23, and ennp1 zebrafish mutants have consistently shown an increase in osteoclast activity around mineralized soft tissues, hinting at the existence of osteoclasts that develop as a response to ectopic calcifications [30,34,55,56]. ...
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Ectopic calcification refers to the pathological accumulation of calcium ions in soft tissues and is often the result of a dysregulated action or disrupted function of proteins involved in extracellular matrix mineralization. While the mouse has traditionally been the go-to model organism for the study of pathologies associated with abnormal calcium deposition, many mouse mutants often have exacerbated phenotypes and die prematurely, limiting the understanding of the disease and the development of effective therapies. Since the mechanisms underlying ectopic calcification share some analogy with those of bone formation, the zebrafish (Danio rerio) - a well-established model for studying osteogenesis and mineralogenesis - has recently gained momentum as a model to study ectopic calcification disorders. In this review, we outline the mechanisms of ectopic mineralization in zebrafish, provide insights into zebrafish mutants that share phenotypic similarities with human pathological mineralization disorders, list the compounds capable of rescuing mutant phenotypes, and describe current methods to induce and characterize ectopic calcification in zebrafish.
... Concerning the zebrafish heart, age-associated changes in electrical function, pathophysiological changes and an increase of sinus arrest episodes have been described [29][30][31]. Additionally, in recent studies, the use of genetic manipulations in zebrafish has helped in defining and investigating molecular drivers of aging or longevity, such as celsr1a, klotho and rag1, encoding a non-classical cadherin, a hormone and a lymphoid-specific endonuclease, respectively [32][33][34]. ...
... We compared the Ensembl IDs of our 1233 DEGs in old vs. young ventricles with the Ensembl IDs of 4927 genes identified as differentially expressed in hearts of klotho mutants compared to wild types [32] using R. Results were visualized with the package VennDiagram and ggplot2. Over-represented GO terms of the overlapping genes were determined as described above. ...
... To assess the relevance of our gene expression dataset, we next compared our 1233 DEGs in old versus young ventricles with genes identified as differentially expressed in the zebrafish klotho mutant, a genetic aging model [32]. The hormone alpha-Klotho regulates lifespan in mammals and zebrafish, as respective mutants prematurely age [32,46]. ...
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Age-associated organ failure and degenerative diseases have a major impact on human health. Cardiovascular dysfunction has an increasing prevalence with age and is one of the leading causes of death. In contrast to humans, zebrafish have extraordinary regeneration capacities of complex organs including the heart. In addition, zebrafish has recently become a model organism in research on aging. Here, we have compared the ventricular transcriptome as well as the regenerative capacity after cryoinjury of old and young zebrafish hearts. We identified the immune system as activated in old ventricles and found muscle organization to deteriorate upon aging. Our data show an accumulation of immune cells, mostly macrophages, in the old zebrafish ventricle. Those immune cells not only increased in numbers but also showed morphological and behavioral changes with age. Our data further suggest that the regenerative response to cardiac injury is generally impaired and much more variable in old fish. Collagen in the wound area was already significantly enriched in old fish at 7 days post injury. Taken together, these data indicate an ‘inflammaging’-like process in the zebrafish heart and suggest a change in regenerative response in the old.
... Singh et al. showed that a loss of α-Klotho, a protective factor that regulates mineral homeostasis in mammals, results in reduced zebrafish lifespans and vascular calcification in the bulbus arteriosus. In addition, the calcification is associated with the ectopic activation of osteoblast differentiation (e.g., ectonucleoside triphosphate diphosphohydrolase 5a, RUNX2b, CLEC11a, and ITGA11a), suggesting that the loss of α-Klotho is related to aging, giving a rise to ectopic calcification [141,142]. ...
