Effect of inhibition of CBP-coactivated β-catenin-mediated Wnt signalling in uremic rats with vascular calcifications
Effect of inhibition of CBP-coactivated β- catenin-mediated Wnt signalling in uremic rats with vascular calcifications
Eva Gravesen 0 1 2
Anders Nordholm 2
Maria Mace 2
Marya Morevati 0 2
Estrid Høgdall 2
Carsten Nielsen 2
Andreas Kjñr 2
Klaus Olgaard 0 1 2
Ewa Lewin 2
0 Nephrological Department P , Rigshospitalet, Copenhagen , Denmark , 3 Nephrological Department B, Herlev Hospital , Copenhagen, Denmark, 4 Molecular Unit , Department of Pathology, Herlev Hospital , Copenhagen, Denmark, 5 Cluster for molecular imaging , Department of Biomedical Sciences and Department of Clinical Physiology, Nuclear Medicine & PET, Rigshospitalet and University of Copenhagen , Denmark
1 Department of Clinical Medicine, Faculty of Health Sciences, University of Copenhagen , Denmark
2 Editor: Jaap A. Joles, University Medical Center Utrecht , NETHERLANDS
Uremic vascular calcification is a regulated cell-mediated process wherein cells in the arterial wall transdifferentiate to actively calcifying cells resulting in a process resembling bone formation. Wnt signalling is established as a major driver for vessel formation and maturation and for embryonic bone formation, and disturbed Wnt signalling might play a role in vascular calcification. ICG-001 is a small molecule Wnt inhibitor that specifically targets the coactivator CREB binding protein (CBP)/β-catenin-mediated signalling. In the present investigation we examined the effect of ICG-001 on vascular calcification in uremic rats. Uremic vascular calcification was induced in adult male rats by 5/6-nephrectomy, high phosphate diet and alfacalcidol. The presence of uremic vascular calcification in the aorta was associated with induction of gene expression of the Wnt target gene and marker of proliferation, cyclinD1; the mediator of canonical Wnt signalling, β-catenin and the matricellular proteins, fibronectin and periostin. Furthermore, genes from fibrosis-related pathways, TGF-β and activin A, as well as factors related to epithelial-mesenchymal transition, snail1 and vimentin were induced. ICG-001 treatment had significant effects on gene expression in kidney and aorta from healthy rats. These effects were however limited in uremic rats, and treatment with ICG-001 did not reduce the Ca-content of the uremic vasculature.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This research was supported by grant
from The Lundbeck Foundation (grant no:
R1262012-12320) (KO). This work was also supported
by the Kirsten and Freddy Johansen Foundation,
the University of Copenhagen, The Danish Kidney
Association and The Danish Nephrological Society.
The funders had no role in study design, data
Patients with chronic kidney disease (CKD) suffer from severe vascular calcification (VC) [
and excess cardiovascular morbidity and mortality [
] that are not fully explained by the
presence of traditional risk factors such as hypertension, diabetes and old age . Disorders in
the mineral homeostasis and bone are today recognized as having a fundamental role in the
cardiovascular complications of CKD. This syndrome has been named CKD-mineral and
collection and analysis, decision to publish, or
preparation of the manuscript.
bone disorder (CKD-MBD) [
]. Although VC is associated with mineral disturbances and the
proinflammatory uremic state, the precise mechanisms behind the development of uremic VC
are still unknown [
]. Research suggests that VC is a product of highly regulated cell-mediated
processes wherein cells in the arterial wall transdifferentiate and become actively calcifying
cells, resulting in a process resembling bone formation [
]. Wnt signalling is established as a
major driver for vessel formation and maturation [
] and for embryonic bone formation [
Although Wnt signalling by and large is silenced in the mature vascular system, it is involved
in adult bone homeostasis, increasing osteoblast maturation which results in increased bone
]. Research suggests, that reactivation of Wnt signalling might be involved in
different organ diseases and cancer forms [
] as well as in development of fibrosis and
epithelial-mesenchymal transition (EMT) [
]. Further, evidence from several research groups
indicates that both canonical (β-catenin-dependent) and non-canonical Wnt signalling are
involved in such cardiovascular diseases as atherosclerosis and VC . As VC resembles
bone formation it is an interesting thought that disturbed Wnt signalling might play a role in
the pathogenesis of VC. In a previous investigation we used high throughput RNA deep
sequencing (RNAseq) on the calcified aorta from uremic rats to study the transcriptional
changes that occur in the aorta between the normal and the uremic, calcified condition [
In the RNAseq study, we found that the expression of a number of Wnt modulators,
Wntinducible genes and Wnt ligands was altered (S1 Table). Induction of canonical Wnt signalling
can result in both proliferation and differentiation. This divergence might be explained by the
binding of β-catenin to one of two different transcriptional coactivators, CBP and p300 [
In the RNAseq investigation we found increased expression of Ccnd1 (coding for cyclinD1)
and decreased expression of Jun in the calcified aorta from uremic rats. CyclinD1 is a marker
of increased proliferation that might be a target of CBP-coactivated β-catenin signalling [
whereas Jun might be a target of p300-coactivated signalling [
]. Our previous results
therefore may suggest a potential involvement of CBP-coactivated Wnt signalling in the uremic
The Wnt inhibitor ICG-001 has previously been shown to inhibit development of fibrosis
in several organs, such as lungs and kidneys [
]. It specifically binds to the β-catenin
coactivator CREB binding protein (CBP) at the β-catenin binding site, and thereby hinders
CBP/βcatenin-mediated signalling [
In the present investigation we examined the effects of ICG-001 on the vasculature, kidneys
and bone in uremic rats with focus on the potential reversibility of uremic vascular
calcification by inhibition of the CBP-coactivated β-catenin-mediated Wnt signalling.
Induction of CRF and associated disturbances in mineral metabolism
Chronic renal failure (CRF) was induced in adult male rats by 5/6-nephrectomy and ICG-001
was administered daily as intraperitoneal (ip) injections in a treatment protocol after the
induction of vascular calcification with high phosphate diet and alfacalcidol (CRF-D) as
outlined in Fig 1. A control group of uremic rats that were not treated with alfalcalcidol (CRF)
was used to examine the potential effects of ICG-001 on uremia and on the fibrotic remnant
kidney of the 5/6-nephrectomy model, and a control group of normal age-matched rats (Ctrl)
were kept in parallel and allocated to either ICG-001 or vehicle.
