Dr. Patni goes over an excellent journal club on a basic science article on FGF23 and bone metabolism.

1. FGF23 Fails to Inhibit
Uremic Parathyroid
Glands
Hitesh Patni, MD
Hofstra North Shore LIJ

2.  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

3.  Calcium acting through the CaSR is the major
regulator
◦ PTH transcription
◦ PTH secretion
◦ Parathyroid gland hyperplasia

5.  Calcitriol acts on VDR in the parathyroid gland
◦ Suppress PTH transcription
◦ However physiologic role may be subordinate of
Calcium.
◦ VDR deficient mice: secondary hyperparathyroidism and
bone abnormalities can be corrected by normalizing
serum calcium concentration

6.  Direct effects on parathyroid hormone production
◦ Regulates PTH mRNA levels
◦ Possibly by increasing post transcriptional PTH mRNA
message stability
 Indirectly by lowering serum calcium by chelation
 By suppressing 1-alpha hydroxylase

7.  Recently been shown to target parathyroid gland
function via FGFR-Klotho complex and suppress
PTH production

8. 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
Aug;4(4):493-500.
 Department of Basic Sciences, Marquette University School of
Dentistry, Milwaukee, WI 53233.

9.  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
abnormalities
 They have been used as a model.

10.  Pilot experiment to test immunologic compatibility
 Skin transplantation was performed between normal and
Hyp mice.
 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
same mouse.

12.  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.

13.  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
changed.
 Separation of normal-Hyp pairs 3 or 6 weeks after
parabiosis caused the normal mice to achieve normal
plasma phosphate levels within 24 h.

14.  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
hypophosphatemia.

15.  The renal phosphate transport defect in normal mice
parabiosed to X-linked hypophosphatemic mice persists
after parathyroidectomy.
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.

16.  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.

17.  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
antagonist.
 Qiang C et al, N Engl J Med 1994; 330:1645-1649 June 9, 1994

18.  Abnormal regulation of sodium phosphate cotransport in the
proximal tubules
 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)
doi:10.1038/ng1095-130

19.  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
hypothesized
 ADHR Consortium. Nat Genet. 2000 Nov;26(3):345-8.

20.  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
22;98(11):6500-5

21.  In cultured renal proximal epithelial cells,
opossum kidney cells.
 FGF-23 activated the mitogen-activated protein
kinase pathway
 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.

22.  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

23.  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.

24. Copyright ©2010 American Society of Nephrology
Gutierrez, O. M. Clin J Am Soc Nephrol 2010;5:1710-1716
Figure 1. Classic versus more contemporary renditions of the "trade-off" hypothesis

25. Copyright ©2010 American Society of Nephrology
Wolf, M. J Am Soc Nephrol 2010;21:1427-1435
Figure 2. Physiologic actions of FGF23

26. Copyright ©2010 American Society of Nephrology
Wolf, M. J Am Soc Nephrol 2010;21:1427-1435
Figure 3. Temporal aspects of disordered phosphorus metabolism in progressive CKD and after kidney
transplantation

27. FGF23 Fails to Inhibit Uremic
Parathyroid Glands
The article, JASN 2010The article, JASN 2010

28. • Male Wistar rats (250 g) from University of Cordoba
(Spain).
• 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
dissecting microscope.
• Normal bovine parathyroid tissue, in small pieces
(<1 mm3
) were used when measurementsrequired,
large amount of tissue protein.

30. • 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
Ca2
/pH Analyzer
• Intact rat PTH levels : Elisa Kit
• Creatinine & phosphate : Spectrophotometry
• FGF23 levels : ELISA Kit

31. • 10 intact rat parathyroidglands or small (1 mm3
) pieces
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
treatments.

32. • The incubation mediumwas replaced hourly, and
the medium removed was stored for biochemical
determinations.
• 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.

33.  Cell viability : Mechanically dispersed cells
 Labelled with Green fluordiacetate (Live) and red
propidium iodine(Dead)
 Assessed by flow cytometry
 Only tissue sampleswith >90% viable cells were
used in the experiments.

34. • 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
analysis.
• A total of 10,000 clean cellnuclei were acquired
• The percentage of cellsin the S phase was used as a
marker of cell proliferation.

35.  Total RNA was extracted (acid guanidinium
thiocyanate-phenol-chloroform mixture)
 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
real-time RT–PCR.

39. Copyright ©2010 American Society of Nephrology
Figure 1. FGF23 reduces PTH secretion in normal rat parathyroid glands
A) Parathyroid glands are incubated in 1.25 mM Ca and then shifted to 0.8
mM to show a functional response. The effect of addition of rat FGF23 is
evaluated in low-Ca (0.8 mM) and high-Ca (1.5 mM) concentrations.

40. 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.

41. Copyright ©2010 American Society of Nephrology
Figure 2. FGF23 increases VDR in normal rat parathyroid
glands

42. Copyright ©2010 American Society of Nephrology
Figure 2. FGF23 increases ERK1/2 phosphorylation in normal rat
parathyroid glands

43. Copyright ©2010 American Society of Nephrology
Figure 2. FGF23 increases CaR expression in normal rat parathyroid
glands

44. Copyright ©2010 American Society of Nephrology
Figure 3. FGF23 increases ERK1/2 phosphorylation in
bovine parathyroid tissue

45. Copyright ©2010 American Society of Nephrology
Figure 4. FGF23 decreases parathyroid cell proliferation in
normal rat parathyroid glands in low-Ca medium

46. Copyright ©2010 American Society of Nephrology
Figure 4. FGF23 decreases parathyroid cell proliferation in
normal rat parathyroid glands in low-Ca medium

47. Control rats Uremic Rats
S Creatinine 0.58 +- 0.02 1.40 +- 0.04
S Phosphate 6.4 +- 0.3 15.5 +- 0.4
S I Calcium 1.24 +- 0.03 0.83 +- 0.07
FGF23 325 +- 59 1835 +- 694

48. Copyright ©2010 American Society of Nephrology
Figure 5. FGF23 reduces PTH secretion and
synthesis in normal but not in uremic parathyroid
glands
In Vivo In Vitro

49. In Vivo

50. Copyright ©2010 American Society of Nephrology
Figure 6. FGF23 has no effect on parathyroid cell proliferation in uremic rats

51. Copyright ©2010 American Society of Nephrology
Figure 6. FGF23 has no effect on parathyroid cell
proliferation in uremic rats

52. Copyright ©2010 American Society of Nephrology
Figure 6. FGF23 has no effect on parathyroid cell proliferation in uremic rats

53. Copyright ©2010 American Society of Nephrology
Figure 7. FGF23 does not increase VDR expression or activate ERK1/2 in parathyroid glands from uremic rats

54. Copyright ©2010 American Society of Nephrology
Figure 8. FGF23 increases VDR expression and activates ERK1/2 in
normal rats in vivo

55. Copyright ©2010 American Society of Nephrology
Figure 9. FGFR1 and Klotho expression are reduced in parathyroid glands from uremic rats in vivo

56. Copyright ©2010 American Society of Nephrology
Figure 10. Effect of extracellular Ca (A), phosphate (B), and FGF23 (C and D) on FGFR1
and Klotho expression in parathyroid glands from uremic rats in vitro

57. Copyright ©2010 American Society of Nephrology
Figure 10. Effect of extracellular Ca (A), phosphate (B), and FGF23 (C
and D) on FGFR1 and Klotho expression in parathyroid glands from
uremic rats in vitro

58.  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.