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Chronic kidney disease (CKD) is an increasing health care problem. About 10% of the general population is affected by CKD, representing the sixth cause of death in the world. Cardiovascular events are the main mortality cause in CKD, with a cardiovascular risk 10 times higher in these patients than the rate observed in healthy subjects. The gradual decline of the kidney leads to the accumulation of uremic solutes with a negative effect on every organ, especially on the cardiovascular system. Mammalian models, sharing structural and functional similarities with humans, have been widely used to study cardiovascular disease mechanisms and test new therapies, but many of them are rather expensive and difficult to manipulate. Over the last few decades, zebrafish has become a powerful non-mammalian model to study alterations associated with human disease. The high conservation of gene function, low cost, small size, rapid growth, and easiness of genetic manipulation are just some of the features of this experimental model. More specifically, embryonic cardiac development and physiological responses to exposure to numerous toxin substances are similar to those observed in mammals, making zebrafish an ideal model to study cardiac development, toxicity, and cardiovascular disease.
... Finally, zebrafish feature all three systems at advanced stages of development including a well-developed regularly beating heart (Jopling et al., 2010). In particular, the zebrafish turned out to be an important model for human heart development and a series of heart diseases including aorta calcification (Singh et al., 2019). Furthermore, the zebrafish allows to examine heart development with a speed and information not achievable even in mouse models. ...
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Two pairs of biological systems acting over long distances have recently been defined as major participants in the regulation of physiological and pathological tissue reactions: i) the nervous and vascular systems form various blood-brain barriers and control axon growth and angiogenesis; and ii) the nervous and immune systems emerge as key players to direct immune responses and maintain blood vessel integrity. The two pairs have been explored by investigators in relatively independent research areas giving rise to the concepts of the rapidly expanding topics of the neurovascular link and neuroimmunology, respectively. Our recent studies on atherosclerosis led us to consider a more inclusive approach by conceptualizing and combining principles of the neurovascular link and neuroimmunology: we propose that the nervous system, the immune system and the cardiovascular system undergo complex crosstalks in tripartite rather than bipartite interactions to form neuroimmune cardiovascular interfaces (NICIs).
... However, the pathological outcomes of hyperphosphatemia in obese individuals have no longer been explored in equal intensity ( Table 2). Investigators found that the prevalence of obesity-associated renal impairment is rising worldwide; along with this, experiments showed vast soft tissue and vessel ossification in the renal system, great vessels, lungs, and other organs in genetically modified hyperphosphatemic obese experimental animals [181]. Phosphate is an important constituent of our everyday foodstuffs and is primarily acquired from proteins. ...
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Abstract Regular intake of ready-to-eat meals is related to obesity and several noninfectious illnesses, such as cardiovascular diseases, hypertension, diabetes mellitus (DM), and tumors. Processed foods contain high calories and are often enhanced with excess refined sugar, saturated and trans fat, Na+ and phosphatecontaining taste enhancers, and preservatives. Studies showed that monosodium glutamate (MSG) induces raised echelons of oxidative stress, and excessive hepatic lipogenesis is concomitant to obesity and type 2 diabetes mellitus (T2DM). Likewise, more than standard salt intake adversely affects the cardiovascular system, renal system, and central nervous system (CNS), especially the brain. Globally, excessive utilization of phosphate-containing preservatives and additives contributes unswervingly to excessive phosphate intake through food. In addition, communities and even health experts, including medical doctors, are not well-informed about the adverse effects of phosphate preservatives on human health. Dietary phosphate excess often leads to phosphate toxicity, ultimately potentiating kidney disease development. The mechanisms involved in phosphate-related adverse effects are not explainable. Study reports suggested that high blood level of phosphate causes vascular ossification through the deposition of Ca2+ and substantially alters fibroblast growth factor-23 (FGF23) and calcitriol.
... However, the pathological outcomes of hyperphosphatemia in obese individuals have no longer been explored in equal intensity ( Table 2). Investigators found that the prevalence of obesity-associated renal impairment is rising worldwide; along with this, experiments showed vast soft tissue and vessel ossification in the renal system, great vessels, lungs, and other organs in genetically modified hyperphosphatemic obese experimental animals [181]. Phosphate is an important constituent of our everyday foodstuffs and is primarily acquired from proteins. ...