Total plasma biochemistry and body weight is available in S2 Table. CRF and CRF-D rats
had significantly increased plasma levels of urea, creatinine, parathyroid hormone (PTH) and
intact fibroblast growth factor 23 (iFGF23) compared to Ctrl rats (Fig 2A±2D). In the group of
CRF-D rats sacrificed at 8 weeks, the body weight was lower (P<0.05), total Ca was elevated
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Fig 1. Experimental design. 5/6 Nx = 5/6 nephrectomy, Ctrl = control, CRF = chronic renal failure, HP = high
phosphate, NP = normal phosphate, D = alfacalcidol, ICG = ICG-001, Veh = Vehicle, 8w = 8weeks. After four weeks of
uremia CRF rats were allocated to five experimental groups according to weight and plasma urea. CRF-D rats were
treated with alfacalcidol to establish VC. At eight weeks the alfacalcidol was stopped and subsequently rats received
daily injections of either ICG-001 or vehicle for four weeks. Rats were sacrificed after 12 weeks of uremia. A group of
alfacalcidol treated CRF rats was sacrificed at 8 weeks to examine the alfacalcidol-induced mineral metabolism
disturbances at baseline. CRF rats not treated with alfalcalcidol and Ctrl rats were allocated to four weeks of either
ICG-001 or vehicle and sacrificed after 8 weeks.
(P<0.05), PTH was suppressed and FGF23 was extremely elevated (P<0.0001) compared to
Ctrl rats (Fig 2C and 2D). A significant decrease in iFGF23 levels was seen in
ICG-001-administered Ctrl rats compared to vehicle-administered Ctrl rats (P<0.05) (Fig 2C). A significantly
higher mean plasma phosphate was seen in ICG-001-administered CRF rats compared to
vehicle-administered CRF rats (P<0.05) (Fig 2F). No other significant differences were seen
between ICG-001- and vehicle-administered rats.
Effects of ICG-001 on kidney gene and protein expression
To ensure the bioavailability and effect of ICG-001, a short-term experiment was conducted
wherein the effect of ICG-001 on kidney gene expression was examined in a model of acutely
induced renal fibrosis, the unilateral ureteral obstruction (UUO) model. Three days of UUO
was accompanied by a significant induction in the gene expression of fibronectin (Fn1),
inhibin-βa (Inhba) and vimentin (Vim) in the obstructed kidney. Administration of ICG-001
abolished the expression of Fn1 and Inhba which was induced by the ureteral obstruction and
reduced the expression of the Wnt target gene, Snai1 (S1 Fig).
As ICG-001 previously has been reported to have anti-fibrotic effects, the effects of
ICG001 on gene- and protein expression in the remnant kidney of the 5/6-Nx model was
examined, as a reduction in kidney damage potentially might have indirect beneficial effects on the
To examine the potential effects of ICG-001 on established kidney fibrosis, the gene
expression of β-catenin (Ctnnb1), the target genes of Wnt/β-catenin; cyclinD1 (Ccnd1) and c-Jun
(Jun) and factors related to EMT; snail1 (Snai1) and vimentin (Vim), as well as the
extracellular matrix associated gene fibronectin (Fn1) and genes from the related pathways; TGF-β
(Tgfb1) and activin A (Inhba) were all examined by qPCR in kidneys obtained from Ctrl and
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Fig 2. Plasma biochemistry. Plasma levels of (a) creatinine, (b) urea, (c) intact fibroblast growth factor 23 (iFGF23), (d) PTH, (e) Ca2+ and (f) phosphate
measured at sacrifice. CRF and CRF-D rats had a mild to moderate reduction in kidney function with increased levels of plasma creatinine, urea, iFGF23
and PTH. In the CRF-D rats sacrificed at 8 weeks the iFGF23 levels were extremely elevated, PTH was suppressed, and a trend towards an increase in Ca2+
was seen. iFGF23 levels were slightly lower in ICG-001-administered Ctrl compared to vehicle-administered Ctrl and plasma phosphate was markedly
higher in the ICG-001-administered CRF compared to the vehicle-administered CRF. Data is presented as mean ± SD, and PTH as median and
interquartile range. n = 6±9. Vehicle-administered Ctrl, CRF and CRF-D rats were compared by one-way ANOVA and Dunnets multiple comparison with
P<0.05, P<0.001 and P<0.0001 vs Ctrl. ICG-001- and vehicle-administered groups were compared using two-tailed t-test with #P <0.05 vs vehicle.
CRF rats. Fn1 and Vim were expressed at low levels and Inhba was not expressed in kidneys
from Ctrl rats (Fig 3A). ICG-001 administration resulted in significant reductions in Snai1
and Vim expression (P<0.05), whereas Fn1 expression was unchanged by ICG-001 (Fig 3A).
In Ctrl rats ICG-001 further resulted in a significant decrease in kidney gene expression of
Ctnnb1, Ccnd1 and Tgfb1 (P<0.05) while the expression of Jun was unaffected (Fig 3A). The
effect of ICG-001 on protein levels of cyclinD1, total β-catenin and active β-catenin was
examined by Western blot. ICG-001-administered Ctrl rats had significantly lower levels of
cyclinD1 and active β-catenin (P<0.05) (Fig 3B) compared to vehicle-administered Ctrl rats.
In kidneys from CRF rats, the gene expression of Fn1, Inhba, Vim and Tgfb1 were higher
(P<0.05) compared to Ctrl rats, while the gene expression of Ccnd1 was reduced (P<0.05).
No differences in gene expression were seen between kidneys from ICG-001- and
vehicleadministered CRF rats (Fig 3C). Likewise, no differences were seen in the protein levels of
cyclinD1 or total and active β-catenin between kidneys from ICG-001- and
vehicle-administered CRF rats examined by Western blot (Fig 3D).
Effects of ICG-001 on aorta gene expression
Aorta gene expression of genes related to Wnt signalling Ctnnb1, Ccnd1 and Jun (Fig 4A),
factors related to epithelial-mesenchymal transition Snai1 and Vim (Fig 4B) as well as
extracellular matrix proteins Fn1 and Postn (Fig 4C) and ligands from the TGF-β superfamily Tgfb1 and
Inhba (Fig 4D) were examined by qPCR. The gene expression levels of Ccnd1, Fn1 and Tgfb1
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Fig 3. Kidney gene and protein expression. Kidney gene expression of factors related to fibrosis and EMT as well as Wnt signalling was examined in Ctrl
rats (a) and in CRF rats (c). In Ctrl rats ICG-001 resulted in a decrease in the expression of Snai1 (snail1), Vim (vimentin), Ctnnb1 (β-catenin), Ccnd1
(cyclinD1) and Tgfb1 (TGF-β), while these effects were eliminated in CRF rats. Protein levels of total β-catenin (TBC), active β-catenin (ABC) and cyclinD1
were examined by WB in kidneys from Ctrl (b) and CRF rats (d). In accordance with the gene expression results, protein levels of active β-catenin and
cyclinD1 were reduced in kidneys from ICG-001-administered Ctrl rats while no differences in protein levels were seen between ICG-001- and
vehicleadministered CRF rats. Blots cropped from different parts of the same gel are presented together. Uncropped blots are provided in S2 Fig. Data is presented
as mean ± SD. n = 6±9. P<0.05 vs Ctrl by unpaired two-tailed t-test, #P <0.05 vs vehicle by unpaired two-tailed t-test.
were low in aortae from both Ctrl and CRF rats. No differences were seen in the aorta gene
expression of Ctnnb1, Ccnd1, Jun, Snai1, Vim, Fn1, Postn, Inhba and Tgfb1 between Ctrl and
CRF rats. The alfacalcidol-treated CRF-D rats had significantly increased aorta gene
expression of Ctnnb1, Ccnd1, Tgfb1, Snai1, Vim, Fn1, Inhba and Postn compared to Ctrl (P<0.05),
whereas Jun expression was unaffected.
