Journal Club: FGF23 Fails to Inhibit Uremic Parathyroid Glands

FGF23 Fails to Inhibit Uremic Parathyroid Glands

Hitesh Patni, MDHofstra North Shore LIJ Medical School

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 regulator◦ 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

Calcium.◦ 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 message stability

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 PTH production

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.

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.

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.

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 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.

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.

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.

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 antagonist.

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

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

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.

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

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.

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.

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


Wolf, M. J Am Soc Nephrol 2010;21:1427-1435

Figure 2. Physiologic actions of FGF23


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

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 food (Ca 0.6%, P 0.6%, 100

IU/100 g vitamin D). • 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 measurements

required, large amount of tissue protein.

• Experimental diet contained normal Ca (0.6%) and high phosphate (1.2%).

• Sham rats fed a normal phosphorus diet (0.6%) was used as a control.

• Serum ionized Ca levels : Ciba-Corning 634 ISE Ca2/pH Analyzer

• Intact rat PTH levels : Elisa Kit• Creatinine & phosphate :

Spectrophotometry

• FGF23 levels : ELISA Kit

• 10 intact rat parathyroid glands or small (1 mm3) pieces of bovine parathyroid tissue : Incubated in buffer solution inside a nylon basket in individual wells with constant shaking at 37°C in a humid atmosphere.

• 1.25 mM CaCl2 / 1 mM phosphate (NaH2PO4:Na2HPO4, 1:2 ratio) concentrations in the medium.

• After incubation period: glands were transferred to various Ca concentrations with specific experimental treatments.

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

• In other experiments, parathyroid glands

were maintained in culture for 6 or 24 hours in a Ca concentration of 0.8 or 1.5 mM with or without the addition FGF23 or other

compounds.

Cell viability : Mechanically dispersed cells Labelled with Green fluordiacetate (Live)

and red propidium iodine (Dead) Assessed by flow cytometry Only tissue samples with >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 the

glands under the inverted microscope using tips followed by gentle pipetting in a 50-µl volume of PBS

• Cells were processed immediately for flow cytometric analysis.

• A total of 10,000 clean cell nuclei were acquired• The percentage of cells in the S phase was used

as a marker of cell proliferation.

Total RNA was extracted (acid guanidinium thiocyanate-phenol-chloroform mixture)

RNA was dissolved in nuclease-free water 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.


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.

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.


Figure 2. FGF23 increases VDR in normal rat parathyroid glands


Figure 2. FGF23 increases ERK1/2 phosphorylation in normal rat parathyroid glands


Figure 2. FGF23 increases CaR expression in normal rat parathyroid glands


Figure 3. FGF23 increases ERK1/2 phosphorylation in bovine parathyroid tissue


Figure 4. FGF23 decreases parathyroid cell proliferation in normal rat parathyroid glands in low-Ca medium


Figure 4. FGF23 decreases parathyroid cell proliferation in normal rat parathyroid glands in low-Ca medium

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


Figure 5. FGF23 reduces PTH secretion and synthesis in normal but not in uremic parathyroid

glands

In Vivo In Vitro

In Vivo


Figure 6. FGF23 has no effect on parathyroid cell proliferation in uremic rats






Figure 7. FGF23 does not increase VDR expression or activate ERK1/2 in parathyroid glands from uremic rats


Figure 8. FGF23 increases VDR expression and activates ERK1/2 in normal rats in vivo


Figure 9. FGFR1 and Klotho expression are reduced in parathyroid glands from uremic rats in vivo


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


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

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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Journal Club: FGF23 Fails to Inhibit Uremic Parathyroid Glands