Full-text available
Regular intake of ready-to-eat meals is related to obesity and several noninfectious illnesses, such as cardiovascular diseases, hypertension, diabetes mellitus (DM), and tumors. Processed foods contain high calories and are often enhanced with excess refined sugar, saturated and trans fat, Na+ andphosphate-containing taste enhancers, and preservatives. Studies showed that monosodium glutamate (MSG) induces raised echelons of oxidative stress, and excessive hepatic lipogenesis is concomitant to obesity and type 2 diabetes mellitus (T2DM). Likewise, more than standard salt intake adversely affects the cardiovascular system, renal system, and central nervous system (CNS), especially the brain. Globally, excessive utilization of phosphate-containing preservatives and additives contributes unswervingly to excessive phosphate intake through food. In addition, communities and even health experts, including medical doctors, are not well-informed about the adverse effects of phosphate preservatives on human health. Dietary phosphate excess often leads to phosphate toxicity, ultimately potentiating kidney disease development. The mechanisms involved in phosphate-related adverse effects are not explainable. Study reports suggested that high blood level of phosphate causes vascular ossification through the deposition of Ca2+ and substantially alters fibroblast growth factor-23 (FGF23) and calcitriol.
... Conventional calcification detection methods have used histochemical stainings including Alcian blue, Alizarin red, and Von Kossa. The widespread calcification in vasculature could be observed in the α-klotho knockout zebrafish at five months old [46]. However, it is also confirmed that Alcian blue and Alizarin red are not sensitive enough to recognize the calcified bone structure in zebrafish embryos [24]. ...
Full-text available
Primary familial brain calcification (PFBC) is a neurogenetic disorder characterized by bilateral calcified deposits in the brain. We previously identified that MYORG as the first pathogenic gene for autosomal recessive PFBC, and established a Myorg -KO mouse model. However, Myorg -KO mice developed brain calcifications until nine months of age, which limits their utility as a facile PFBC model system. Hence, whether there is another typical animal model for mimicking PFBC phenotypes in an early stage still remained unknown. In this study, we profiled the mRNA expression pattern of myorg in zebrafish, and used a morpholino-mediated blocking strategy to knockdown myorg mRNA at splicing and translation initiation levels. We observed multiple calcifications throughout the brain by calcein staining at 2–4 days post-fertilization in myorg- deficient zebrafish, and rescued the calcification phenotype by replenishing myorg cDNA. Overall, we built a novel model for PFBC via knockdown of myorg by antisense oligonucleotides in zebrafish, which could shorten the observation period and replenish the Myorg -KO mouse model phenotype in mechanistic and therapeutic studies.
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Control of tissue metabolism and growth involves interactions between organs, tissues, and cell types, mediated by cytokines or direct communication through cellular exchanges. Indeed, over the past decades, many peptides produced by adipose tissue, skeletal muscle and bone named adipokines, myokines and osteokines respectively, have been identified in mammals playing key roles in organ/tissue development and function. Some of them are released into the circulation acting as classical hormones, but they can also act locally showing autocrine/paracrine effects. In recent years, some of these cytokines have been identified in fish models of biomedical or agronomic interest. In this review, we will present their state of the art focusing on local actions and inter-tissue effects. Adipokines reported in fish adipocytes include adiponectin and leptin among others. We will focus on their structure characteristics, gene expression, receptors, and effects, in the adipose tissue itself, mainly regulating cell differentiation and metabolism, but in muscle and bone as target tissues too. Moreover, lipid metabolites, named lipokines, can also act as signaling molecules regulating metabolic homeostasis. Regarding myokines, the best documented in fish are myostatin and the insulin-like growth factors. This review summarizes their characteristics at a molecular level, and describes both, autocrine effects and interactions with adipose tissue and bone. Nonetheless, our understanding of the functions and mechanisms of action of many of these cytokines is still largely incomplete in fish, especially concerning osteokines (i.e., osteocalcin), whose potential cross talking roles remain to be elucidated. Furthermore, by using selective breeding or genetic tools, the formation of a specific tissue can be altered, highlighting the consequences on other tissues, and allowing the identification of communication signals. The specific effects of identified cytokines validated through in vitro models or in vivo trials will be described. Moreover, future scientific fronts (i.e., exosomes) and tools (i.e., co-cultures, organoids) for a better understanding of inter-organ crosstalk in fish will also be presented. As a final consideration, further identification of molecules involved in inter-tissue communication will open new avenues of knowledge in the control of fish homeostasis, as well as possible strategies to be applied in aquaculture or biomedicine.
