1. Sandro Mazzaferro Il Trattamento dei Disturbi del Metabolismo Minerale nell’IRC AGGIORNAMENTI IN NEFROLOGIA CLINICA XIII Incontro Teramo, 11-12 ottobre 2013 Aula Convegni II Lotto Aspetti fisiopatologici ed epidemiologici (Manoppello – PE)

2. Disturbances of Mineral Metabolism in CRF involve … … 2 A possible «rule of … … …»

3. 3 IONS At least …

4. 1. Serum Calcium Intra- vs Extra-cellular Concentrations Blood stream Cell Ca++ = 100 nmol/l Intracellular Ca++: ~10.000 fold lower!! Ca++ = 1.10 mmol/l 4 There must be some mechanism that lowers intracellular Ca++!

5. Ca++ as Second Messenger Smajilovic S. Br J Pharma 2011 CaSR: its discovery has changed the status of Ca 5 Ca++ = 100 nmol/l Ca++ = 1.10 mmol/l Ca++ as First Messenger

6. Ca derangements in CRF Hypo-Calcemia and/or negative Ca balance: –Anxiety, Paresthesias, Cramps, Tetany, Seizures, Abdominal pain, Congestive heart failure, Calcifications, etc. –High PTH, High turnover bone, Osteopenia Hyper-Calcemia and/or positive Ca balance: –Weakness, Nausea, Vomiting, Polyuria, Polydipsia, Hypertension, Lethargy, Coma –Low PTH, Low turnover bone, Calcifications 6

7. 2. Serum Phosphate Intra- vs Extra-cellular Concentrations Intracellular P is ~50 times HIGHER Blood stream Cell Remember, intracellular Ca is 10.000 times LOWER! (1.0 mmol/l vs 0.0001 mmol/l) There must be some mechanism that increases intracellular P. (!?!) 7 Pi = 1.5 mmol/l Pi = 70 mmol/l

8. Pi Receptor? 8 No Pi Receptor identified so far.

9. Derangements of Pi in CRF Hypo-Phosphatemia and/or negative Pi balance: –Low cardiac output, Arrhytmia, Hypotension, Astenia, Confusion, Seizures, Coma –High insulin, Low PTH, Pseudofractures, Osteomalacia Hyper-Phosphatemia and/or positive Pi balance: –Hypocalcemia, Hypotension, Excitability, –High PTH, High turnover, Calcification 9

10. 3. Serum H+ Intra- vs Extra-cellular Concentrations Blood stream Cell H+ = 40 nmol/l In general intra- and extra- cellular H+ are quite similar H+ = 40 nmol/l 10 Please note that: pH 7.30 = 50 nmol/l pH 7.50 = 30 nmol/l

11. Derangements of H+ in CRF Acidosis and/or Acidemia: –Fatigue, tachipnea, sleepness, confusion, coma –Osteopenia/Malacia Alkalosis and/or Alkalemia: –Anxiety, nausea, tremor, twitching, spasm, numbness, dizziness, coma –Calcifications 11

12. 12 HORMONES At least …

13. 1. PTH hormone & fragments 1-115 1-90 1-84 7-84

14. PTH fragments are produced in the cytosol, and are influenced by Ca++ and CaSR! Friedmann P AmJPhy-RP 2006

15. PTH-Type I Expressed in: –Kidney, –Osteoblasts –Smooth muscle, –Brain, –Fetal tissues, Binds: –NH-term PTH (1-84); –PTH-rP PTH- Type II Expressed in: –Brain, –Endothelium –Smooth muscle, –GI tract endocrine cells, –sperm Not in Obl or kidney Binds: –TIP39, –NH-term PTH (1-84) PTH Receptors

16. D’Amour P. Clin Biochem 2012 Type I and Type II Receptors C-term PTH Receptor

17. Derangements of PTH in CRF Hypoparathyroidism: –Low Ca, high Pi, low AP –Low turnover bone, Ectopic calcification Hyperparathyroidism: –High Ca, high Pi, high AP –High turnover bone, Ectopic calcification 17

18. 2. Vitamin D Metabolites & Hormone 18

19. 19 Vitamin D Receptor: expressed in almost every tissue

20. Derangements of Vitamin D in CRF Hypovitaminosis D: –HypoCa, high PTH, high AP –Bone Resorption/Osteomalacia/Penia –Other (pleiotropic?) Hypervitaminosis D: –Hyper Ca, low-high PTH, low-high AP –Bone mineralization, low turnover –Ectopic Calcifications –Other (pleiotropic?) 20