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Fig 4. Aorta gene expression. Gene expression was examined in aortae from Ctrl, CRF and CRF-D rats by qPCR. The expression of genes related to (a)
Wntsignaling: Ctnnb1(β-catenin), Ccnd1 (cyclinD1), and Jun (c-Jun), (b) Epithelial-mesenchymal transition, EMT: Snai1 (snail1) and Vim (vimentin), (c)
Extracellular matrix proteins: Fn1 (fibronectin) and Postn (periostin), and (d) genes from the TGF-β superfamily ligands: Tgfb1 (TGF-β) and Inhba (activin A) is
shown. The expression of genes related to Wnt signalling; β-catenin and cyclinD1, and to EMT; snail1 and vimentin, and to extracellular matrix proteins;
fibronectin and periostin as well as genes from the TGF-β superfamily ligands, TGF-β and activin A were all significantly induced in the aortae from CRF-D rats,
while Jun was not changed. No differences in gene expression were seen between Ctrl and CRF rats, and the gene expression of cyclinD1, TGF-β and fibronectin
was very low in these groups. The expression of Jun was higher in ICG-001-administered Ctrl compared to vehicle-administered Ctrl, while no other differences
were noted between ICG-001- and vehicle-administered Ctrl, CRF or CRF-D rats. Data is presented as mean ± SD. n = 6±9. Vehicle-administered Ctrl, CRF and
CRF-D rats were compared by one-way ANOVA and Dunnets multiple comparison with P<0.05 vs Ctrl. ICG-001- and vehicle-treated groups were compared
using two-tailed t-test with #P <0.05 vs vehicle.
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ICG-001-administered Ctrl rats had significantly increased aorta gene expression of Jun
compared to vehicle-administered Ctrl (P<0.05). No other differences were seen in gene
expression between aortae from ICG-001- and vehicle-administered Ctrl. Likewise, no
differences in aorta gene expression were seen between ICG-001- and vehicle-administered CRF
and CRF-D rats.
Effects of ICG-001 on protein expression and vascular calcification in the
uremic aorta and in the aorta from control rats
VC in the proximal thoracic aorta was examined by von Kossa staining and the total
Ca-content was determined. Ca-content of the proximal thoracic aorta was markedly increased in
CRF-D rats compared to Ctrl rats (P = 0.0001), whereas no significant difference was seen in
Ca-content between aorta from CRF and Ctrl rats (Fig 5A). No differences in Ca-content
between ICG-001- and vehicle-administered rats were noted. A subgroup of CRF-D rats had
manifest calcifications on the von Kossa staining (Fig 5B and 5C). The protein expression of
cyclinD1 was examined by immunohistochemistry (IHC), and positive cyclinD1 staining was
observed in the nucleus of the vascular smooth muscle cells (VSMC). CyclinD1 staining was
negative in the majority of Ctrl rats, although weak staining was noted in a few rats. A positive
cyclinD1 staining was however seen in 50% of the CRF rats, but with no difference between
ICG-001- and vehicle-administered rats. All vehicle-administered CRF-D rats stained positive,
whereas in the ICG-001-administered CRF-D group two out of six rats stained negative, while
four out of six rats stained positive (Fig 5D). As the IHC-staining only provided a
semi-quantitative measure, aorta protein expression of cyclinD1 was further quantified by Western blot.
Thus, the protein levels of cyclinD1, as well as total and active β-catenin were examined by
Western blot in aorta from vehicle-administered Ctrl rats and from ICG-001- and
vehicleadministered CRF-D rats. In agreement with the IHC result, a significant increase in cyclinD1
protein levels was seen in aorta from CRF-D rats compared to Ctrl (P<0.01), and significant
increases in protein levels of both total and active β-catenin were seen in aortae from CRF-D
rats compared to Ctrl (P<0.05) (Fig 5E). ICG-001-administered CRF-D rats had unchanged
protein levels of total and active β-catenin compared to vehicle treated CRF-D rats (Fig 5F).
ICG-001 administration in CRF-D rats resulted in significantly lower cyclinD1 protein levels
compared to vehicle-administered CRF-D rats (P<0.05) (Fig 4F).
Characterization of the disturbed gene expression in the uremic calcified
To further examine the disturbed gene expression in the calcified aorta, CRF-D rats with
positive VK stain was compared to CRF-D rats with negative VK stain and further compared to
CRF and Ctrl. As no differences were noted in aorta gene expression between ICG-001- and
vehicle-administered rats, ICG-001 and vehicle groups were pooled. The presence of manifest
calcifications as defined by positive VK stain was accompanied by an enormous shift in gene
expression (P<0.0001), which was blunted in the CRF-D rats with negative VK stain (Fig 6A).
The genes with the highest relative increases (>5-fold vs Ctrl) were Fn1, Ccnd1, Postn, Tgfb1
and Inhba. The rats with positive VK stain were further characterized by higher plasma levels
of creatinine and urea (P<0.05), whereas no differences in plasma phosphate, Ca2+ or total Ca
were seen between the groups (Fig 6B). A heatmap of the aorta gene expression was created.
Aorta gene expression was correlated to the rat with the highest expression of fibronectin
(Fn1) (rat ID = 2), and ranked according to the Pearson correlation coefficient. The heatmap
demonstrates that a subset of the examined genes was capable of distinguishing the rats with
positive VK stain from the remainder of the rats. The Ctrl rats were grouped opposed to the
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Fig 5. Aorta calcification and protein expression. Ca-content was determined in the proximal thoracic aorta. A marked increase in Ca-content was seen in
CRF-D rats, while no difference was seen between CRF and Ctrl. No differences were seen in Ca-content between ICG-001- and vehicle-administered rats
(a). Representative pictures of von Kossa and cyclinD1 stains are presented in (b). No Ctrl or CRF rats stained positive by VK. Three rats stained positive in
both the ICG-001- and vehicle-administered CRF-D groups (c), and noticeably all rats staining positive by VK had massive, confluent staining, both in the
proximal thoracic (right side of the image) and distal abdominal aorta (left side of the image). Aortic protein expression of cyclinD1 was examined by IHC
(d). The majority of Ctrl rats stained negative and approximately half of the CRF rats stained positive for cyclinD1. No differences were noted between
ICG001- and vehicle-administered Ctrl and CRF rats. All vehicle-administered CRF-D rats stained positive for cyclinD1, while two ICG-001-administered
CRF-D rats stained negative. CyclinD1, total β-catenin (TBC) and active β-catenin (ABC) protein levels were examined by WB in aortae from
vehicleadministered Ctrl and CRF-D rats (e) and in aortae from ICG-001 and vehicle-administered CRF-D rats (f). Aortic protein levels of cyclinD1, total and active
β-catenin were increased in vehicle-administered CRF-D rats compared to Ctrl, and protein levels of cyclinD1 were reduced in ICG-001-admininstered
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CRF-D rats compared to vehicle-administered CRF-D rats. Blots cropped from different parts of the same gel are presented together. Uncropped blots are
provided in S1 Fig. Data is presented as mean ± SD. n = 5±9. Vehicle-treated Ctrl, CRF and CRF-D rats were compared by one-way ANOVA and Dunnets
multiple comparison with P<0.001 vs Ctrl. ICG-001- and vehicle-administered groups were compared using unpaired two-tailed t-test with #P <0.05 vs
vehicle. In WB (e) Ctrl and CRF-D rats were compared by unpaired two-tailed t-test with P<0.05 and P<0.001 vs Ctrl.
