Transcript FGF23

Evidence For A Novel Bone-Kidney Axis
Regulating Systemic Phosphate Homeostasis
L. Darryl Quarles, M.D.
Summerfield Endowed Professor of Nephrology
University Of Kansas Medical Center
Learning Objectives
• Examine the role of hyperphosphatemia in
vascular calcifications and mortality
• Discuss the functions of:
– Phex
– FGF23
– Klotho
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Novel Genes Regulating
Phosphate Homeostasis
• Propose a model showing how these factors may
participate in a novel bone-kidney axis regulating
systemic phosphate homeostasis and
mineralization.
• Examine the role of FGF23 in the pathogenesis of
disordered mineral homeostasis in CKD.
Soft-Tissue Calcifications
Are Widespread in Dialysis Patients
Soft Tissue Calcification
Calciphylaxis
Soft Tissue Calcification
• Whole-body scan—dark areas
indicate calcium uptake
Kok M, et al. Clin Nucl Med. 2003;28:144-145.
Image from Richardson MS. 1999. Available at
ttp://www.rad.washington.edu/maintf/cases/unk39/answers.html..
Image from Block GA. 2004.
Coronary-Artery Calcification Is Prevalent in ESRD
A
B
Goodman WG et al N Engl J Med.342:1478-83, 2000.
Arterial Calcification Status Increases All-Cause (A) and
Cardiovascular Mortality (B) in ESRD Patients
London, G. M. et al. Nephrol. Dial. Transplant. 2003 18:1731-1740
Uncertain Pathogenesis of Vascular
Calcification in CKD
• Risk factors include:
70% of increased CV risk accounted for by traditional factors
– Age
– Hypertension
– Diabetes mellitus
– Inflamation-C-reactive protein
CKD-related Factors
–
–
–
–
Time on dialysis
Hyperphosphataemia
Calcium intake
Treatment with Vitamin D?
Disturbances in Mineral Metabolism Are Associated With
Increased Risk of Mortality in Hemodialysis
Serum Calcium
Serum Phosphorus
2.2
Relative Risk of Death
(n=40,538)
2.0
Relative Risk of Death
(n=40,538)
1.8
Referent group
1.8
1.6
1.4
1.2
1.0
0.6
0.0
Referent group
1.6
1.4
1.2
1.0
0.8
0.6
0.0
Serum Phosphorus (mg/dL)
Adapted from Block GA, et al. J Am Soc Nephrol. 2004;15:2208-2218.
Corrected Serum Calcium (mg/dL)
Inorganic Phosphate But Not Calcium
Concentration Affects Mineralization
[Ca]
[Pi]
[Pi]
[Ca]
von Kossa
Alizarin red
Murshed M, et al Unique coexpression in osteoblasts of broadly expressed genes accounts
for the spatial restriction of ECM mineralization to bone. Genes Dev. 19:1093-104, 2005.
Genetic Rescue of the Mgp -/- Phenotype
Murshed M, et al Unique coexpression in osteoblasts of broadly expressed genes accounts
for the spatial restriction of ECM mineralization to bone. Genes Dev. 19:1093-104, 2005.
Vascular Calcification &
Morbidity/Mortality in CKD
• Consensus regarding role of
hyperphosphatemia, but lack of
prospective studies demonstrating that
interventions to lower serum phosphate
improves survival.
• Understanding of the hormonal cascades
regulating phosphate homeostasis may
provide insights into additional pathways
affecting the systemic complications of
hyperphosphatemia.
Regulation of Phosphorus Homeostasis
Is there a hormonal cascade
regulating serum phosphorus
concentrations independent of PTH?
PTH-Independent
Hypophosphatemic Disorders
• Tumor-Induced Osteomalacia (TIO).
• Autosomal Dominant Hypo-phosphatemic
Rickets (ADHR).
• McCune-Albright/Bone Fibrous Dysplasia.
• X-Linked Hypophosphatemic Rickets
(XLH).
• Linear Nevus Sebaceous Syndrome.
X-Linked Hypophosphatemic Rickets
(XLH):Clinical Features
• Most common inherited form of rickets.
• X-linked dominant disorder.
• Phenotype:
– Renal
• Decreased renal tubular reabsorption of phosphate.
