Journal Club: FGF23 Fails to Inhibit Uremic Parathyroid Glands
FGF23 Fails to Inhibit
Hitesh Patni, MD
Hofstra North Shore LIJ
Four molecular targets regulating parathyroid
functions have been identified:
◦ G protein coupled calcium sensing receptor
◦ Vitamin D receptor
◦ Extracellular phosphate sensor
◦ FGF 23 : FGFR-Klotho complex
Calcium acting through the CaSR is the major
◦ PTH transcription
◦ PTH secretion
◦ Parathyroid gland hyperplasia
Calcitriol acts on VDR in the parathyroid gland
◦ Suppress PTH transcription
◦ However physiologic role may be subordinate of
◦ VDR deficient mice: secondary hyperparathyroidism and
bone abnormalities can be corrected by normalizing
serum calcium concentration
Direct effects on parathyroid hormone production
◦ Regulates PTH mRNA levels
◦ Possibly by increasing post transcriptional PTH mRNA
Indirectly by lowering serum calcium by chelation
By suppressing 1-alpha hydroxylase
Recently been shown to target parathyroid gland
function via FGFR-Klotho complex and suppress
Parabiosis suggests a humoral factor is involved in X-
linked hypophosphatemia in mice.
Meyer RA Jr, Meyer MH, Gray RW. J Bone Miner Res. 1989
Department of Basic Sciences, Marquette University School of
Dentistry, Milwaukee, WI 53233.
X Linked Hypophosphatemia is the most common cause
of human vitamin D-resistant rickets.
It is characterized by
◦ low renal tubular reabsorption of phosphate,
◦ low plasma phosphate,
◦ absence of elevated 1,25-dihydroxyvitamin D
◦ osteomalacic bone
The Hyp mutation in mice produces the same
They have been used as a model.
Pilot experiment to test immunologic compatibility
Skin transplantation was performed between normal and
In each mouse two circular patches of skin were
removed from the lateral abdomen, and was exchanged
with another mouse, and the other was reattached to the
At 4 weeks of age.
A lateral incision was made through the skin from the
pelvic to the pectoral girdle. A transverse incision was
made through the abdominal muscles parallel to the
edge of the ribs. The margins of the muscles of the two
mice were joined with silk suture (5-0) to create a tunnel
connecting the peritoneal cavities. Finally, the margins of
the skin of the two mice were joined.
The normal mice joined to Hyp mice showed a
progressive diminution of plasma phosphate over the
next 3 weeks to approach the level of the Hyp mice.
These normal mice had a greater renal phosphate
excretion index than normal-normal pairs
This change is specific since the urinary losses of
potassium and magnesium were not significantly
Separation of normal-Hyp pairs 3 or 6 weeks after
parabiosis caused the normal mice to achieve normal
plasma phosphate levels within 24 h.
Presence of a phosphaturic factor in the Hyp mice
that can cross a parabiotic union into normal mice
and induce many of the symptoms of X-linked
The renal phosphate transport defect in normal mice
parabiosed to X-linked hypophosphatemic mice persists
Meyer RA Jr et al, Bone Miner Res. 1989 Aug;4(4):523-32.
Department of Basic Sciences, School of Dentistry, Marquette
University, Milwaukee, WI 53233.
Hyp mouse phenotype is neither corrected nor
transferred by renal transplantation.
The phosphate transport defect in Hyp mice, and likely
X-linked hypophosphatemia, is the result of a humoral
factor, and is not an intrinsic renal abnormality.
Nesbitt t et al, J Clin Invest. 1992 May;89(5):1453-9. Department of
Medicine, Duke University Medical Center, Durham, North Carolina 27710.
In tumor-induced osteomalacia, hypophosphatemia,
hyperphosphaturia, low plasma 1,25-dihydroxyvitamin D
concentrations, and osteomalacia, all biochemical and pathological
abnormalities disappear when the tumor is removed.
The medium in which sclerosing hemangioma cells from a patient
with oncogenic osteomalacia were cultured, inhibited phosphate
transport, without increasing cellular concentrations of cAMP.
The medium had PTH - like immunoreactivity but no PTH-related
protein immunoreactivity, and its action was not blocked by a PTH
Qiang C et al, N Engl J Med 1994; 330:1645-1649 June 9, 1994
Abnormal regulation of sodium phosphate cotransport in the
By positional cloning, they isolated a candidate gene from the HYP
region in Xp22.1.
This gene exhibits homology to a family of endopeptidase genes,
members of which are involved in the degradation or activation of a
variety of peptide hormones.