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This thesis aimed to understand the role that the hypothalamus-pituitary-thyroid (HPT) axis plays in appetite regulation of goldfish (Carassius auratus). I altered nutritional and thyroid statuses to measure the response of thyroid axis components and appetite-regulating peptides. I predicted that fasting would downregulate the thyroid axis and trigger an orexigenic response, while overfeeding would upregulate the thyroid axis and trigger an anorexigenic response. Additionally, I predicted that hyperthyroid conditions would lead to negative feedback of the thyroid axis and an orexigenic response, whilst opposite under hypothyroid conditions. I uncovered for both experiments that the thyroid axis in goldish is most responsive to overfeeding and hyperthyroidism. Overfeeding led to a time-dependent increase in central thyroid transcripts while fasting decreased thyroid hormone degradation peripherally with no central response, no treatment altered levels of thyroid hormone in circulation. Hyperthyroidism resulted in negative feedback to the pituitary, but not hypothalamus, and did not lead to an increase in food intake despite an increase in the levels of thyroxine. The thyroid inhibitor, propylthiouracil, did not induce hypothyroidism or alter the expression of any thyroid axis transcript. Appetite-regulating peptides correlated weakly to changes in the thyroid, suggesting an overall poor association in goldfish between appetite regulation and thyroid status.
Heart disease is the leading cause of death worldwide. Despite decades of research, most heart pathologies have limited treatments, and often the only curative approach is heart transplantation. Thus, there is an urgent need to develop new therapeutic approaches for treating cardiac diseases. Animal models that reproduce the human pathophysiology are essential to uncovering the biology of diseases and discovering therapies. Traditionally, mammals have been used as models of cardiac disease, but the cost of generating and maintaining new models is exorbitant, and the studies have very low throughput. In the last decade, the zebrafish has emerged as a tractable model for cardiac diseases, owing to several characteristics that made this animal popular among developmental biologists. Zebrafish fertilization and development are external; embryos can be obtained in high numbers, are cheap and easy to maintain, and can be manipulated to create new genetic models. Moreover, zebrafish exhibit an exceptional ability to regenerate their heart after injury. This review summarizes 25 years of research using the zebrafish to study the heart, from the classical forward screenings to the contemporary methods to model mutations found in patients with cardiac disease. We discuss the advantages and limitations of this model organism and introduce the experimental approaches exploited in zebrafish, including forward and reverse genetics and chemical screenings. Last, we review the models used to induce cardiac injury and essential ideas derived from studying natural regeneration. Studies using zebrafish have the potential to accelerate the discovery of new strategies to treat cardiac diseases.
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We previously discovered a new osteogenic growth factor that is required to maintain adult skeletal bone mass, Osteolectin/Clec11a. Osteolectin acts on Leptin Receptor+ (LepR+) skeletal stem cells and other osteogenic progenitors in bone marrow to promote their differentiation into osteoblasts. Here we identity a receptor for Osteolectin, integrin a11, which is expressed by LepR+ cells and osteoblasts. a11b1 integrin binds Osteolectin with nanomolar affinity and is required for the osteogenic response to Osteolectin. Deletion of Itga11 (which encodes a11) from mouse and human bone marrow stromal cells impaired osteogenic differentiation and blocked their response to Osteolectin. Like Osteolectin deficient mice, Lepr-cre; Itga11fl/fl mice appeared grossly normal but exhibited reduced osteogenesis and accelerated bone loss during adulthood. Osteolectin binding to a11b1 promoted Wnt pathway activation, which was necessary for the osteogenic response to Osteolectin. This reveals a new mechanism for maintenance of adult bone mass: Wnt pathway activation by Osteolectin/a11b1 signaling.