• Aberrant regulation of 1,25 (OH)2 Vitamin D3
production.
– Skeletal
• Defective calcification of cartilage (rickets) and bone
(osteomalacia).
• Growth retardation.
XLH:Genetic Abnormality
• PHEX gene (PHosphate regulating gene homologous to
Endopeptidases on X Chr).
• Disease gene encodes a member of M13 family of Type
II transmembrane zinc metallo-endopeptidase.
• Mutations have been identified in 86% of familial and
57% of sporadic cases of XLH.
• Phex substrates are likely responsible for renal and
skeletal phenotypes in XLH.
• Phex could convert a prohormone to a phosphateconserving factor, or inactivate a phosphaturic
hormone and/or mineralization inhibitor (most likely).
Phex Function: Lesions from the Hyp
mouse homologue of XLH
• Expresses the major phenotypic features of XLH.
• Mouse Phex cDNA sequence is highly homologous to
that of humans.
• Hyp has a 3' Phex deletion creating a truncated
endopeptidase lacking the catalytic domain.
• Systemic/humoral phosphaturic factor
(“Phosphatonin”) identified by parabiosis and crosstransplant studies.
• Autocrine/paracrine nascent defect in Hyp-derived
osteoblasts leading to impaired mineralization
independent of hypophosphatemia, caused by inhibitor
of mineralization (“Minhibin”).
Shared Pathophysiology of
Hypophosphatemic Disorders?
Human
Diseases
Mouse
Homologue
Abnormal Gene
Expression/Activity
ADH
None
FGF23
TIO/OHO
None
FGF23, PHEX, MEPE,
DMP-1, HSP-90, Osteopontin
MAS
None
(GNAS1 )-activating mutations,
FGF23
XLH
Hyp, Gy
PHEX/Phex, MEPE, FGF23
FGF23: A Candidate for Phosphatonin?
•
•
•
•
FGF23 is a ~32 kDa (251 amino
acids) protein with an N-terminal
region containing the FGF
homology domain and a novel 71
aa C-terminus that has
phosphaturic activity in vivo.
FGF23 is overproduced by tumors
causing tumor-induced
osteomalacia (TIO).
Autosomal dominant
hypophosphatemic rickets (ADHR)
is caused by missense mutations
of the 176-RXXR-179 motif in FGF23 preventing its processing into
inactive N- and C-terminal
fragments.
FGF23 is proposed to be a
substrate for PHEX
Phex-Dependent Cleavage And Inactivation Of
The Phosphaturic Hormone FGF23 Hypothesis
Is FGF23 Phosphatonin?
• To determine whether FGF23 is involved in the
pathogenesis of XLH we:
– Examined FGF23 levels in XLH and Hyp.
– Confirmed that FGF23 has phosphaturic activity.
– Determined whether FGF23 deficiency rescues
the hypophosphatemia in Hyp mice.
– Assessed if FGF23 is a substrate for Phex.
Serum Phosphorus (A), Serum FGF23 (B) And
Their Correlation (C) In Subjects With XLH
Weber T. Liu S, Quarles LD J Bone Mineral Res. 2003.
Increased Circulating fgf23 Levels in Hyp
Administration of FGF23 to Mice
Induces Hypophosphatemia
Is FGF23 The Phosphaturic Factor
In XLH/Hyp?
• To determined if FGF23 deficiency
rescues the hypophosphatemia in
Hyp mice, we:
– Generated FGF23 null mice,
– Transferred FGF23 deficiency onto the
Hyp mouse background to determine if
superimposed FGF23 deficiency
rescued the hypophosphatemia in Hyp
mice.