21. 3. FGF23-Klotho Why together? FGF23 null vs Klotho null transgenic mice 21

22. FGF23 null vs Klotho null 22 PhenotypeFGF23-/- Serum PHigh 1,25-D levelHigh Serum CaHigh Bone Mine- ralization Defective Ectopic Calcification Present Arterio- sclerosis Present LifespanShort Kl -/- High Defective Present Short

23. FGF23 Receptors, FGF23 and Klotho Razzaque MS Trends in Mol Med 2006 Klotho (local and circulating) is essential for activity and selectivity of FGF23

24. Blood [ H + ] [ Ca ++ ] [ P ++ ] [1,25D] [PTH] From Kidney to Bone and viceversa Klotho FGF23 Mazzaferro S. et al Arch. Biochem. Biophys. (2010)

25. Possible differences between the two? 25

26. FGF23 regulation Systemic factors: –Pi ( = ); –1,25D ( = ) Local factors (bone derived): –DMP1 deletion = increased FGF23; –Phex inactivation = increased FG23; –MEPE administration = increased FGF23 (Transcriptional and post-transcriptional mechanisms) ? Feed-back system!

27. Blood [ 1,25D] [ P ++ ] From Bone to Kidney FGF23 MEPE- ASARM cleavage DMP1 PHEX ASARM Klotho FGF23 FGF23 synthesis is affected not only by serum Pi and 1,25D, but also by the metabolism of other bone proteins Mazzaferro S. et al Arch. Biochem. Biophys. (2010) Klotho

28. Klotho functions outside FGF23 OrganActivityEffect KidneyNaPi2a/2c inactivationHypophosphatemia Renal TRPV5/6 ActivationCa reabsorption Renal ROMK1 ActivationPotassium secretion Na/K ATPase ActivationCa homeostasis ParathyroidsNa/K ATPase ActivationPTH stimulation Choroid Plexus Na/K ATPase ActivationCa homeostasis SytemicNaPi3 ReductionVascular Calcification inhibition Insulin/IGF-1 inhibitionInsulin resistance – Anti-age Wnt suppressionAnti-age Endothelial NO Up- regulation Endothelial dysfunction protection Pi Ca K PTH VC Ins Age

29. 29 S-Klotho, pg/mlFGF23, pg/ml N CKD 2CKD 3 CKD 4 CKD 2CKD 3CKD 4 * * * (*) p<0.005 vs Normal $ N § ($) p<0.001 vs N (§) p<0.006 vs N, CKD-2, CKD-3 Mazzaferro S et al. Soluble alpha Klotho in Chronic renal failure. ISN 2012 Derangements of FGF23/Klotho in CRF

30. Low Klotho/High FGF23 Human Syndromes Human Klotho inactivating gene mutation –HyperPi, HyperCa, High 1,25D, High FGF23, Calcinosis. FGF23 excess related syndromes –Primary: ADHR, ARHR, XLH, TIO [Rickets, Osteomalacia] –Secondary: Chronic Renal Failure [CV disease and mortality] 30

31. 31 ORGANS At least …

32. 1. Intestine (Absorption, Secretion, Excretion of Ca, Pi, H) CarriersReceptorsHormones TRP-V5,6Vit. D Receptor1,25 D NaPi-2a,bPTH Receptor IIPTH Ca Sensing ReceptorFGF23/Klotho Pi Receptor (?) Intestinal Endocrine functions: -several GI hormones, -possibly a Phosphatonin

33. 2. Kidney (Filtration, Reabsorption, Excretion of Ca, Pi, H) CarriersReceptorsHormones TRP-V5,6Vit. D Receptor1,25 D NaPi-2a,bPTH Receptor IPTH Ca Sensing ReceptorFGF23/Klotho Pi Receptor (?) Renal Endocrine functions: -Renin, Epo, Calcitriol, -Klotho

34. 3. Bone (Resorption, Deposition, Ca, Pi, H) CarriersReceptorsHormones TRP-V5,6Vit. D Receptor1,25 D NaPi-2a,bPTH Receptor IPTH Ca Sensing ReceptorFGF23/Klotho Pi Receptor (?) Bone Endocrine functions: -FGF23 -Osteocalcin

35. Bone cells lineage (modern view) Hematopoetic stem cell Pre-osteoclast Macrophage Osteoclast Pre-osteoblast Osteoblast Runx2 Stromal cell Lining cell Osteocyte Systemic Hormones: -PTH, 1,25D, Estrogens, etc Cytokines: - ILs, PGs, TGFb, etc OPG/RANK/ RANKL Myocyte MRF4 35 No-more inactive! + Remodeling = mineral metabolism Growth & Modeling

