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Evidence For A Novel Bone-Kidney Axis Regulating Systemic Phosphate Homeostasis

Evidence For A Novel Bone-Kidney Axis Regulating Systemic Phosphate Homeostasis. L. Darryl Quarles, M.D. Summerfield Endowed Professor of Nephrology University Of Kansas Medical Center. Learning Objectives. Examine the role of hyperphosphatemia in vascular calcifications and mortality

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Evidence For A Novel Bone-Kidney Axis Regulating Systemic Phosphate Homeostasis

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  1. Evidence For A Novel Bone-Kidney Axis Regulating Systemic Phosphate Homeostasis L. Darryl Quarles, M.D. Summerfield Endowed Professor of Nephrology University Of Kansas Medical Center

  2. Learning Objectives • Examine the role of hyperphosphatemia in vascular calcifications and mortality • Discuss the functions of: • Phex • FGF23 • Klotho • Propose a model showing how these factors may participate in a novel bone-kidney axis regulating systemic phosphate homeostasis and mineralization. • Examine the role of FGF23 in the pathogenesis of disordered mineral homeostasis in CKD. } Novel Genes Regulating Phosphate Homeostasis

  3. Soft-Tissue CalcificationsAre Widespread in Dialysis Patients Calciphylaxis Soft Tissue Calcification Soft Tissue Calcification • Whole-body scan—dark areas indicate calcium uptake Image from Richardson MS. 1999. Available at ttp://www.rad.washington.edu/maintf/cases/unk39/answers.html.. Image from Block GA. 2004. Kok M, et al. Clin Nucl Med. 2003;28:144-145.

  4. Coronary-Artery Calcification Is Prevalent in ESRD A B Goodman WG et al N Engl J Med.342:1478-83, 2000.

  5. Arterial Calcification Status Increases All-Cause (A) and Cardiovascular Mortality (B) in ESRD Patients London, G. M. et al. Nephrol. Dial. Transplant. 2003 18:1731-1740

  6. Uncertain Pathogenesis of Vascular Calcification in CKD • Risk factors include: 70% of increased CV risk accounted for by traditional factors • Age • Hypertension • Diabetes mellitus • Inflamation-C-reactive protein CKD-related Factors • Time on dialysis • Hyperphosphataemia • Calcium intake • Treatment with Vitamin D?

  7. 1.8 2.2 2.0 1.6 1.8 1.4 1.6 1.2 1.4 1.0 1.2 0.8 1.0 0.6 0.6 0.0 0.0 Serum Calcium Serum Phosphorus Referent group Referent group Relative Risk of Death (n=40,538) Relative Risk of Death (n=40,538) < 3.0 > 9.0 > 11.0 < 8.0 5.0–6.0 9.0–9.5 6.0–7.0 9.5–10.0 3.0–4.0 8.0–8.5 8.0–9.0 10.5–11.0 4.0–5.0 8.5–9.0 7.0–8.0 10.0–10.5 Serum Phosphorus (mg/dL) Corrected Serum Calcium (mg/dL) Disturbances in Mineral Metabolism Are Associated With Increased Risk of Mortality in Hemodialysis Adapted from Block GA, et al. J Am Soc Nephrol. 2004;15:2208-2218.

  8. Inorganic Phosphate But Not Calcium Concentration Affects Mineralization [Ca] [Pi] [Ca] [Pi] von Kossa Alizarin red Murshed M, et al Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev. 19:1093-104, 2005.

  9. Genetic Rescue of the Mgp -/- Phenotype Murshed M, et al Unique coexpression in osteoblasts of broadly expressed genes accounts for the spatial restriction of ECM mineralization to bone. Genes Dev. 19:1093-104, 2005.

  10. Vascular Calcification & Morbidity/Mortality in CKD • Consensus regarding role of hyperphosphatemia, but lack of prospective studies demonstrating that interventions to lower serum phosphate improves survival. • Understanding of the hormonal cascades regulating phosphate homeostasis may provide insights into additional pathways affecting the systemic complications of hyperphosphatemia.

  11. Regulation of Phosphorus Homeostasis Is there a hormonal cascade regulating serum phosphorus concentrations independent of PTH?

  12. PTH-Independent Hypophosphatemic Disorders • Tumor-Induced Osteomalacia (TIO). • Autosomal Dominant Hypo-phosphatemic Rickets (ADHR). • McCune-Albright/Bone Fibrous Dysplasia. • X-Linked Hypophosphatemic Rickets (XLH). • Linear Nevus Sebaceous Syndrome.