Targeting Strategy Used To Disrupt Fgf23
And Genotyping of Fgf23 Deficient Mice
5’ Flanking
A
S
Fgf23 Gene
Targeting
Construct
N
Exon1
S
Exon2
Exon3
H
H
TK
Neo
H
Exon2
EGFP
Short arm
5’ Flanking
Targeted
Allele
Exon3
Long arm
Exon2
Neo
Exon3
S H
S
H
H
EGFP
B
Neo (640bp)
Fgf23 (266 bp)
Gross Appearance of 3-Week Old WildType, Fgf23 Hetererzygous and KO mice
Fgf23+/+
Fgf23+/-
Fgf23-/-
Serum Pi, 1,25(OH)2D3 and Fgf23 levels in
Wild-Type, Heterozygous and Homozygous
Fgf23-Deficient Mice
16
b
14
12
a
10
8
6
4
2
0
WT
Het
Genotypes
KO
C
b
800
a
a,b
600
400
200
0
Serum Fgf23 Concentrations (pg/ml)
c
B 1000
Serum 1,25(OH)2D3 Concentrations (pM)
Serum Phosphorus Concentration (mg/dL)
A 18
50
40
30
20
10
0
WT
Het
KO
Genotypes
WT
Het
Genotypes
KO
Breeding Strategy/Study Design
Fgf23+/-/XY
(Heterozygous fgf23-KO males)
Wild-type
Hyp
Fgf23+/+ Fgf23+/+/Hyp
X
Fgf23+/-/HypX
(Heterozygous fgf23-KO /Hyp females)
Fgf23-KO
Fgf23-KO/Hyp
Fgf23-/-
Fgf23-/-/Hyp
3 weeks
Serum Phosphate, Calcium, fgf23,
1,25(OH)2D3, MicroCT
Serum Phosphorus Concentration (mg/dL)
Serum Pi levels in Wild-Type, Fgf23-, Phex-,
and combined Fgf23/Phex-Deficient Mice
18
c
n>5
c
16
b
14
12
a
10
8
d
d
6
4
2
0
WT
Het
KO
Hyp
Genotypes
Het/Hyp
KO/Hyp
Serum 1,25 (OH)2 D3 Concentrations (pM)
Serum 1,25(OH)2D3 Levels in Wild-Type, Fgf23-,
Phex-, and combined Fgf23/Phex-KO Mice
n=4
1000
b
b
800
a
a,b
600
400
c
200
0
c
WT
Het
KO
Hyp
Genotypes
Het/Hyp
KO/Hyp
Serum Fgf23 levels in Wild-Type, Fgf23-, Phex-,
and combined Fgf23/Phex-Deficient Mice
Serum Fgf23 Levels (pg/ml)
2500
n>5
b
2000
c
1500
1000
500
a
0
WT
a
Het
KO
Hyp
Genotypes
Het/Hyp
KO/Hyp
Relationship Between Serum Pi and Fgf23
Levels as a Function of Genotype
16
KO and KO/Hyp
14
Het
12
WT
10
8
Het/Hyp
Hyp
6
Threshold
Serum Phosphate Levels (mg/dL)
Serum Phosphate Levels (mg/dL)
16
14
KO
and
12 KO/Hyp
10
Het
WT
8
Het/Hyp
6
Hyp
4
4
0
500
1000
1500
2000
Serum Fgf23 Levels (pg/ml)
2500
1
10
100
1000
Serum Fgf23 Levels (pg/ml, log scale)
10000
Failure Of Phex-Dependent Cleavage Of FGF23:
Cotransfection Studies
Expression of fgf23 mRNA levels in normal
and Hyp-derived bone and osteoblasts
Inactivating Phex mutations in Hyp increase FGF23 gene expression in bone
of heterozyogous FGF23 knock-out/GFP knock-in mice
Fgf23 +/+
GP
CB
Hyp/Fgf23 +/-
Fgf23 +/-
GP
GP
CB
CB
BM
BM
BM
Role of fgf23 in Phex Deficiency
• Superimposed fgf23 deficiency rescues
hypophoshatemia in Hyp mice.
• Fgf23 does not appear to be a substrate of Phex.
• Phex-deficiency increases fgf23 expression through
unknown mechanisms.
• An alternative hypothesis is needed to explain
increments in circulating fgf23 levels in association
with inactivating Phex mutations.
What Is The Physiological
Role of FGF23?
• Regulation of FGF23 expression.
• End-organ effects of FGF23:
– Kidney.
– Parathyroid gland.
– Other tissues.
• Role in CKD
Physiological Function of FGF23: Lesions
From Studies of the Fgf23 Promoter
•
•
•
Isolation and characterization of mouse
Fgf23 promoter.
Evaluation of potenital regulators of Fgf23
promoter activity, including PTH, Vitamin D,
calcium and phosphorus in vitro.
