FGF23 Fails to Inhibit
Uremic Parathyroid
Glands
Hitesh Patni, MD
Hofstra North Shore LIJ
 Four molecular targets regulating parathyroid
functions have been identified:
◦ G protein coupled calcium sensing recept...
 Calcium acting through the CaSR is the major
regulator
◦ PTH transcription
◦ PTH secretion
◦ Parathyroid gland hyperplas...
 Calcitriol acts on VDR in the parathyroid gland
◦ Suppress PTH transcription
◦ However physiologic role may be subordina...
 Direct effects on parathyroid hormone production
◦ Regulates PTH mRNA levels
◦ Possibly by increasing post transcription...
 Recently been shown to target parathyroid gland
function via FGFR-Klotho complex and suppress
PTH production
Parabiosis suggests a humoral factor is involved in X-
linked hypophosphatemia in mice.
 Meyer RA Jr, Meyer MH, Gray RW. ...
 X Linked Hypophosphatemia is the most common cause
of human vitamin D-resistant rickets.
 It is characterized by
◦ low ...
 Pilot experiment to test immunologic compatibility
 Skin transplantation was performed between normal and
Hyp mice.
 I...
 At 4 weeks of age.
 A lateral incision was made through the skin from the
pelvic to the pectoral girdle. A transverse i...
 The normal mice joined to Hyp mice showed a
progressive diminution of plasma phosphate over the
next 3 weeks to approach...
 Presence of a phosphaturic factor in the Hyp mice
that can cross a parabiotic union into normal mice
and induce many of ...
 The renal phosphate transport defect in normal mice
parabiosed to X-linked hypophosphatemic mice persists
after parathyr...
 Hyp mouse phenotype is neither corrected nor
transferred by renal transplantation.
 The phosphate transport defect in H...
 In tumor-induced osteomalacia, hypophosphatemia,
hyperphosphaturia, low plasma 1,25-dihydroxyvitamin D
concentrations, a...
 Abnormal regulation of sodium phosphate cotransport in the
proximal tubules
 By positional cloning, they isolated a can...
 Identification of genes responsible for inherited disorders involving
disturbances in phosphate homeostasis may provide ...
 Overproduction of FGF23 causes TIO
 Mutations in the FGF23 gene result in autosomal
dominant hypophosphatemic rickets p...
 In cultured renal proximal epithelial cells,
opossum kidney cells.
 FGF-23 activated the mitogen-activated protein
kina...
 Klotho, a senescence-related molecule,
generates the FGF23 receptor.
 FGFR1(IIIc), was directly converted by Klotho
int...
 FGF23 acts directly on the parathyroid through the
MAPK pathway to decrease serum PTH.
 Ben-dov IZ et al, J Clin Invest...
Copyright ©2010 American Society of Nephrology
Gutierrez, O. M. Clin J Am Soc Nephrol 2010;5:1710-1716
Figure 1. Classic v...
Copyright ©2010 American Society of Nephrology
Wolf, M. J Am Soc Nephrol 2010;21:1427-1435
Figure 2. Physiologic actions o...
Copyright ©2010 American Society of Nephrology
Wolf, M. J Am Soc Nephrol 2010;21:1427-1435
Figure 3. Temporal aspects of d...
FGF23 Fails to Inhibit Uremic
Parathyroid Glands
The article, JASN 2010The article, JASN 2010
• Male Wistar rats (250 g) from University of Cordoba
(Spain).
• 12-hour/12-hour light/dark cycle
• Ad libitum access to f...
• Experimental diet contained normalCa (0.6%) and
high phosphate (1.2%).
• Sham rats fed a normal phosphorus diet (0.6%)
w...
• 10 intact rat parathyroidglands or small (1 mm3
) pieces
of bovine parathyroid tissue : Incubated in buffer
solution ins...
• The incubation mediumwas replaced hourly, and
the medium removed was stored for biochemical
determinations.
• In other e...
 Cell viability : Mechanically dispersed cells
 Labelled with Green fluordiacetate (Live) and red
propidium iodine(Dead)...
• Intact rat parathyroid glands.
• At the end of the experiment(in vivo or in vitro),
isolated cells were obtained from th...
 Total RNA was extracted (acid guanidinium
thiocyanate-phenol-chloroform mixture)
 RNA was dissolved in nuclease-freewat...
Copyright ©2010 American Society of Nephrology
Figure 1. FGF23 reduces PTH secretion in normal rat parathyroid glands
A) P...
Figure 1. FGF23 reduces PTH secretion in normal rat parathyroid glands
(B) Regulation of PTH, VDR, and CaR mRNA levels by ...
Copyright ©2010 American Society of Nephrology
Figure 2. FGF23 increases VDR in normal rat parathyroid
glands
Copyright ©2010 American Society of Nephrology
Figure 2. FGF23 increases ERK1/2 phosphorylation in normal rat
parathyroid ...
Copyright ©2010 American Society of Nephrology
Figure 2. FGF23 increases CaR expression in normal rat parathyroid
glands
Copyright ©2010 American Society of Nephrology
Figure 3. FGF23 increases ERK1/2 phosphorylation in
bovine parathyroid tiss...
Copyright ©2010 American Society of Nephrology
Figure 4. FGF23 decreases parathyroid cell proliferation in
normal rat para...
Copyright ©2010 American Society of Nephrology
Figure 4. FGF23 decreases parathyroid cell proliferation in
normal rat para...
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 +- 0.0...
Copyright ©2010 American Society of Nephrology
Figure 5. FGF23 reduces PTH secretion and
synthesis in normal but not in ur...
In Vivo
Copyright ©2010 American Society of Nephrology
Figure 6. FGF23 has no effect on parathyroid cell proliferation in uremic r...
Copyright ©2010 American Society of Nephrology
Figure 6. FGF23 has no effect on parathyroid cell
proliferation in uremic r...
Copyright ©2010 American Society of Nephrology
Figure 6. FGF23 has no effect on parathyroid cell proliferation in uremic r...
Copyright ©2010 American Society of Nephrology
Figure 7. FGF23 does not increase VDR expression or activate ERK1/2 in para...
Copyright ©2010 American Society of Nephrology
Figure 8. FGF23 increases VDR expression and activates ERK1/2 in
normal rat...
Copyright ©2010 American Society of Nephrology
Figure 9. FGFR1 and Klotho expression are reduced in parathyroid glands fro...
Copyright ©2010 American Society of Nephrology
Figure 10. Effect of extracellular Ca (A), phosphate (B), and FGF23 (C and ...
Copyright ©2010 American Society of Nephrology
Figure 10. Effect of extracellular Ca (A), phosphate (B), and FGF23 (C
and ...
 In normal rats, FGF23 reduces parathyroid cell
function and parathyroid cells proliferation
 In hyperplastic parathyroi...
Journal Club: FGF23 Fails to Inhibit Uremic Parathyroid Glands
Journal Club: FGF23 Fails to Inhibit Uremic Parathyroid Glands
Journal Club: FGF23 Fails to Inhibit Uremic Parathyroid Glands
Journal Club: FGF23 Fails to Inhibit Uremic Parathyroid Glands
Journal Club: FGF23 Fails to Inhibit Uremic Parathyroid Glands
Journal Club: FGF23 Fails to Inhibit Uremic Parathyroid Glands
Journal Club: FGF23 Fails to Inhibit Uremic Parathyroid Glands
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Journal Club: FGF23 Fails to Inhibit Uremic Parathyroid Glands