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Motivation: In RNA-seq differential expression analysis, investigators aim to detect those genes with changes in expression level across conditions, despite technical and biological variability in the observations. A common task is to accurately estimate the effect size, often in terms of a logarithmic fold change (LFC). Results: When the read counts are low or highly variable, the maximum likelihood estimates for the LFCs has high variance, leading to large estimates not representative of true differences, and poor ranking of genes by effect size. One approach is to introduce filtering thresholds and pseudocounts to exclude or moderate estimated LFCs. Filtering may result in a loss of genes from the analysis with true differences in expression, while pseudocounts provide a limited solution that must be adapted per dataset. Here, we propose the use of a heavy-tailed Cauchy prior distribution for effect sizes, which avoids the use of filter thresholds or pseudocounts. The proposed method, Approximate Posterior Estimation for GLM, apeglm, has lower bias than previously proposed shrinkage estimators, while still reducing variance for those genes with little information for statistical inference. Availability: The apeglm package is available as an R/Bioconductor package at, and the methods can be called from within the DESeq2 software. Supplementary information: Supplementary data are available at Bioinformatics online.
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Autophagy increases the lifespan of model organisms; however, its role in promoting mammalian longevity is less well-established1,2. Here we report lifespan and healthspan extension in a mouse model with increased basal autophagy. To determine the effects of constitutively increased autophagy on mammalian health, we generated targeted mutant mice with a Phe121Ala mutation in beclin 1 (Becn1F121A/F121A) that decreases its interaction with the negative regulator BCL2. We demonstrate that the interaction between beclin 1 and BCL2 is disrupted in several tissues in Becn1 F121A/F121A knock-in mice in association with higher levels of basal autophagic flux. Compared to wild-type littermates, the lifespan of both male and female knock-in mice is significantly increased. The healthspan of the knock-in mice also improves, as phenotypes such as age-related renal and cardiac pathological changes and spontaneous tumorigenesis are diminished. Moreover, mice deficient in the anti-ageing protein klotho 3 have increased beclin 1 and BCL2 interaction and decreased autophagy. These phenotypes, along with premature lethality and infertility, are rescued by the beclin 1(F121A) mutation. Together, our data demonstrate that disruption of the beclin 1-BCL2 complex is an effective mechanism to increase autophagy, prevent premature ageing, improve healthspan and promote longevity in mammals.
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Cardiovascular calcification was originally considered a passive, degenerative process, however with the advance of cellular and molecular biology techniques it is now appreciated that ectopic calcification is an active biological process. Vascular calcification is the most common form of ectopic calcification, and aging as well as specific disease states such as atherosclerosis, diabetes, and genetic mutations, exhibit this pathology. In the vessels and valves, endothelial cells, smooth muscle cells, and fibroblast-like cells contribute to the formation of extracellular calcified nodules. Research suggests that these vascular cells undergo a phenotypic switch whereby they acquire osteoblast-like characteristics, however the mechanisms driving the early aspects of these cell transitions are not fully understood. Osteoblasts are true bone-forming cells and differentiate from their pluripotent precursor, the mesenchymal stem cell (MSC); vascular cells that acquire the ability to calcify share aspects of the transcriptional programs exhibited by MSCs differentiating into osteoblasts. What is unknown is whether a fully-differentiated vascular cell directly acquires the ability to calcify by the upregulation of osteogenic genes or, whether these vascular cells first de-differentiate into an MSC-like state before obtaining a “second hit” that induces them to re-differentiate down an osteogenic lineage. Addressing these questions will enable progress in preventative and regenerative medicine strategies to combat vascular calcification pathologies. In this review, we will summarize what is known about the phenotypic switching of vascular endothelial, smooth muscle, and valvular cells.
