Evidence For A Novel Bone-Kidney Axis Regulating Systemic Phosphate Homeostasis

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

• 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.

Novel Genes Regulating Phosphate Homeostasis}

Soft-Tissue CalcificationsAre Widespread in Dialysis Patients

Calciphylaxis

Image from Richardson MS. 1999. Available at ttp://www.rad.washington.edu/maintf/cases/unk39/answers.html..Image from Block GA. 2004.

Soft Tissue Calcification

• Whole-body scan—dark areas indicate calcium uptake

Kok M, et al. Clin Nucl Med. 2003;28:144-145.

Soft Tissue Calcification

Coronary-Artery Calcification Is Prevalent in ESRD

A

B

Goodman WG et al N Engl J Med.342:1478-83, 2000.

London, G. M. et al. Nephrol. Dial. Transplant. 2003 18:1731-1740

Arterial Calcification Status Increases All-Cause (A) and Cardiovascular Mortality (B) in ESRD Patients

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

Adapted from Block GA, et al. J Am Soc Nephrol. 2004;15:2208-2218.

< 3.0

Serum Phosphorus (mg/dL)

Referent group

Serum Phosphorus

3.0–4.04.0–5.0

5.0–6.06.0–7.0

7.0–8.08.0–9.0 > 9.0

0.00.6

1.2

1.4

1.6

1.8

2.0

2.2

1.0

Rel

ativ

e R

isk

of D

eath

(n=4

0,53

8)

0.00.6

0.8

1.0

1.2

1.4

1.6

1.8

< 8.0

Corrected Serum Calcium (mg/dL)

Referent group

Serum Calcium

8.0–8.58.5–9.0

9.0–9.5

9.5–10.0

10.0–10.5

10.5–11.0> 11.0

Rel

ativ

e R

isk

of D

eath

(n=4

0,53

8)

Inorganic Phosphate But Not Calcium Concentration Affects Mineralization

[Ca]

von Kossa

[Ca]

[Pi]

Alizarin red

[Pi]

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.

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

• 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.

Vascular Calcification & Morbidity/Mortality in CKD

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 phosphate- conserving 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 cross-transplant 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?

HumanDiseases

Mouse Homologue

Abnormal Gene Expression/Activity

ADH

TIO/OHO

XLH

None

None

Hyp, Gy

FGF23

FGF23, PHEX, MEPE,DMP-1, HSP-90, Osteopontin

PHEX/Phex, MEPE, FGF23

MAS None (GNAS1 )-activating mutations,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 FGF-23 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.

Exon1 Exon3Exon2H

5’ Flanking

Fgf23 Gene

TK Neo

Short armEGFP

Long arm

Exon3Exon2H H

Targeted Allele

EGFP

Neo Exon3Exon2H H

5’ Flanking

N

SS

S S H

Targeting Strategy Used To Disrupt Fgf23 And Genotyping of Fgf23 Deficient Mice

TargetingConstruct

M

Neo (640bp)

Fgf23 (266 bp)

-/-+/-+/+ H2O

A

B

Fgf23+/+

Fgf23-/-

Gross Appearance of 3-Week Old Wild-Type, Fgf23 Hetererzygous and KO mice

Fgf23+/-

Genotypes

WT Het KO

Se

rum

1,2

5(O

H) 2

D3 C

on

ce

ntr

ati

on

s (

pM

)

0

200

400

600

800

1000

aa,b

b

B

WT Het KO

Se

rum

Ph

os

ph

oru

s C

on

ce

ntr

ati

on

(m

g/d

L)

0

2

4

6

8

10

12

14

16

18

a

b

c

Serum Pi, 1,25(OH)2D3 and Fgf23 levels in Wild-Type, Heterozygous and Homozygous

Fgf23-Deficient Mice

Genotypes

A

Se

rum

Fg

f23

Co

nc

en

tra

tio

ns

(p

g/m

l)

0

10

20

30

40

50

WT Het KO

Genotypes

C

Breeding Strategy/Study Design

Serum Phosphate, Calcium, fgf23, 1,25(OH)2D3, MicroCT

3 weeks

Fgf23+/+ Fgf23-/-

Fgf23-KO

Fgf23+/+/Hyp

Hyp

Fgf23-/-/Hyp

Fgf23-KO/HypWild-type

Fgf23+/-/XY (Heterozygous fgf23-KO males)

Fgf23+/-/HypX (Heterozygous fgf23-KO /Hyp females)

X

Serum Pi levels in Wild-Type, Fgf23-, Phex-, and combined Fgf23/Phex-Deficient Mice

WT Het KO Hyp Het/Hyp KO/Hyp

Ser

um

Ph

osp

ho

rus

Co

nce

ntr

atio

n (

mg

/dL

)

0

2

4

6

8

10

12

14

16

18

Genotypes

a

b

c c

dd

n > 5

Serum 1,25(OH)2D3 Levels in Wild-Type, Fgf23-, Phex-, and combined Fgf23/Phex-KO Mice

WT Het KO Hyp Het/Hyp KO/HypSer

um

1,2

5 (O

H)2

D3

Co

nce

ntr

atio

ns

(pM

)

0

200

400

600

800

1000

aa,b

b b

cc

Genotypes

n=4

Genotypes

WT Het KO Hyp Het/Hyp KO/Hyp

Ser

um

Fg

f23

Lev

els

(pg

/ml)

0

500

1000

1500

2000

2500

a a

b

c

n > 5

Serum Fgf23 levels in Wild-Type, Fgf23-, Phex-, and combined Fgf23/Phex-Deficient Mice

Serum Fgf23 Levels (pg/ml)

0 500 1000 1500 2000 2500

Se

rum

Ph

os

ph

ate

Le

ve

ls (

mg

/dL

)