36. Organic Matrix - Non Collagenous Proteins (11%) 1.GAGs: - Proteoglycans, Hyaluronan; 2.Glycoproteins: - ON, SIBLINGs (OPN; BSP; DMP1; SPP1; MEPE), OPG, SOST, RDG contaning proteins, Thrombospondin 3.Γ-Carboxylic Acid containing: - MGP, OC 4.Other Proteins: - Growth factors (BMPs, FGFs), Enzymes (AP, TRAP, PHEX, MMPs), Absorbed from circulation (Albumin, Fetuin) 36 Non-collagenous bone protein contribute to Ca and Pi fluxes from and into bone

38. Normal CKD Stage -2 FGF23FGF23 DMP1DMP1 MEPEMEPE La disfunzione della «ghiandola osso» inizia già nelle fasi precoci della CKD

39. 39 A possible «rule of … … …» IONS ORGANS HORMONES Systemic (CV) involvement or CKD-MBD

40. Targets for Mineral Metabolism Control in CRF PTH 1,25D FGF23/ Klotho Ca P H

41. Available drugs for CKD-MBD 1.Phosphate binders 2.Vitamin D and analogs 3.Calcimimetics 41 (again … … … )

42. Epidemiologic aspects (in Dialysis)

43. Aim of the study: To evaluate - Drug prescription policy, - Biochemical control of SH, with new available drugs (from 2006 to 2008)

44. 1. Drug Prescription policy: (a.) PTH inhibitors

45. Calcitriol p.o. Calcitriol i.v. Paricalcitol i.v. Cinacalcet p.o. Any Vitamin D from 50.8% to 58.2% (p<.001) ~2ug/w~3.5ug/w~45mg/d

46. 1. Drug Prescription policy: (b.) Phosphate Binders

47. Changes in P binders prescription policy Any Ca Acetate Ca CarbonateSevelamer Aluminum

48. Average doses of P binders Ca AcetateCa CarbonateSevelamerAluminum ~2g/day ~4g/day

49. 2. Biochemical control of SH

50. Mean (SD) Values of PTH, Ca, and P During the Survey. BaselineMonth 6 Month 12 Month 18 P value PTH, pg/mL 310.3 (292.4) 287.8 (252.2) 286.4 (248.1) 279.5 (250.1)0.0002 Ca, mg/dL 9.1 (0.8) 9.0 (1.0) 9.0 (0.7) 9.0 (0.7)0.383 P, mg/dL 5.0 (1.4) 4.9 (1.3) 5.0 (1.3) 5.0 (1.4)0.085 Ca=calcium, P=phosphate, PTH=parathyroid hormone.

51. Patients at target (K/DOQI)? PTH Ca P All three

52. Conclusions CKD-MBD = complex metabolic derangement of renal failure, with significant systemic involvement and morbidity/mortality. Its therapeutic management is rather difficult, even with new drugs, The «rule of 3» seems cute, but insufficient to help manage it properly 52

53. EVOLVE: Study Design Screening Phase Up to 30 Days Titration Phase (Visits Q2 Wks) Follow up Phase (Visits Q8 Wks) Follow-up period = ~ 2.5 yearsEnrollment = ~ 1.5 years Day 1Week 20Week 52 Event-driven study that concludes when approximately 1,882 subjects have experienced a primary composite event Placebo plus standard of care(n = 1,900) Cinacalcet plus standard of care (n = 1,900) Adapted from: Chertow GM, et al. Clin J Am Soc Nephrol. 2007;2:898-905. All patients could receive vitamin D sterols and phosphate binders, as necessary, at the discretion of the physician. Trial Population Hemodialysis iPTH  300 pg/mL Ca  8.4 mg/dL Ca x P  45 mg 2 /dL 2

54. Primary Composite Endpoint (ITT) not met: Non-significant* 7% Reduction in the Risk of Death or Nonfatal Cardiovascular Events in Patients with SHPT Proportion Event-free 0.0 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Time (months) 04812162024283236404448525660 Hazard ratio, 0.93 (95% CI, 0.85, 1.02) Log-rank test, P = 0.11* Subjects at risk: 1948 1935 1842 1804 1739 1693 1638 1579 1556 1476 1472 1384 1312 1303 1224 1230 1160 1177 1109 1115 1053 1051 996 989 940 679 650 399 404 113 114 Cinacalcet Placebo Kaplan-Meier plot of the time to the primary composite endpoint (death, myocardial infarction, hospitalization for unstable angina, heart failure, or peripheral vascular event) in EVOLVE™. Adapted from Chertow GM, et al. N Engl J Med. Epub 2012 Nov 3; DOI: 10.1056/NEJMoa1205624 * The trial did not meet its primary endpoint in the unadjusted intent-to-treat analysis

55. CKD-MBD Working Group ERA-EDTA 55

56. 56