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Purpose of review: α-Klotho (Klotho) occurs in three isoforms, a membrane-bound form acting as a coreceptor for fibroblast growth factor-23 (FGF23) signalling, a shed soluble form consisting of Klotho's large ectodomain thought to act as an enzyme or a hormone, and a secreted truncated form generated by alternative splicing of the Klotho mRNA with unknown function. The purpose of this review is to highlight the recent advances in our understanding of Klotho's function in mineral homeostasis. Recent findings: A number of seminal discoveries have recently been made in this area, shifting existing paradigms. The crystal structure of the ternary FGF receptor (FGFR)-1c/Klotho/FGF23 complex has been uncovered, revealing how the ligand FGF23 interacts with FGFR1c and the coreceptor Klotho at atomic resolution. Furthermore, it was shown that soluble Klotho lacks any glycosidase activity and serves as a bona fide coreceptor for FGF23 signalling. Experiments with a combination of Klotho and Fgf23-deficient mouse models demonstrated that all isoforms of Klotho lack any physiologically relevant, FGF23-independent functions in mineral homeostasis or ageing. Finally, it was demonstrated that the alternatively spliced Klotho mRNA is degraded and is not translated into a secreted Klotho protein isoform in humans. Summary: Taken together, there is now overwhelming evidence that the main physiological function of transmembrane and soluble Klotho for mineral homeostasis is their role as coreceptors mediating FGF23 actions. In light of these findings, the main pathophysiological consequence of the downregulation of Klotho observed in acute and chronic renal failure may be the induction of renal FGF23 resistance.
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Mechanisms of metabolic hormones The endocrine fibroblast growth factors (FGF19, FGF21 and FGF23) are circulating hormones that regulate important metabolic and physiological functions in vertebrates. Canonical FGFs require heparan sulfate proteoglycans to activate FGF receptors, but endocrine FGFs instead depend on klotho proteins for this process. There are two klothos, encoded by different genes: β-klotho is essential for FGF19- and FGF21-dependent signaling, whereas α-klotho is required for FGF23-dependent signalling. In this issue, Joseph Schlessinger and colleagues report crystal structures of the β-klotho extracellular domain, in ligand-free form and bound to a C-terminal peptide of FGF21. Moosa Mohammadi and colleagues report the atomic structure of a 1:1:1 ternary complex, which consists of the extracellular domain that is shed from membrane-anchored α-klotho into body fluids, the FGFR1c ligand-binding domain and FGF23. These hormones and their receptors are highly desirable drug targets owing to their central role in metabolism and physiology. Their structures offer the first glimpse of klotho and provide long-awaited mechanistic insights into the signalling pathways that are regulated by endocrine FGFs.
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Bone marrow stromal cells maintain the adult skeleton by forming osteoblasts throughout life that regenerate bone and repair fractures. We discovered that subsets of these stromal cells, osteoblasts, osteocytes, and hypertrophic chondrocytes secrete a C-type lectin domain protein, Clec11a, which promotes osteogenesis. Clec11a-deficient mice appeared developmentally normal and had normal hematopoiesis but reduced limb and vertebral bone. Clec11a-deficient mice exhibited accelerated bone loss during aging, reduced bone strength, and delayed fracture healing. Bone marrow stromal cells from Clec11a-deficient mice showed impaired osteogenic differentiation, but normal adipogenic and chondrogenic differentiation. Recombinant Clec11a promoted osteogenesis by stromal cells in culture and increased bone mass in osteoporotic mice in vivo. Recombinant human Clec11a promoted osteogenesis by human bone marrow stromal cells in culture and in vivo. Clec11a thus maintains the adult skeleton by promoting the differentiation of mesenchymal progenitors into mature osteoblasts. In light of this, we propose to call this factor Osteolectin.