CRF-D rats with positive VK stain, and strikingly, the CRF-D rats with negative VK stain were
not readily separated from the CRF rats in the heatmap (Fig 6C).
Effects of ICG-001 on bone morphology and bone gene expression
As Wnt signalling is active in adult bone, the potential off-target effects of ICG-001 on bone
morphology, trabecular bone mineral density (BMD) and gene expression were examined by
Fig 6. VC-associated disturbances in aorta gene expression. Gene expression in aortae with manifest calcifications by VK stain were compared with aortae
from CRF-D rats with negative VK stain and further gene expression in aortae from CRF and Ctrl rats were compared to CRF-D rats with negative VK (a).
The presence of manifest calcifications was associated with a massive shift in gene expression, which was dampened in aortae from CFR-D rats with negative
VK. The rats with positive VK were further characterized by increased plasma levels of creatinine and urea (b), while no differences in plasma levels of
phosphate, Ca2+ and TCa were noted between VK positive and VK negative CRF-D rats (b). A heatmap was generated, where rats were ranked according to
the Pearson correlation coefficient and compared to the rat with the highest expression of Fn1 (ID = 2) (c). In the heatmap the rats with positive VK stain were
grouped together and Ctrl rats were located in the opposite end, while the CRF-D rats with negative VK and CRF rats were found in the middle, and were not
readily separated. Data is presented as mean ± SD. n = 8±17. Groups were compared by one-way ANOVA and Dunnets multiple comparison with P<0.05
and P = 0.0001 vs CRF-D with neg. VK.
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Fig 7. Bone μCT data. Bone microstructure was examined by μCT scan. Representative pictures are presented in (a). Trabecular bone mineral density
(BMD), bone volume/total volume (BV/TV), bone surface area/BV (BSa/BV) and cortical cross-sectional area (CCSa) is presented in (b). Trabecular
thickness (Tb/th), number (Tb/no) and spacing (Tb/sp) is presented in (c). Trabecular BMD was decreased in CRF rats while the trabecular BMD and bone
volume were increased in CRF-D rats. The ICG-001 administration resulted in a further decrease in trabecular BMD in CRF rats. In both CRF and CRF-D
rats bone surface area was reduced and trabecular thickness was increased compared to Ctrl. Trabecular number was reduced in CRF rats and a trend
towards an increase in trabecular spacing was seen in CRF, and these changes were reduced in CRF-D rats. Data is presented as mean ± SD. n = 3±7.
Vehicle-administered Ctrl, CRF and CRF-D rats were compared by one-way ANOVA and Dunnets multiple comparison with P<0.05 and P<0.001 vs
Ctrl. ICG-001- and vehicle-administered groups were compared using unpaired two-tailed t-test with ##P <0.01 vs vehicle.
microCT (μCT) and qPCR. Representative μCT pictures are presented in Fig 7A. CRF rats had
decreased trabecular BMD (P<0.001) and unchanged bone volume fraction (BV/TV),
compared to Ctrl, whereas an increase in trabecular BMD and bone volume fraction was seen
CRF-D rats (P<0.001), compared to Ctrl. Bone surface area (BSa/BV) was decreased in both
CRF and CRF-D rats (P<0.001) compared to Ctrl and no differences were seen between
groups in cortical cross-sectional area (CCSa) (Fig 7B). Trabecular thickness (Tb/th) was
increased both in CRF and CRF-D rats compared to Ctrl (P<0.001), whereas trabecular
number (Tb/no) was decreased in CRF (P<0.05) and not significantly different in CRF-D rats
compared to Ctrl (Fig 7C). No significant differences were seen in trabecular spacing (Tb/sp)
between groups (Fig 7C). ICG-001-administered CRF rats had significantly lower trabecular
BMD compared to vehicle-administered CRF rats (P<0.01), while no differences in trabecular
BMD were seen between ICG-001- and vehicle-administered Ctrl or CRF-D rats.
Furthermore, no differences between ICG-001- and vehicle-administered rats were found in the bone
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volume fraction, bone surface area, cortical cross-sectional area or in trabecular thickness,
number or spacing.
mRNA levels of the early osteoblast marker runt-related transcription factor (Runx2), the
mature osteocyte marker sclerostin (Sost) and the osteoclast differentiation factor rank ligand
(Tnfsf11), as well as mRNA levels of β-catenin (Ctnbb1), cyclinD1 (Ccnd1) and Jun were
determined in femoral cortical bone tissue. The data are presented in S3 Fig. mRNA-levels of
Runx2 and Sost were decreased in CRF rats compared to Ctrl (P<0.05). Likewise, Runx2
mRNA levels were decreased in CRF-D rats compared to Ctrl (P<0.05). A significant increase
in Rankl mRNA levels was seen in CRF-D rats compared to Ctrl (P<0.05). No significant
differences were seen in mRNA levels of Ctnbb1, Ccnd1 and Jun between Ctrl rats and CRF or
CRF-D rats. ICG-001-administered CRF-D rats had significantly decreased levels of Rankl
(P<0.05), increased levels of β-catenin (P<0.05) and decreased levels of Jun mRNA (P<0.01)
compared to vehicle-administered CRF-D rats. No significant differences were found in
mRNA levels between ICG-001- or vehicle-administered Ctrl and CRF rats.
The involvement of Wnt signalling and the effect of Wnt inhibition on established uremic
vascular calcification were examined in the present study. The small molecule compound
ICG001 was used to inhibit Wnt signalling. ICG-001 specifically targets the β-catenin binding site
on CBP, and thereby hinders CBP-activated β-catenin signalling [
]. Vascular calcification
was induced by long term uremia, high phosphate diet and alfacalcidol treatment in 5/
6-nephrectomized rats. The Wnt pathway was significantly disturbed in the uremic, calcified
aorta. Treatment with ICG-001 ameliorated the expression of cyclinD1. Surprisingly,
administration of ICG-001 did not reverse the established vascular calcifications.