4

6

8

10

12

14

16

HypHet/Hyp

WT

Het

KO and KO/Hyp

1 10 100 1000 10000

Se

rum

Ph

os

ph

ate

Le

ve

ls (

mg

/dL

)

4

6

8

10

12

14

16

Serum Fgf23 Levels (pg/ml, log scale)

Hyp

Het/Hyp

WT

Het

KO and KO/Hyp

Relationship Between Serum Pi and Fgf23 Levels as a Function of Genotype

Threshold

Failure Of Phex-Dependent Cleavage Of FGF23:

Cotransfection Studies

Expression of fgf23 mRNA levels in normal

and Hyp-derived bone and osteoblasts

Fgf23 +/+

BM

CB

GP

CB

BM

GP

Hyp/Fgf23 +/-

BM

GP

CB

Fgf23 +/-

Inactivating Phex mutations in Hyp increase FGF23 gene expression in bone of heterozyogous FGF23 knock-out/GFP knock-in mice

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

• 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.

Physiological Function of FGF23: Lesions From Studies of the Fgf23 Promoter

1 mM 2 mM 3 mM 4 mM

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Phosphate Concentration

1 mM 2 mM 3 mM 5 mM0.0

0.2

0.4

0.6

0.8

1.0

1.2

Calcium Concentration

Luci

fera

se A

ctiv

ities

(F

irefly

/Ren

illa)

Luci

fera

se A

ctiv

ities

(F

irefly

/Ren

illa)

Effect of Extracellular Calcium and Phosphate on Fgf23 Promoter Activity

Lu

cife

rase

Act

ivit

ies

(Fir

efly

/Ren

illa)

1,25-(OH)2-vitamin D 3 Concentration

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 M 10-10 M 10-9 M 10-8 M

**

Effect of 1,25‑(OH)2D3 on Fgf23 Promoter Activity

Lu

cife

rase

Act

ivit

ies

(Fir

efly

/Ren

illa)

PTH Concentration

0 mM 1 mM 10 mM 100 mM

0.0

0.2

0.4

0.6

0.8

1.0

* * *

Effect of PTH on Fgf23 Promoter Activity

Vitamin D Stimulated Fgf23 Transcripts in Ros17/2.8 Cells

vehicle

Re

lati

ve

Co

nc

en

tra

tio

n o

f F

gf2

3 (

Fg

f23

/Pp

ia)

0

100

200

300

400

1,25-(OH)2D3.

8 Hours 24 Hours

2.40.7 16.00.9 (7 fold) 1.80.3 264.348.9 (147 fold)

P<0.01

P<0.01

Ser

um

In

tact

PT

H

(p

g/m

l)

Vehicle Calcitriol0

10

20

30

40

*

Vehicle Calcitriol

Ser

um

Cal

ciu

m

(mg

/dL

)

0

2

4

6

8

10

Vehicle Calcitriol

Ser

um

Ph

osp

ho

rus

(

mg

/dL

)

0

2

4

6

8

10

Ser

um

FG

F2

3

(p

g/m

l)

0

20

40

60

80

100

120

140

160

Vehicle Calcitriol

*A B

DC

Effect of 1,25‑(OH)2D3 on Serum Levels of Fgf23, PTH, Phosphorus and Calcium

100 ng/g/BW Calcitriol IP

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?

Bone

Kidney

Reabsorption of CaReabsorption of Ca2+ 2+

1,25(OH)1,25(OH)22vitamin D synthesisvitamin D synthesis

POPO442-2-absorptionabsorption

Parathyroid glands

PTHCa2+ absorption

Gut

PO42-absorption

Serum Serum POPO44

2-2-

FGF-23

Serum Ca2+

1,25(OH)1,25(OH)22vitamin Dvitamin D

Uncertain Role of FGF23 in CKD

• Circulating levels of FGF23 are increased in CKD.

100

101

102

103

104

105

106

FG

F-2

3 (R

U/m

l)

Control XLH Unknown ESRD

0

10

100

1000

10,000

100,000

4 5 6 7 8 9 10 11

-20x103

0

20x103

40x103

60x103

80x103

100x103

Phosphorus (mg/dl)

FG

F-2

3 (R

U/L

)

FGF-23 = -34456.23 + 7315.06 * PR-Square = 0.23

Circulating FGF23 Levels In ESRD (C-terminal assay)

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.

Intact FGF23 in ESRD 14 to 98,646 ng/L (nl 27.8 ± 9.0 ng/L )

Circulating FGF23 Levels In ESRD (Intact assay)

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

Sevelamer Hydrochloride and Calcium Bicarbonate Reduce Serum Fibroblast Growth Factor 23 Levels in Dialysis Patients

Vitamin D Treatment Is Associated With Increased FGF23 Levels In Dialysis Patients

Shohei N et al. Kidney Intern 67:1171-1178, 2005


• 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.

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

FGF23 Mitigates Hyperphosphatemia in CKD

Gutierrez O JASN 2204-2205, 2005

FGF23 Accentuates Calcitriol Deficiency in CKD

Factors Associated with calcitriol

Full multivariate β P R2

model .57 log FGF23 -20.4 0.008 sP -0.01 0.961 log PTH 0.17 0.280 sCa 0.16 0.396 25-OHD3 0.02 0.043 eGFR 0.01 0.067

Gutierrez O JASN 2204-2205, 2005




• 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

PO4 7.9-8.9 mg/dlTMP/GFR 3.5 mM/l 1,25(OH2)D3 65-85 pg/ml C-terminal FGF23 >1,800 Ru/mluCa 4.6 mg/kg BW

Tissue Calcification Serum Biochemistries

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




• 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 non-responding 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.

Documents

Evidence For A Novel Bone-Kidney Axis Regulating Systemic Phosphate Homeostasis