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

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Journal Club: FGF23 Fails to Inhibit Uremic Parathyroid Glands

  1. 1. FGF23 Fails to Inhibit Uremic Parathyroid Glands Hitesh Patni, MD Hofstra North Shore LIJ
  2. 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. 3.  Calcium acting through the CaSR is the major regulator ◦ PTH transcription ◦ PTH secretion ◦ Parathyroid gland hyperplasia
  4. 4.  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
  5. 5.  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
  6. 6.  Recently been shown to target parathyroid gland function via FGFR-Klotho complex and suppress PTH production
  7. 7. 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.
  8. 8.  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.
  9. 9.  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.
  10. 10.  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.
  11. 11.  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.
  12. 12.  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.
  13. 13.  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.
  14. 14.  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.
  15. 15.  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
  16. 16.  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
  17. 17.  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.
  18. 18.  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
  19. 19.  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.
  20. 20.  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
  21. 21.  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.
  22. 22. 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
  23. 23. Copyright ©2010 American Society of Nephrology Wolf, M. J Am Soc Nephrol 2010;21:1427-1435 Figure 2. Physiologic actions of FGF23
  24. 24. 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
  25. 25. FGF23 Fails to Inhibit Uremic Parathyroid Glands The article, JASN 2010The article, JASN 2010
  26. 26. • 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.
  27. 27. • 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
  28. 28. • 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.
  29. 29. • 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.
  30. 30.  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.
  31. 31. • 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.
  32. 32.  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.
  33. 33. 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.
  34. 34. 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.
  35. 35. Copyright ©2010 American Society of Nephrology Figure 2. FGF23 increases VDR in normal rat parathyroid glands
  36. 36. Copyright ©2010 American Society of Nephrology Figure 2. FGF23 increases ERK1/2 phosphorylation in normal rat parathyroid glands
  37. 37. Copyright ©2010 American Society of Nephrology Figure 2. FGF23 increases CaR expression in normal rat parathyroid glands
  38. 38. Copyright ©2010 American Society of Nephrology Figure 3. FGF23 increases ERK1/2 phosphorylation in bovine parathyroid tissue
  39. 39. Copyright ©2010 American Society of Nephrology Figure 4. FGF23 decreases parathyroid cell proliferation in normal rat parathyroid glands in low-Ca medium
  40. 40. Copyright ©2010 American Society of Nephrology Figure 4. FGF23 decreases parathyroid cell proliferation in normal rat parathyroid glands in low-Ca medium
  41. 41. 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
  42. 42. 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
  43. 43. In Vivo
  44. 44. Copyright ©2010 American Society of Nephrology Figure 6. FGF23 has no effect on parathyroid cell proliferation in uremic rats
  45. 45. Copyright ©2010 American Society of Nephrology Figure 6. FGF23 has no effect on parathyroid cell proliferation in uremic rats
  46. 46. Copyright ©2010 American Society of Nephrology Figure 6. FGF23 has no effect on parathyroid cell proliferation in uremic rats
  47. 47. Copyright ©2010 American Society of Nephrology Figure 7. FGF23 does not increase VDR expression or activate ERK1/2 in parathyroid glands from uremic rats
  48. 48. Copyright ©2010 American Society of Nephrology Figure 8. FGF23 increases VDR expression and activates ERK1/2 in normal rats in vivo
  49. 49. Copyright ©2010 American Society of Nephrology Figure 9. FGFR1 and Klotho expression are reduced in parathyroid glands from uremic rats in vivo
  50. 50. 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
  51. 51. 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
  52. 52.  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.

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