Collective behaviors of groups of animals, such as schooling and shoaling of fish, are central to species survival, but genes that regulate these activities are not known. Here we parsed collective behavior of groups of adult zebrafish using computer vision and unsupervised machine learning into a set of highly reproducible, unitary, several hundred millisecond states and transitions, which together can account for the entirety of relative positions and postures of groups of fish. Using CRISPR-Cas9 we then targeted for knockout 35 genes associated with autism and schizophrenia. We found mutations in three genes had distinctive effects on the amount of time spent in the specific states or transitions between states. Mutation in immp2l (inner mitochondrial membrane peptidase 2-like gene) enhances states of cohesion, so increases shoaling; mutation in in the Nav1.1 sodium channel, scn1lab+/- causes the fish to remain scattered without evident social interaction; and mutation in the adrenergic receptor, adra1aa-/-, keeps fish close together and retards transitions between states, leaving fish motionless for long periods. Motor and visual functions seemed relatively well-preserved. This work shows that the behaviors of fish engaged in collective activities are built from a set of stereotypical states. Single gene mutations can alter propensities to collective actions by changing the proportion of time spent in these states or the tendency to transition between states. This provides an approach to begin dissection of the molecular pathways used to generate and guide collective actions of groups of animals.
Aging, the main risk factor for cardiovascular disease (CVD), is becoming progressively more prevalent in our societies. A better understanding of how aging promotes CVD is therefore urgently needed to develop new strategies to reduce disease burden. Atherosclerosis and heart failure contribute significantly to age-associated CVD-related morbimortality. CVD and aging are both accelerated in patients suffering from Hutchinson-Gilford progeria syndrome (HGPS), a rare genetic disorder caused by the prelamin A mutant progerin. Progerin causes extensive atherosclerosis and cardiac electrophysiological alterations that invariably lead to premature aging and death. This review summarizes the main structural and functional alterations to the cardiovascular system during physiological and premature aging and discusses the mechanisms underlying exaggerated CVD and aging induced by prelamin A and progerin. Because both proteins are expressed in normally aging non-HGPS individuals, and most hallmarks of normal aging occur in progeria, research on HGPS can identify mechanisms underlying physiological aging. Expected final online publication date for the Annual Review of Physiology Volume 80 is February 10, 2018. Please see for revised estimates.
Vascular calcification in chronic kidney disease is a very complex process traditionally explained in multifactorial terms. Here we sought to clarify relevance of the diverse agents acting on vascular calcification in uremic rats and distinguish between initiating and complicating factors. After 5/6 nephrectomy, rats were fed a 1.2% phosphorus diet and analyzed at different time points. The earliest changes observed in the aortic wall were noticed 11 weeks after nephrectomy: increased Wnt inhibitor Dkk1 mRNA expression and tissue non-specific alkaline phosphatase (TNAP) expression and activity. First deposits of aortic calcium were observed after 12 weeks in areas of TNAP expression. Increased mRNA expressions of Runx2, BMP2, Pit1, Pit2, HOXA10, PHOSPHO1, Fetuin-A, ANKH, OPN, Klotho, cathepsin S, MMP2, and ENPP1 were also found after TNAP changes. Increased plasma concentrations of activin A and FGF23 were observed already at 11 weeks post-nephrectomy, while plasma PTH and phosphorus only increased after 20 weeks. Plasma pyrophosphate decreased after 20 weeks, but aortic pyrophosphate was not modified, nor was the aortic expression of MGP, Msx2, several carbonic anhydrases, osteoprotegerin, parathyroid hormone receptor-1, annexins II and V, and CD39. Thus, increased TNAP and Dkk1 expression in the aorta precedes initial calcium deposition, and this increase is only preceded by elevations in circulating FGF23 and activin A. The expression of other agents involved in vascular calcification only changes at later stages of chronic kidney disease, in a complex branching pattern that requires further clarification.