FGF23 and Klotho in Bone Mineralization in CKD
Katherine Wesseling Perry, MD MSCRAssociate Professor of Pediatrics
David Geffen School of Medicine at UCLA3/9/2019
Objectives
• 1. To understand the direct effects of FGF23 on mineral metabolism
• 2. To describe changes in FGF23 and Klotho levels in CKD
• 3. To understand complexities of the studies evaluating the direct effect of FGF23 and Klotho on bone in human CKD
FGF23: what it doesToo much: hypophosphatemic rickets
Too little: tumoral calcinosis
Is there a direct effect of FGF23 on bone?
FGF23: where it’s made
BoneCorrelation with blood levels (anuric):r=0.7; p
Klotho is expressed in osteocytes;Klotho null mice have osteoporosis
Kuro-o M et al Nature 1997
Rhee Y et al Bone 2011
Wild type Klotho-/-
Mineralization defects co-exist with increased FGF23 in early CKD
% o
f Sub
ject
s w
ith A
bnor
mal
Val
ues
for
Bon
e Tu
rnov
eran
d M
iner
aliz
atio
n
0
20
40
60
80
100
CKD Stage2 3 4
Wesseling-Perry K et al CJASN 2013
Bone turnoverMineralization
Bone FGF23 (osteocytes/Bone Area)
OS/BS r = - 0.60, p
Direct effects of FGF23 and Klotho: confounding effects of 1,25D
Andrukhova O et al JBMR 2017
1,25D directly inhibits bone mineralization
Lieben L et al J Clin Invest 2012
FGF23 null fetuses have normal phosphate and skeletons
Ma Y et al Endocrinology 2017
Developmental change in receptors or a sign of very little effect on bone?
Klotho null fetuses also have normal skeletons
Ma Y et al Endocrinology 2017
Developmental change in receptors or a sign of very little effect on bone?
Forced FGF23 over-expression suppresses osteoblast
differentiation and mineralization
Wang H et al JBMR 2008
Osteocyte-specific Klotho deletion increases bone formation and mass
Komaba H et al Kidney Int 2017
Overexpression of Klotho inhibits mineralization of MC3T3 cells
Komaba H et al Kidney Int 2017
Exogenous FGF23 and Klotho together inhibit osteoblast
mineralization
Shalhoub V et al Calcif Tissue Int 2011
MC3T3.E1
FGF23 and Klotho inhibit osteoblastic Wnt signaling in CKD
Carillo-Lopez N et al Kidney Int 2016
Whole animal UMR106-01
UMR106-01
UMR106-01
Klotho is down-regulated in CKD bone; particularly in high phosphate
conditions
Komaba H et al Kidney Int 2017
CKD mimics the effect of Klotho-/-; there is no added
effect on bone
Komaba H et al Kidney Int 2017
Healthy controls 12
Dialysis patients 21
0
0.5
1
1.5
2
Aliz
arin
Red
(abs
orba
nce)
1 week 2 weeks 3 weeks
*
Primary osteoblasts from dialysis patients mineralize poorly in culture
Pereira RC et al Kidney Int 2018
0
4
8
12
16Alkaline phosphatase
base
*
1 week 2 weeks
*
**
Gen
e ex
pres
sion
(fol
d co
ntro
l at b
asel
ine)
Gen
e ex
pres
sion
(fol
d co
ntro
l at b
asel
ine)
Gen
e ex
pres
sion
(fol
d co
ntro
l at b
asel
ine)
0.1
2
40
800
base 2 weeks
**
1 week
Runx2
ControlCKD
A B
C
0
2
4
6
8
10
*
**Osteocalcin
base 2 weeks1 week
… this equates to defective maturation
Pereira RC Kidney Int 2018
Pereira RC et al Kidney Int 2018
0
0.2
0.4
0.6
0 100
FGF23 (nm)
Aliz
arin
Red
(abs
orba
nce)
* *
ControlCKD
Phosphate, but not FGF23, plays a role in osteoblast maturation
0
0.2
0.4
0.6
0.8
1
0 2 5 10
β-glycerol-phosphate (mM)
Aliz
arin
Red
(abs
orba
nce)
**
*
Pereira RC Kidney Int 2018
DAPI FGF23 e11/gp38 merge
BMTB
BMTB
BMTB
BMTB
A
In human CKD, FGF23 is a marker of early osteocytes
Pereira RC et al Kidney Int 2018
A
#1
#2
#3
#4
FGF23
FGF2320.89
21.40
17.86
24.16
FGF23
time
Cal
cium
con
tent
#1
#2 #3#4 Active
boneformation
B C
D E F
FGF23-expressing osteocytes characterize early 2o mineralization
Pereira RC et al Kidney Int 2018
Summary• FGF23 and Klotho both affect phosphate and 1,25D and
both are regulated by 1,25D
• Klotho (and maybe FGF23+Klotho) may suppress osteoblast maturation
• While CKD bone disease mimics the effects of Klotho deficiency, primary human osteoblasts in vitro maintain intrinsic defects in maturation
• FGF23 may be a marker of early osteocytes in CKD bone
FGF23 and Klotho in Bone Mineralization in CKD ObjectivesFGF23: what it doesFGF23: where it’s madeKlotho is expressed in osteocytes;�Klotho null mice have osteoporosisMineralization defects co-exist with increased FGF23 in early CKDDirect effects of FGF23 and Klotho: confounding effects of 1,25D1,25D directly inhibits bone mineralizationFGF23 null fetuses have normal phosphate and skeletonsKlotho null fetuses also have normal skeletonsForced FGF23 over-expression suppresses osteoblast differentiation and mineralizationOsteocyte-specific Klotho deletion increases bone formation and massOverexpression of Klotho inhibits mineralization of MC3T3 cellsExogenous FGF23 and Klotho together inhibit osteoblast mineralizationFGF23 and Klotho inhibit osteoblastic Wnt signaling in CKDKlotho is down-regulated in CKD bone; particularly in high phosphate conditionsCKD mimics the effect of Klotho-/-; there is no added effect on boneSlide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Summary