To examine the bioavailability and the antifibrotic effects of ICG-001, a short-term
experiment was first conducted in the unilateral ureteral obstruction (UUO) model. Renal fibrosis is
the final common pathway of all progressive renal diseases [
]. Several studies and previous
results from our group have shown that kidney injury could reactivate developmental
programs involved in nephrogenesis, attempting renal repair [24±27]. Wnt family members and
factors controlling Wnt function are critical morphogens in the developing kidney .
Although Wnt signalling have been implied in the regeneration of injured of kidney tissue,
persistent activation and dysregulation of the Wnt pathways underlie fibrosis and progressive
renal failure . In agreement with previously reported results from our group, UUO was
associated with induction of expression of genes related to fibrosis [
]. ICG-001 abolished the
induced expression of pro-fibrotic genes in the UUO experiment, confirming the
bioavailability and effect of the compound, in agreement with previous results by Hao et al [
]. The UUO
model is an experimental model of unilateral progressive renal fibrosis in the obstructed
kidney, which takes place in a non-uremic environment due to the functioning untouched
contralateral kidney [
]. In the 5/6-nephrectomy model, the remnant kidney is morphologically
characterized by progressive glomerulosclerosis and tubulointerstitial fibrosis[
], but in
contrast to the UUO model, this takes place in an uremic milieu. ICG-001 could potentially have
anti-fibrotic effects on the remnant kidney in the 5/6-nephrectomy model and hereby improve
kidney function, and could therefore theoretically both have direct and indirect beneficial
effects on the vasculature. Therefore, the effects of ICG-001 on kidney gene and protein
expression were examined in CRF rats and in Ctrl rats from our vascular calcification study. In
the kidneys from Ctrl rats ICG-001 resulted in a decrease in the gene expression of snail 1 and
vimentin, as well as in a decrease in the gene expression of TGF-β, β-catenin and cyclinD1
compared to vehicle-administered Ctrl. These results are in agreement with the significant
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effects seen in the UUO model in the present study. The ICG-001-induced decrease in
cyclinD1 and β-catenin gene expression in kidneys from Ctrl rats was confirmed at the protein
level by Western blot. No effects of ICG-001 were however found on either gene or protein
expression in kidneys from CRF rats, despite the significant effects of ICG-001 on kidney gene
expression in both the UUO model and in Ctrl rats from the vascular calcification model.
The reason for this lack of effect of ICG-001 on the fibrotic remnant uremic kidney remains
uncertain. Even though the genes related to fibrosis were induced in kidneys from CRF rats,
βcatenin gene expression was similar to that of Ctrl rats, and cyclinD1 gene expression was
suppressed compared to Ctrl rats. The suppressed cyclinD1 levels might be due to cell cycle arrest
in the established fibrosis, as previously described [
]. Furthermore, even though the
ICG-001 had an antifibrotic effect when given as a preventive treatment in the UUO model,
the ICG-001 was administered at a time when established kidney fibrosis was already present
in the vascular calcification model. Thus, potentially Wnt signalling was involved in the
development of kidney fibrosis, but selective targeting the CBP/β-catenin-mediated Wnt signalling
was insufficient to amend the established fibrotic changes. Furthermore, uremia by itself
might modify the effects of ICG-001. In this context it should be noted that the plasma levels
of the circulating Wnt inhibitors sclerosin (Sost) and dickkopf-related protein 1 (Dkk1) are
increased in uremia  and advanced uremia might per se be associated with a general state
of Wnt inhibition, which potentially could make pharmacological inhibition of Wnt signalling
Several studies have identified Wnt signalling as being a key player in the development of
vascular pathology [
]. This emphasizes the relevance of therapeutic strategies targeting
components of the Wnt signalling pathway in the treatment of uremic vascular calcification.
VSMC in the vascular lamina media normally exhibit a low proliferation rate [
results in the activation and dedifferentiation of VSMC toward a synthetic, osteochondrogenic
phenotype with the capability to proliferate, migrate and increase the production of
extracellular matrix [
]. The effect of β-catenin on VSMC proliferation is well-established [
βCatenin/TCF signalling can upregulate the expression of proliferative genes such as cyclinD1
]. In the present model of uremic vascular calcification, the CRF-D rats had increased aorta
Ca-content, and a subgroup of CRF-D rats stained positive by von Kossa as a sign of a more
advanced calcification process. Aorta gene and protein expression of β-catenin and cyclinD1, a
reported target of CBP-coactivated Wnt signalling, were induced in the CRF-D rats, while the
expression of the Jun gene, a reported target of p300-coactivated Wnt signalling, was
unchanged. These results are in agreement with those of our previous RNAseq study [
might suggest an activation of CBP-coactivated β-catenin-mediated Wnt signalling in the
aorta from these rats. In Ctrl rats ICG-001 administration resulted in an induction of Jun gene
expression, which is consistent with a shift towards p300-coactivated signalling [
VSMC are quiescent in healthy vasculature [
], cyclinD1 gene and protein expression were
very low in aorta from Ctrl rats and there was no detectable effect of ICG-001 on cyclinD1 in
Ctrl aortas. In aortae from CRF-D rats no effects of ICG-001 were seen on gene expression of
Wnt-related genes and markers of fibrosis, EMT and matricellular proteins. This finding is
surprising as fibronectin, periostin and snail1 are reported to be targets of Wnt signalling [35±
37], and further surprising as we previously have shown that the pro-fibrotic gene expression
induced in the uremic, calcified aorta in the present model is modifiable, and does respond to
treatment with the TGF-β antagonist, BMP7 [
]. In our previous study BMP7 treatment
resulted in a reduction in the expression of fibronectin, periostin, inhibin-βa and snail1, while
the expression of these genes was unaffected by ICG-001 treatment in the present
investigation. Although TGF-β and Wnt signalling are closely intertwined [
], the examined genes
were apparently more receptive to modulation of the TGF-β signalling pathway.
12 / 21
Although cyclinD1 gene expression was similar in the aorta from ICG-001- and
vehicleadministered CRF-D rats, a number of the ICG-001-administered CRF-D rats stained negative
for aorta cyclinD1 by immunohistochemistry staining. Therefore, the protein levels of
cyclinD1 were quantified by Western blot, which demonstrated a significant reduction in
protein levels of cyclinD1 in aortae from ICG-001 treated CRF-D rats compared to
vehicle-administered CRF-D rats. ICG-001 did not reduce expression of Wnt-related genes and markers of
fibrosis, EMT and matricellular proteins in CRF-D rats except for cyclinD1. These results
might indicate that other signalling pathways such as TGF-β and activin could be involved in
the transcriptional alterations induced in the present model of advanced vascular calcification.
Furthermore, research from several groups indicates a possible role of non-canonical Wnt
signalling in atherosclerosis [
], and non-canonical Wnt signalling might potentially also be
involved in uremic vascular calcification. Noticeably, the most upregulated Wnt ligand in the
vasculature was Wnt16 (please, see S1 Table), that has been shown to induce both
non-canonical and canonical Wnt signalling [
]. Although our data suggest that there is activation of
canonical Wnt signalling in uremic vascular calcification, the data does not exclude a
concurrent induction of non-canonical Wnt signalling with potential pro-calcific effects.