Confirmation that these factors regulate
serum Fgf23 levels in vivo.
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1 mM
2 mM
3 mM
4 mM
Phosphate Concentration
Luciferase Activities (Firefly/Renilla)
Luciferase Activities (Firefly/Renilla)
Effect of Extracellular Calcium and Phosphate
on Fgf23 Promoter Activity
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1 mM
2 mM
3 mM
5 mM
Calcium Concentration
Luciferase Activities (Firefly/Renilla)
Effect of 1,25-(OH)2D3 on Fgf23 Promoter Activity
3.0
*
*
2.5
2.0
1.5
1.0
0.5
0.0
0M
10-10 M
10-9 M
10-8 M
1,25-(OH)2-vitamin D 3 Concentration
Luciferase Activities (Firefly/Renilla)
Effect of PTH on Fgf23 Promoter Activity
1.0
0.8
*
*
*
0.6
0.4
0.2
0.0
0 mM
1 mM
10 mM
PTH Concentration
100 mM
Vitamin D Stimulated Fgf23 Transcripts
in Ros17/2.8 Cells
Relative Concentration of Fgf23 (Fgf23/Ppia)
2.40.7
16.00.9 (7 fold)
1.80.3
264.348.9 (147 fold)
400
P<0.01
vehicle
1,25-(OH)2D3.
300
200
P<0.01
100
0
8 Hours
24 Hours
Effect of 1,25-(OH)2D3 on Serum Levels of Fgf23, PTH,
Phosphorus and Calcium
A
*
160
B
40
100 ng/g/BW Calcitriol IP
Serum Intact PTH
(pg/ml)
120
(pg/ml)
Serum FGF23
140
100
80
60
40
30
20
10
*
20
0
0
Serum Phosphorus
(mg/dL)
C
Vehicle
Calcitriol
10
D
8
Serum Calcium
(mg/dL)
Vehicle
6
4
10
8
6
4
2
2
0
Calcitriol
Vehicle
Calcitriol
0
Vehicle
Calcitriol
Characteristics of Fgf23 Promoter
•
The mouse Fgf23 promoter is characterized by:
- A transcription start site 123 bp upstream of the initial ATG.
- A TATA box 35 bp upstream of the transcription start site.
- 67% homology with the human promoter over the first 800 bps.
•
The 3.5kb 5' flanking region of the mouse Fgf23 gene
has promoter activity in vitro.
•
In ROS 17/2.8 osteoblasts, 1,25(OH)2D3 stimulates
activity of the Fgf23 promoter/reporter construct, and
alterations of extracellular phosphorus and calcium
concentrations have no effect.
•
Injection of calcitriol into wild-type mice increases
serum Fgf23 levels from a basal level 90.0±8.9 pg/ml to
136.4 ± 8.7pg/ml (Mean ± SEM) at 24 hours after
injection.
Regulation of Phosphate Homeostasis By FGF23:
Counter Regulatory Hormone for Vitamin D-Induced
Hyperphosphatemia?
Parathyroid glands
PTH
1,25(OH)2vitamin D
Serum
Ca2+
Kidney
Reabsorption of Ca2+
1,25(OH)2vitamin D synthesis
PO42-absorption
Gut
Ca2+ absorption
PO42-absorption
Serum
PO42-
FGF-23
Bone
Uncertain Role of FGF23 in CKD
• Circulating levels of FGF23 are increased in CKD.
Circulating FGF23 Levels In ESRD
(C-terminal assay)
106
FGF-23 = -34456.23 + 7315.06 * P
R-Square = 0.23
100x103
100,000
10
FGF-23 (RU/L)
FGF-23 (RU/ml)
5
10
10,000
4
1000
10
3
100
10
2
80x103
60x103
40x103
20x103
0
10
10
1
-20x103
0
100
4
Control XLH Unknown ESRD
5
6
7
8
9
10
11
Phosphorus (mg/dl)
Weber TJ, Liu S, Indridason OS, Quarles LD. Serum FGF23 levels in normal and disordered phosphorus homeostasis.
J Bone Miner Res. 2003 Jul;18(7):1227-34.