  13. X-Linked Hypophosphatemic Rickets (XLH):Clinical Features • Most common inherited form of rickets. • X-linked dominant disorder. • Phenotype: • Renal • Decreased renal tubular reabsorption of phosphate. • Aberrant regulation of 1,25 (OH)2 Vitamin D3 production. • Skeletal • Defective calcification of cartilage (rickets) and bone (osteomalacia). • Growth retardation.

  14. XLH:Genetic Abnormality • PHEX gene (PHosphate regulating gene homologous to Endopeptidases on X Chr). • Disease gene encodes a member of M13 family of Type II transmembrane zinc metallo-endopeptidase. • Mutations have been identified in 86% of familial and 57% of sporadic cases of XLH. • Phex substrates are likely responsible for renal and skeletal phenotypes in XLH. • Phex could convert a prohormone to a phosphate- conserving factor, or inactivate a phosphaturic hormone and/or mineralization inhibitor (most likely).

  15. Phex Function: Lesions from the Hyp mouse homologue of XLH • Expresses the major phenotypic features of XLH. • Mouse Phex cDNA sequence is highly homologous to that of humans. • Hyp has a 3' Phex deletion creating a truncated endopeptidase lacking the catalytic domain. • Systemic/humoral phosphaturic factor (“Phosphatonin”) identified by parabiosis and cross-transplant studies. • Autocrine/paracrine nascent defect in Hyp-derived osteoblasts leading to impaired mineralization independent of hypophosphatemia, caused by inhibitor of mineralization (“Minhibin”).

  16. Shared Pathophysiology of Hypophosphatemic Disorders? Human Diseases Mouse Homologue Abnormal Gene Expression/Activity ADH FGF23 None TIO/OHO FGF23, PHEX, MEPE, DMP-1, HSP-90, Osteopontin None MAS None (GNAS1 )-activating mutations, FGF23 PHEX/Phex, MEPE, FGF23 Hyp, Gy XLH

  17. FGF23: A Candidate for Phosphatonin? • FGF23 is a ~32 kDa (251 amino acids) protein with an N-terminal region containing the FGF homology domain and a novel 71 aa C-terminus that has phosphaturic activity in vivo. • FGF23 is overproduced by tumors causing tumor-induced osteomalacia (TIO). • Autosomal dominant hypophosphatemicrickets (ADHR) is caused by missense mutations of the 176-RXXR-179 motif in FGF-23 preventing its processing into inactive N- and C-terminal fragments. • FGF23 is proposed to be a substrate for PHEX Phex-Dependent Cleavage And Inactivation Of The Phosphaturic Hormone FGF23 Hypothesis

  18. Is FGF23 Phosphatonin? • To determine whether FGF23 is involved in the pathogenesis of XLH we: • Examined FGF23 levels in XLH and Hyp. • Confirmed that FGF23 has phosphaturic activity. • Determined whether FGF23 deficiency rescues the hypophosphatemia in Hyp mice. • Assessed if FGF23 is a substrate for Phex.

  19. Serum Phosphorus (A), Serum FGF23 (B) And Their Correlation (C) In Subjects With XLH Weber T. Liu S, Quarles LD J Bone Mineral Res. 2003.

  20. Increased Circulating fgf23 Levels in Hyp

  21. Administration of FGF23 to Mice Induces Hypophosphatemia

  22. Is FGF23 The Phosphaturic Factor In XLH/Hyp? • To determined if FGF23 deficiency rescues the hypophosphatemia in Hyp mice, we: • Generated FGF23 null mice, • Transferred FGF23 deficiency onto the Hyp mouse background to determine if superimposed FGF23 deficiency rescued the hypophosphatemia in Hyp mice.

  23. Exon1 Exon2 Exon3 Targeting Strategy Used To Disrupt Fgf23 And Genotyping of Fgf23 Deficient Mice A 5’ Flanking S H H S Fgf23 Gene TK Neo Exon2 Exon3 N H Targeting Construct H EGFP Long arm Short arm Exon2 5’ Flanking Neo Exon3 S Targeted Allele H S H EGFP +/+ -/- H2O M +/- B Neo (640bp) Fgf23 (266 bp)

  24. Gross Appearance of 3-Week Old Wild-Type, Fgf23 Hetererzygous and KO mice Fgf23+/+ Fgf23+/- Fgf23-/-

  25. 18 50 c C B 16 1000 b b 40 14 a 12 800 30 a,b 10 a Serum Phosphorus Concentration (mg/dL) Serum Fgf23 Concentrations (pg/ml) 600 8 20 6 Serum 1,25(OH)2D3 Concentrations (pM) 400 4 10 2 200 0 0 WT Het KO WT Het KO 0 WT Het KO Genotypes Genotypes Serum Pi, 1,25(OH)2D3 and Fgf23 levels in Wild-Type, Heterozygous and Homozygous Fgf23-Deficient Mice A Genotypes