ICG-001 administration significantly increased the expression of jun in aortae from Ctrl
rats, while there was no effect in uremic rats. This is in parallel with the effects of ICG-001 on
kidneys from Ctrl rats, and with no effect on kidneys from CRF rats. As noted above, uremia is
associated with increased circulating levels of Wnt inhibitors, which could modify the effects
of ICG-001. It should also be noted, that the present model of uremic vascular calcification is
associated with an increased expression of the Wnt inhibitor sclerostin in the aorta (S1 Table).
This might imply the presence of local endogenous Wnt inhibitors in the vasculature.
Sclerostin is primarily expressed in osteocytes and chondrocytes and it inhibits bone formation by
osteoblasts. Thus, the increased levels of sclerostin in the calcified aorta might have a protective
effect against osteochondrogenic transformation in the lamina media. The precise role of
sclerostin in the vasculature has however not yet been established. Interestingly, the increased
circulating levels of the Wnt inhibitors sclerostin and Dkk1 have in uremia been suggested to be
involved in the pathogenesis of the CKD-MBD [
], and a role of neutralizing antibodies
against circulating Wnt-inhibitors has been proposed for treatment of CKD-MBD [
The disturbed gene expression associated with the presence of severe calcifications in aortae
from von Kossa positive CRF-D rats was compared with gene expression in aortae from von
Kossa negative CRF-D rats. The presence of von Kossa positive calcifications was associated
with a massive induction of the examined genes; fibronectin, cyclinD1, periostin, TGF-β,
inhibin-βa, snail1, vimentin and β-catenin, except for Jun, and this induction was blunted in
the CRF-D rats with negative von Kossa staining. This finding might suggest that the
incorporation of calcium and phosphorus deposits in the vasculature is associated with changes in the
VSMC phenotype that at some point intensifies or accelerates. This notion is further supported
by the fact, that all rats with positive von Kossa had massive calcifications throughout most of
the medial layer, and no sporadic calcifications were seen. Noticeably, the rats with positive
von Kossa stain were slightly more uremic compared to von Kossa negative rats, whereas no
differences in plasma calcium and phosphate were noted.
The main purpose of the present study was to investigate the reversibility of uremic vascular
calcification by treatment with ICG-001. No effect of ICG-001 was seen on the Ca-content of
the aorta, and a comparable number of rats stained positive by von Kossa in ICG-001- and
vehicle-administered CRF-D rats. Even though we previously have shown, that the profibrotic
gene expression in the calcified aorta can be ameliorated by treatment with BMP7, the
established vascular calcification was not reversible by the BMP7 treatment or by the ICG-001 used
in the present investigation. These findings indicate that the accumulation of calcium and
13 / 21
phosphorus in the vascular wall is not readily reversible and will potentially require the
presence of specialized cells able to digest the calcium and phosphate crystals [
Wnt signalling is active in adult bone; consequently, caution should be taken with regard to
bone health when systemically administering a Wnt inhibitor. Therefore, the potential effects on
bone microstructure were examined by μCT and a negative impact of ICG-001 on bone in CRF
rats was revealed. CRF rats had decreased trabecular BMD compared to Ctrl rats, and the
ICG001 resulted in a further reduction in BMD in CRF rats probably due to further inhibition of Wnt
signalling in bone, in addition to the well-known resistance to PTH [
]. ICG-001 did not reduce
BMD in the heavily calcified, adynamic bone from CRF-D rats. Of importance, ICG-001 had no
impact on bone microstructure in normal control rats. This potential functional redundancy and
difference in effects between the coactivators CBP and p300 in the regulation of Wnt signalling in
bone has not previously been described. Although much focus has been drawn to CBP and p300 a
number of other β-catenin coactivators exist [
], which potentially could be recruited when the
CBP/β-catenin interaction is blocked. By specifically targeting the CBP/β-catenin interaction, it is
possible that the risk of potential off-targets effects on normal bone was reduced.
CBP/β-catenin antagonists are currently used in preclinical and clinical investigations as
treatment for advanced solid tumors and advanced myeloid malignancies. The use of ICG-001
in clinical studies creates a huge significant requirement not only for knowing the effects, but
also for studying the potential side effects of this compound. The results of the present study,
which show a clear effect of ICG-001 on bone, but also on normal vasculature and kidneys
should be considered in this context. The limited effect of ICG-001 on the calcified vasculature
and the lack of effect on long term kidney fibrosis in uremia are surprising. In the present
investigation a rather high dose of the compound was used, which previously has been shown
to be effective in different models of organ fibrosis [
19, 20, 44, 45
]. The effect of increasing
doses of ICG-001 will be subject for future experimental studies.
In conclusion, the presence of uremic vascular calcification is associated with a massive
shift in aorta gene expression, and in induction of the expression of Wnt-related genes and
proteins, including β-catenin and cyclinD1. Although Wnt inhibition by ICG-001 had
significant effects on protein and gene expression in kidneys and aortae from normal control rats,
these effects were limited in uremia, and ICG-001 treatment did not reduce the Ca-content of
the uremic aorta. The present results indicate induction of Wnt signalling in the calcified
aorta, although the specific inhibition of CBP-coactivated β-catenin signalling did not reduce
the degree of vascular calcification. Therefore, the lack of reversibility of aorta calcification in
the current study stresses the importance of preventing the development of vascular
calcification in chronic kidney disease.
Materials and methods
Animals and experimental models
Inbred adult male Dark Agouti (DA) rats weighing 200g (Envigo, The Netherlands) were used
in the vascular calcification study. Rats were housed in a temperature controlled environment
with a 12-hour light/dark cycle and ad libitum access to water and diet. Chronic renal failure
(CRF) was induced by 5/6-nephrectomy. The 5/6-nephrectomy was performed as a one-step
procedure through an incision in the back, as previously described [
]. Rats were
anaesthetized with Hypnorm/Midazolam (2μL/g; Panum Institute, Copenhagen, Denmark). Carprofen
(Pfizer, Denmark) was given subcutaneously as pain relief for three days. CRF rats were fed a
high phosphate diet throughout the study (0.9%Ca, 1.2%P, 600IU vitamin D per kg diet;
Altromin Spezialfutter GmbH & Co, Germany) and control rats were fed standard diet (0.9% Ca,
0.7% P, 600IU vitamin D per kg diet; Altromin). To increase the development of VC, CRF rats
14 / 21
were administered alfacalcidol (Leo Pharmaceuticals, Denmark) 80 ng intraperitoneally (ip)
three times weekly for four weeks. In the UUO study adult male Wistar rats (Taconic A/S,
Denmark) were used. Briefly, in the UUO rats the left ureter was ligated under general
anaesthesia and the ligature was kept until sacrifice after 72 hours. ICG-001 (Cat#4505, Tocris,
BioTechne, UK) was administered daily as ip injections at a dose of 5mg/kg, equivalent to the
dosing used in previous studies [
19, 44, 45, 47, 48
]. ICG-001 was dissolved in 96% ethanol and the
injected volume was 50μL/day. Vehicle consisted of 50μL 96% ethanol.