F Francis et al, Nature Genetics 11, 130 - 136 (1995)
Identification of genes responsible for inherited disorders involving
disturbances in phosphate homeostasis may provide insight into the
pathways that regulate phosphate balance.
Positional cloning approach : identify the ADHR gene.
3 missense mutations in four unrelated families affecting two
arginines, supports the speculation that the ADHR phenotype is
caused by a gain-of-function mechanism.
The existence of a phosphaturic factor termed 'phosphatonin' was
ADHR Consortium. Nat Genet. 2000 Nov;26(3):345-8.
Overproduction of FGF23 causes TIO
Mutations in the FGF23 gene result in autosomal
dominant hypophosphatemic rickets possibly by
preventing proteolytic cleavage.
Takashi S et al, Proc Natl Acad Sci U S A. 2001 May
In cultured renal proximal epithelial cells,
opossum kidney cells.
FGF-23 activated the mitogen-activated protein
The inhibitors of the MAPK pathway, PD98059
and SB203580, blocked the activity of FGF-23.
Yamashita t et al, J Biol Chem. 2002 Aug 2;277(31):28265-70. Epub
2002 May 24.
Klotho, a senescence-related molecule,
generates the FGF23 receptor.
FGFR1(IIIc), was directly converted by Klotho
into the FGF23 receptor.
Itaru U et al, Nature 444, 770-774, 7 December 2006
FGF23 acts directly on the parathyroid through the
MAPK pathway to decrease serum PTH.
Ben-dov IZ et al, J Clin Invest. 2007 Dec;117(12):4003-8.
FGF23 Fails to Inhibit Uremic
The article, JASN 2010The article, JASN 2010
• Male Wistar rats (250 g) from University of Cordoba
• 12-hour/12-hour light/dark cycle
• Ad libitum access to food (Ca 0.6%, P 0.6%, 100
IU/100 g vitaminD).
• Anesthetized with pentobarbital (50 mg/kg),and blood
was drained by aortic puncture; within 2 minutes,the
parathyroid glands were dissected under a
• Normal bovine parathyroid tissue, in small pieces
) were used when measurementsrequired,
large amount of tissue protein.
• Experimental diet contained normalCa (0.6%) and
high phosphate (1.2%).
• Sham rats fed a normal phosphorus diet (0.6%)
was used as acontrol.
• Serum ionized Ca levels : Ciba-Corning634 ISE
• Intact rat PTH levels : Elisa Kit
• Creatinine & phosphate : Spectrophotometry
• FGF23 levels : ELISA Kit
• 10 intact rat parathyroidglands or small (1 mm3
of bovine parathyroid tissue : Incubated in buffer
solution inside a nylon basket in individual wells with
constantshaking at 37°C in a humid atmosphere.
• 1.25 mM CaCl2 / 1 mM phosphate (NaH2PO4:Na2HPO4,
1:2 ratio) concentrationsin the medium.
• After incubation period: glands were transferred to
various Ca concentrationswith specific experimental
• The incubation mediumwas replaced hourly, and
the medium removed was stored for biochemical
• In other experiments, parathyroid glandswere
maintained in culture for 6 or 24 hours in a Ca
concentrationof 0.8 or 1.5 mM with or without the
addition FGF23 or othercompounds.
Cell viability : Mechanically dispersed cells
Labelled with Green fluordiacetate (Live) and red
Assessed by flow cytometry
Only tissue sampleswith >90% viable cells were
used in the experiments.
• Intact rat parathyroid glands.
• At the end of the experiment(in vivo or in vitro),
isolated cells were obtained from theglands under the
inverted microscope using tips followed by gentle
pipetting in a 50-µl volume ofPBS
• Cells were processed immediately for flow cytometric
• A total of 10,000 clean cellnuclei were acquired
• The percentage of cellsin the S phase was used as a
marker of cell proliferation.
Total RNA was extracted (acid guanidinium
RNA was dissolved in nuclease-freewater and
heated for 10 minutes at 60°C.
Quantified by spectrophotometry
PTH, VDR, CaR, FGFR1, and Klotho mRNA
levels were determined versus β-actin mRNA by
Figure 1. FGF23 reduces PTH secretion in normal rat parathyroid glands
(B) Regulation of PTH, VDR, and CaR mRNA levels by FGF23 in vitro.
Intact rat parathyroid glands are incubated for 6 hours in Ca concentrations
of 0.8 and 1.5 mM with or without FGF23.
In normal rats, FGF23 reduces parathyroid cell
function and parathyroid cells proliferation
In hyperplastic parathyroid glands from rats with
renal failure, there is a reduction in receptors for
FGF23 and in Klotho
Leads to tissue resistance to the action of FGF23.