  26. Fgf23+/-/HypX (Heterozygous fgf23-KO /Hyp females) Fgf23+/-/XY (Heterozygous fgf23-KO males) X Wild-type Fgf23-KO/Hyp Hyp Fgf23-KO Fgf23+/+/Hyp Fgf23-/- Fgf23-/-/Hyp Fgf23+/+ 3 weeks Serum Phosphate, Calcium, fgf23, 1,25(OH)2D3, MicroCT Breeding Strategy/Study Design

  27. 18 c n > 5 c 16 b 14 a 12 10 Serum Phosphorus Concentration (mg/dL) d 8 d 6 4 2 0 WT Het KO Hyp Het/Hyp KO/Hyp Genotypes Serum Pi levels in Wild-Type, Fgf23-, Phex-, and combined Fgf23/Phex-Deficient Mice

  28. n=4 1000 b b 800 a,b a 600 Serum 1,25 (OH)2 D3 Concentrations (pM) 400 c 200 c 0 WT Het KO Hyp Het/Hyp KO/Hyp Genotypes Serum 1,25(OH)2D3 Levels in Wild-Type, Fgf23-, Phex-, and combined Fgf23/Phex-KO Mice

  29. Serum Fgf23 levels in Wild-Type, Fgf23-, Phex-, and combined Fgf23/Phex-Deficient Mice 2500 n > 5 b 2000 c 1500 Serum Fgf23 Levels (pg/ml) 1000 500 a a 0 WT Het KO Hyp Het/Hyp KO/Hyp Genotypes

  30. 16 KO and KO/Hyp 14 Het 12 WT Serum Phosphate Levels (mg/dL) 10 8 Het/Hyp Hyp 6 4 0 500 1000 1500 2000 2500 Serum Fgf23 Levels (pg/ml) Relationship Between Serum Pi and Fgf23 Levels as a Function of Genotype 16 14 KO and KO/Hyp Het 12 WT Serum Phosphate Levels (mg/dL) 10 8 Het/Hyp 6 Threshold Hyp 4 1 10 100 1000 10000 Serum Fgf23 Levels (pg/ml, log scale)

  31. Failure Of Phex-Dependent Cleavage Of FGF23: Cotransfection Studies

  32. Expression of fgf23 mRNA levels in normal and Hyp-derived bone and osteoblasts

  33. Inactivating Phex mutations in Hyp increase FGF23 gene expression in bone of heterozyogous FGF23 knock-out/GFP knock-in mice Fgf23 +/+ Hyp/Fgf23 +/- Fgf23 +/- GP GP GP CB CB BM CB BM BM

  34. Role of fgf23 in Phex Deficiency • Superimposed fgf23 deficiency rescues hypophoshatemia in Hyp mice. • Fgf23 does not appear to be a substrate of Phex. • Phex-deficiency increases fgf23 expression through unknown mechanisms. • An alternative hypothesis is needed to explain increments in circulating fgf23 levels in association with inactivating Phex mutations.

  35. What Is The Physiological Role of FGF23? • Regulation of FGF23 expression. • End-organ effects of FGF23: • Kidney. • Parathyroid gland. • Other tissues. • Role in CKD

  36. Physiological Function of FGF23: Lesions From Studies of the Fgf23 Promoter • Isolation and characterization of mouse Fgf23 promoter. • Evaluation of potenital regulators of Fgf23 promoter activity, including PTH, Vitamin D, calcium and phosphorus in vitro. • Confirmation that these factors regulate serum Fgf23 levels in vivo.

  37. Effect of Extracellular Calcium and Phosphate on Fgf23 Promoter Activity 1.2 1.2 1.0 1.0 0.8 0.8 0.6 0.6 Luciferase Activities (Firefly/Renilla) Luciferase Activities (Firefly/Renilla) 0.4 0.4 0.2 0.2 0.0 0.0 1 mM 2 mM 3 mM 5 mM 1 mM 2 mM 3 mM 4 mM Calcium Concentration Phosphate Concentration

  38. 3.0 * * 2.5 2.0 1.5 1.0 0.5 0.0 0 M 10-10 M 10-9 M 10-8 M Effect of 1,25‑(OH)2D3 on Fgf23 Promoter Activity Luciferase Activities (Firefly/Renilla) 1,25-(OH)2-vitamin D 3 Concentration