The experimental studies were approved by the Animal Experiments Inspectorate, Denmark
(Reference: 2017-15-0201-01214) and performed in accordance with the NIH Guide for the
Care and Use of Laboratory Animals. The rats were under daily supervision by the researchers
and the animal caretakers from Department of Experimental Medicine, The Panum Institute,
University of Copenhagen, and animals were sacrificed if failure to thrive was noted.
The UUO study was performed in order to examine the effect and bioavailability of the
commercially available ICG-001. In a short-term experiment the effect of ICG-001 on kidney gene
expression was examined in the unilateral ureteral obstruction model (UUO model), where
the contralateral kidney is left untouched. Rats were allocated to Ctrl, UUO/Veh or UUO/
ICG. ICG-001 or vehicle was administered at the time of surgery and daily for three days with
the last dose given in the morning on the day of sacrifice.
The vascular calcification study examined the effect of four weeks treatment with ICG-001
on established VC in chronic uremia. ICG-001 was administered after induction of VC in CRF
rats with high phosphate diet and alfacalcidol (CRF-D). A control group of uremic rats not
treated with alfalcalcidol (CRF) was used to examine the potential effects of Wnt inhibition on
uremia and on the fibrotic remnant kidney of the 5/6-nephrectomy model. After four weeks of
uremia CRF rats were allocated to five experimental groups according to weight and plasma
urea as outlined in Fig 1. A group of normal age-matched rats (Ctrl) were kept in parallel and
allocated to either ICG-001 or vehicle. The last ICG-001 or vehicle injection was administered
in the morning on the day of sacrifice. At sacrifice rats were anaesthetized with pentobarbital
(50μg/kg ip; Nycomed-DAK, Denmark) and eye-blood was drawn. The aorta was dissected,
removing blood and connective tissue, and kidney rudiments from CRF rats and
corresponding segments from Ctrl rats were collected. Cortical bone from the right femur was harvested
for qPCR and the left femur was placed in 70% ethanol and stored at 5ÊC for μCT scan.
Plasma biochemistry and aorta Ca-content
Plasma creatinine, urea, phosphate and total Ca were analyzed by Vitros 250 (Ortho-Clinical
Diagnostics, Raritan, USA). Plasma Ca2+ was measured by ABL800 (Radiometer, Copenhagen,
Denmark). Plasma intact FGF23 (iFGF23) was measured by a human FGF23 ELISA (Kainos
Laboratories, Tokyo, Japan), with an intra-assay coefficient of variation of 2.5% and
interassay coefficient of variation of 5% in our lab [
]. Plasma PTH was measured by a rat
bioactive intact PTH ELISA assay (Immutopics, San Clemente, USA) with an intra-assay coefficient
of variation of 4% and intra-assay variation of 9% in our lab [
]. Aorta Ca-content was
determined by the o-cresolphthalein method and normalized to the dry weight. A small section of
the proximal thoracic aorta was lyophilized for 24 hours to determine the dry weight. After
lyophilisation the aorta section was decalcified in 1M HCl for 24 hours and the Ca-content of
the supernatant was determined using a commercial assay (Sigma-Aldrich, St. Louis, USA).
15 / 21
Thoracic aorta, cortical bone and kidney tissue were manually ground by mortar and pestle
immersed in liquid nitrogen. Total RNA was extracted from the tissue-powder using the
EZNA RNA kit (Omega Bio-tek, GA, USA). First strand cDNA was synthesized from 0.2±
1.5μg of RNA with Superscript III cDNA kit (Invitrogen, MA, USA). Jumpstart
(SigmaAldrich, MO, USA) and Lightcycler 480II (Roche, Basel, Switzerland) were used for qRT-PCR,
with a binding temperature of 59ÊC. The mRNA levels were normalized to the mean of
reference genes Arbp and Rpl13a and reference gene stability was confirmed using Genorm [
Primers are listed in S3 Table.
Protein was extracted from kidney and thoracic aorta in T-PER (Thermo Scientific, Rockford,
IL) with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific). The protein
concentration was determined by the BCA assay (Thermo Scientific). 30 μg of protein was run on
Mini-Protean precast gel (Bio-Rad, Munich, Germany) and transferred onto nitrocellulose
membranes (Bio-Rad). Membranes were blocked with 5% bovine serum albumin (BSA) (Roche,
Mannheim, Germany) in PBS with 0.05% tween. Primary and secondary antibodies were diluted
in PBS with 3% BSA. The antibodies used were monoclonal anti-human cyclinD1 (1:500, Cat#29
78, Cell Signaling Technology), monoclonal anti-mouse total β-catenin (1:2000, Cat#610153, BD
Biosciences), monoclonal active β-catenin (1:500, Cat#05±665, Merck Millipore), polyclonal
antihuman park7 (1:1000000, Cat#ab18257, Abcam). The active β-catenin antibody specifically
detects β-catenin dephosphorylated at Ser37 and Thr41, as this state of β-catenin has been shown
to mediate Wnt-signalling [
]. The total β-catenin antibody binds the C-terminal region of
βcatenin. The secondary antibodies used were HRP-conjugated anti-rabbit (1:2000, Cat#P0448,
Dako) and anti-mouse (1:1000, Cat#P0447, Dako). Blots were visualised by the Amersham ECL
Prime Detection Reagent (GE Healthcare, Freiburg, Germany) using the Chemidoc XRS+ System
(Bio-Rad). Western blot quantifications were performed with ImageJ.
Histology and immunohistochemistry
For von Kossa (VK) and immunohistochemistry (IHC) staining, sections of the proximal
thoracic aorta and distal abdominal aorta were fixed in 10% buffered formalin for 24±48 hours at
room temperature. After formalin fixation tissue sections were dehydrated and embedded into
paraffin blocks. 4μm sections were cut and stored at -20ÊC until staining. VK staining was
performed according to standard protocols. IHC staining was performed using the Dako Link
Autostainer in an automated protocol. Deparaffinization, rehydration and heat-induced
antigen retrieval was performed in EnVision Flex Target Retrieval Solution at pH = 9 in the PT
Link module (Agilent Dako, Copenhagen, Denmark). Endogenous peroxidase activity was
blocked by incubation with EnVision FLEX Peroxidase-Blocking Reagent (Agilent Dako).
Sections were incubated with rabbit anti-human cyclinD1 antibody (Clone EP12, Agilent Dako),
followed by HRP-conjugated secondary anti-rabbit antibody (Agilent Dako) and Flex DAB
Chromogen (Agilent Dako). Slides were counterstained with hematoxylin. Background
staining was determined in sections incubated without primary antibody. Images were acquired on
Axio Imager Z1 with Axiocam (Carl Zeiss, Germany). All specimens were blinded before
scoring. CyclinD1-stained sections were quantified on a scale from 0±2 with 0 = negative, 1 = low
positive, 2 = high positive. VK-stained sections were scored on a scale from 1±6, as previously
], but as almost all sections either scored 1 (negative) or 6 (high), results are
reported as positive or negative.