Circulating FGF23 Levels In ESRD
(Intact assay)
Intact FGF23 in ESRD
14 to 98,646 ng/L
(nl 27.8 ± 9.0 ng/L )
Sevelamer Hydrochloride and Calcium Bicarbonate Reduce
Serum Fibroblast Growth Factor 23 Levels in Dialysis Patients
Fumihiko Koiwa, (2005) Sevelamer Hydrochloride and Calcium Bicarbonate Reduce Serum Fibroblast Growth Factor 23 Levels in
Dialysis Patients. Therapeutic Apheresis and Dialysis 9:4, 336-339
Vitamin D Treatment Is Associated With Increased FGF23
Levels In Dialysis Patients
Shohei N et al. Kidney Intern
67:1171-1178, 2005
Uncertain Role of FGF23 in CKD
• Circulating levels of FGF23 are increased in CKD.
• Evidence for a role in development of secondary HPT.
Cross-sectional clinical observations: Diminishes
1,25(OH)2D3 in kidney and stimulates PTH by
parathyroid gland.
FGF23 Mitigates Hyperphosphatemia in CKD
Factors Associated with FePO4
Full multivariate
β
P
R2
model
.50
log FGF23
5.2
0.009
log PTH
6.0
0.004
eGFR
-.02
0.031
sP
-1.0
0.615
Gutierrez O JASN 2204-2205, 2005
FGF23 Accentuates Calcitriol
Deficiency in CKD
Factors Associated with calcitriol
Full multivariate β
model
log FGF23 -20.4
sP
-0.01
log PTH
0.17
sCa
0.16
25-OHD3
0.02
eGFR
0.01
P
0.008
0.961
0.280
0.396
0.043
0.067
Gutierrez O JASN 2204-2205, 2005
R2
.57
Uncertain Role of FGF23 in CKD
• Circulating levels of FGF23 are increased in CKD.
• Evidence for a role in development of secondary HPT.
Cross-sectional clinical observations: Diminishes
1,25(OH)2D3 in kidney and stimulates PTH by
parathyroid gland.
• Effects on other organ systems (positive vs negative?)
– Vasculature
– Pituitary
– Pancreas?
– Bone?
Hyperphosphatemic Familial Tumoral
Calcinosis (HFTC; MIM211900)
• Autosomal recessive hyperphosphatemic
disorder characterized by the progressive
deposition of calcified masses in cutaneous and
subcutaneous tissues.
• Caused by recessive mutations in at least two
genes
– GALNT3 (ppGalNacT3-mediated O-glycosylation
may protect normal FGF23 from proteolysis)
– FGF23 (destabilizing mutations)
S71G Missense Mutation in FGF23 Causes
Tumoral Calcinosis
Tissue Calcification
Serum Biochemistries
PO4
7.9-8.9 mg/dl
TMP/GFR
3.5 mM/l
1,25(OH2)D3
65-85 pg/ml
C-terminal FGF23 >1,800 Ru/ml
uCa
4.6 mg/kg BW
Mutation
Chefetz I, et al . A novel homozygous missense mutation in FGF23
causes Familial Tumoral Calcinosis associated with disseminated
visceral calcification. Hum Genet. 2005 Sep 7;:1-6
Uncertain Role of FGF23 in CKD
• Circulating levels of FGF23 are increased in CKD.
• Evidence for a role in development of secondary HPT.
Cross-sectional clinical observations: Diminishes
1,25(OH)2D3 in kidney and stimulates PTH by
parathyroid gland.
• Effects on other organ systems (positive vs negative?)
– Vasculature
– Pituitary
– Pancreas?
– Bone?
IS A CO-FACTOR REQUIRED FOR FGF23 ACTIONS?
[Klotho]: Identification of Essential Molecule
Responsible for Tissue Specific FGF23 Signaling
• FGF23 induced ERK/Egr-1 in kidney, parathyroid
and pituitary, but not heart, lung, liver and spleen.
• Klotho purified from kidney as a FGF23
interacting protein.
• FGF23 responsiveness could be imparted to nonresponding cells by overexpression of Klotho.
• Klotho is either a receptor for FGF23 or required
for FGF23 activation of a cell surface receptor.
• FGF23 levels are markedly increased in Klotho
null mice due to end-organ resistance?
Urakawa, I et al ASBMR M132, 2005.