  39. Effect of PTH on Fgf23 Promoter Activity 1.0 0.8 * * * 0.6 Luciferase Activities (Firefly/Renilla) 0.4 0.2 0.0 0 mM 1 mM 10 mM 100 mM PTH Concentration

  40. Vitamin D Stimulated Fgf23 Transcripts in Ros17/2.8 Cells 2.40.7 16.00.9 (7 fold) 1.80.3 264.348.9 (147 fold) 400 P<0.01 vehicle 1,25-(OH)2D3. 300 Relative Concentration of Fgf23 (Fgf23/Ppia) 200 P<0.01 100 0 24 Hours 8 Hours

  41. * Vehicle Calcitriol Effect of 1,25‑(OH)2D3 on Serum Levels of Fgf23, PTH, Phosphorus and Calcium A B 100 ng/g/BW Calcitriol IP 40 160 140 120 30 100 Serum FGF23 (pg/ml) Serum Intact PTH (pg/ml) 80 20 60 40 10 * 20 0 0 Calcitriol Vehicle C 10 10 D 8 8 Serum Calcium (mg/dL) 6 6 Serum Phosphorus (mg/dL) 4 4 2 2 0 0 Calcitriol Vehicle Calcitriol Vehicle

  42. Characteristics of Fgf23 Promoter • The mouse Fgf23 promoter is characterized by: - A transcription start site 123 bp upstream of the initial ATG. - A TATA box 35 bp upstream of the transcription start site. - 67% homology with the human promoter over the first 800 bps. • The 3.5kb 5' flanking region of the mouse Fgf23 gene has promoter activity in vitro. • In ROS 17/2.8 osteoblasts, 1,25(OH)2D3 stimulates activity of the Fgf23 promoter/reporter construct, and alterations of extracellular phosphorus and calcium concentrations have no effect. • Injection of calcitriol into wild-type mice increases serum Fgf23 levels from a basal level 90.0±8.9 pg/ml to 136.4 ± 8.7pg/ml (Mean ± SEM) at 24 hours after injection.

  43. Parathyroid glands PTH Gut Kidney Ca2+absorption Reabsorption of Ca2+ PO42-absorption 1,25(OH)2vitamin D synthesis PO42-absorption Regulation of Phosphate Homeostasis By FGF23: Counter Regulatory Hormone for Vitamin D-Induced Hyperphosphatemia? 1,25(OH)2vitamin D Serum Ca2+ Serum PO42- Bone FGF-23

  44. Uncertain Role of FGF23 in CKD • Circulating levels of FGF23 are increased in CKD.

  45. FGF-23 = -34456.23 + 7315.06 * P R-Square = 0.23 100,000 10,000 FGF-23(RU/L) 1000 FGF-23 (RU/ml) 100 10 0 Phosphorus (mg/dl) Control XLH Unknown ESRD Circulating FGF23 Levels In ESRD (C-terminal assay) Weber TJ, Liu S, Indridason OS, Quarles LD. Serum FGF23 levels in normal and disordered phosphorus homeostasis. J Bone Miner Res. 2003 Jul;18(7):1227-34.

  46. Circulating FGF23 Levels In ESRD (Intact assay) Intact FGF23 in ESRD 14 to 98,646 ng/L (nl 27.8 ± 9.0 ng/L )

  47. Sevelamer Hydrochloride and Calcium Bicarbonate Reduce Serum Fibroblast Growth Factor 23 Levels in Dialysis Patients Fumihiko Koiwa, (2005) Sevelamer Hydrochloride and Calcium Bicarbonate Reduce Serum Fibroblast Growth Factor 23 Levels in Dialysis Patients. Therapeutic Apheresis and Dialysis9:4, 336-339

  48. Vitamin D Treatment Is Associated With Increased FGF23 Levels In Dialysis Patients Shohei N et al. Kidney Intern 67:1171-1178, 2005

  49. Uncertain Role of FGF23 in CKD • Circulating levels of FGF23 are increased in CKD. • Evidence for a role in development of secondary HPT. Cross-sectional clinical observations: Diminishes 1,25(OH)2D3 in kidney and stimulates PTH by parathyroid gland.

  50. FGF23 Mitigates Hyperphosphatemia in CKD Factors Associated with FePO4 Full multivariate β P R2 model .50 log FGF23 5.2 0.009 log PTH 6.0 0.004 eGFR -.02 0.031 sP -1.0 0.615 Gutierrez O JASN 2204-2205, 2005

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