16 / 21
Micro-computed tomography (μCT)
High resolution micro-computed tomography (μCT) was performed on the whole femur. CT
images were acquired on an Inveon Multimodality PET/CT scanner (Siemens, USA) with the
following settings: 361 projections, 60 kV, 500 μA and 1300 ms exposure. Images were
reconstructed with an isotropic voxel size of 32 μm. Image analysis was performed using the Inveon
Software (Siemens). The distal femur growth plate was used as reference. The cortical bone
cross section area (CCSa) was measured mid shaft (10 mm proximal to the reference growth
plate) by manually drawing the outer and inner perimeter, and then subtracting the ellipsoid
areas. For analysis of trabecular bone, a region of interest (ROI) of 0.960 mm along the
longitudinal direction was drawn manually starting at 2.080 mm proximal to the reference growth
plate. The CT images of ROI were segmented into bone and marrow by a visually chosen fixed
threshold for all groups. Bone morphometry was calculated using the Inveon Software based
on the parallel plate model by Parfitt [
]. The following parameters were calculated: the ratio
of total trabecular volume to total tissue volume (BV/TV), the ratio of trabecular bone surface
to trabecular bone volume (BSa/BV), trabecular thickness (Tb/th), trabecular number (Tb/no),
trabecular spacing (Tb/sp). A standard bone phantom (Inveon, Siemens) was calibrated and
applied to calculate the BMD of the trabecular bone.
Data are presented as mean ± standard deviation (SD). One-way analysis of variance
(ANOVA) and Dunnet's post-hoc test was used to test for between-group differences in mean.
Two-tailed unpaired t-test was used to compare means between ICG-001- and
vehicle-administered groups. P<0.05 was considered significant. Calculations were performed in Graphpad
Prism 7.0. Heatmap was generated using the publically available Morpheus Software provided
by Broad Institute (Cambridge, MA) (https://software.broadinstitute.org/morpheus/).
S1 Table. Aortic expression of genes related to the Wnt-signaling pathway. In a previous
study we performed RNA deep sequencing of aortae from Ctrl rats and uremic rats with
vascular calcification [
]. The database was searched for genes related to Wnt-signaling;
specifically, Wnt ligands, intracellular Wnt-signaling transducers, Wnt target genes and Wnt
inhibitors were searched. Only genes with significant differences between uremic and Ctrl rats
S2 Table. Plasma biochemistry and body weight at sacrifice. Data is presented as mean ± SD
and PTH as median and [range]. n = 6±9. Vehicle-treated Ctrl, CRF and CRF-D rats were
compared by one-way ANOVA and Dunnets multiple comparison with P<0.05, P<0.001
and P<0.0001 vs Ctrl. ICG-001- and vehicle-administered groups were compared using
unpaired two-tailed t-test with #P <0.05 vs vehicle.
S3 Table. Primer sequences.
S1 Fig. Effect of ICG-001 on kidney gene expression in unilateral ureteral obstruction
(UUO). ICG-001 was administered at the time of obstruction and subsequently daily at a dose
of 5mg/kg. Rats were sacrificed after three days of UUO. Kidney gene expression was
examined in the obstructed kidney from ICG-001 (UUO-ICG) and vehicle treated rats (UUO-Veh)
17 / 21
as well as in the normal kidney from untouched control rats (Ctrl). UUO resulted in an
induction in kidney expression of profibrotic genes, and ICG-001 administration ameliorated this
response. Data is presented as mean ± SD. Data is presented as mean ± SD. n = 6±9. P<0.05
vs Ctrl by unpaired two-tailed t-test, #P <0.05 vs vehicle by unpaired two-tailed t-test.
S2 Fig. Uncropped WB. (a) WB from Fig 3B, (b) WB from Fig 3D, (c) WB from Fig 4E, (d)
WB from Fig 4F.
S3 Fig. Bone gene expression. Gene expression was examined in cortical femoral bone tissue
by qPCR. Gene expression of the early osteoblast marker Runx2 and the mature osteocyte
marker Sost was decreased in CRF and CRF-D rats compared to Ctrl, whereas the osteoclast
differentiation marker Rankl was increased in CRF-D rats compared to Ctrl. ICG-001
treatment resulted in a decrease in the expression of Rankl in CRF-D rats, and surprisingly an
increase in Ctnnb1 and a decrease in Jun was seen in ICG-001 treated CRF-D rats compared
to vehicle-treated CRF-D rats. Data is presented as mean ± SD. n = 6±9. Vehicle-treated Ctrl,
CRF and CRF-D rats were compared by one-way ANOVA and Dunnets multiple comparison
with P<0.05 and P<0.001 vs Ctrl. ICG-001- and vehicle-treated groups were compared
using unpaired two-tailed t-test with #P <0.05 and ##P <0.01 vs vehicle.
The authors would like to thank the technicians, Nina Sejthen, Christina Grønhøj and Dorte
Skriver-Jensen for their excellent work.
Conceptualization: Eva Gravesen, Anders Nordholm, Maria Mace, Carsten Nielsen, Andreas
Kjñr, Klaus Olgaard, Ewa Lewin.
Data curation: Eva Gravesen, Anders Nordholm, Marya Morevati, Carsten Nielsen, Klaus
Olgaard, Ewa Lewin.
Formal analysis: Eva Gravesen, Maria Mace, Marya Morevati, Carsten Nielsen, Andreas Kjñr,
Klaus Olgaard, Ewa Lewin.
Funding acquisition: Eva Gravesen, Anders Nordholm, Klaus Olgaard, Ewa Lewin.
Investigation: Eva Gravesen, Anders Nordholm, Maria Mace, Marya Morevati, Klaus Olgaard,
Methodology: Eva Gravesen, Anders Nordholm, Maria Mace, Marya Morevati, Estrid
Høgdall, Carsten Nielsen, Andreas Kjñr, Klaus Olgaard, Ewa Lewin.
Project administration: Eva Gravesen, Klaus Olgaard, Ewa Lewin.
Resources: Eva Gravesen, Maria Mace, Marya Morevati, Estrid Høgdall.
Software: Eva Gravesen, Maria Mace, Carsten Nielsen, Andreas Kjñr.
Supervision: Klaus Olgaard, Ewa Lewin.
Validation: Maria Mace, Estrid Høgdall.
Visualization: Eva Gravesen, Marya Morevati, Carsten Nielsen, Andreas Kjñr.
18 / 21
Writing ± original draft: Eva Gravesen.
Writing ± review & editing: Eva Gravesen, Anders Nordholm, Maria Mace, Marya Morevati,
Estrid Høgdall, Carsten Nielsen, Andreas Kjñr, Klaus Olgaard, Ewa Lewin.
19 / 21
20 / 21
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