FGF23 (and Klotho): what s new? Brief introduction to FGF23. Introduction to FGF23. FGF23: phosphorylation pathways. FGF23: phosphorylation pathways

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1 (and Klotho): what s new? Brief introduction to Justine Bacchetta, MD, PhD Reference Center for Rare Renal Diseases Long Beach, CA, 2017 Calcium and phosphate metabolism 1-25 vitamin D Introduction to synthezised by osteocytes FGF-R: tyrosine kinase receptor FGFR-1/2/3/4 Phosphorus Calcium Klotho: cofactor Anti-aging protein Tissue-specific expression Kidney and parathyroid gland PTH Stimulating effect / Inhibiting effect Bacchetta, EMC 2015 Unakawa, Nature 2006; Bonewald JBMR 2010 : phosphorylation pathways Klotho-dependent and Klotho-independent activation of the phosphorylation pathways Erk 1/2 and Akt pathway Calcineurin-NFAT pathway : phosphorylation pathways Klotho-dependent and Klotho-independent activation of the phosphorylation pathways Erk 1/2 and Akt pathway Calcineurin-NFAT pathway Faul, JCI 2011 Faul, JCI

2 : 3 main historical actions : an endocrine FGF with off-target effects Kidney Saito, J Biol Chem 2003 Andrukhova, EMBO 2014 Heart & Cardiomyocytes Faul, JCI 2011 Proximal tubule Parathyroid Parathyroid Hippocampal cells and CNS Npt inhibition 2a-2c 1 α hydroxylase inhibition 24 hydroxylase stimulation PTH inhibition 1 α hydroxylase stimulation Silver, PN 2010 Olauson Plos One 2013 Immune system Hensel, J Neurochem 2016 Other targets? Bacchetta, JBMR 2012 Chonchol, JASN 2015 & JASN 2016 Bone Wang JBMR 2008, Wesseling-Perry JCEM 2009, Allard CTI 2015 Prie, Kidney Int 2009 Hypophosphatemia, PTH and 1-25 OH 2 vitamin D inhibition Cartilage Kawai, JBC 2012 Dentoalveolar complex Chu, Anat Rec 2010 as an inhibitor of bone mineralization New insights: physiology in bone Model of genetic hyperphosphatemia (GALNT 3 mutation) Low intact levels High C-terminal levels Familial tumoral calcinosis Masi CTI 2015 as an inhibitor of bone mineralization Comparison of mineralization in vitro in preosteoblastic cells from a patient with GALNT3 mutation (low intact levels) and from controls Higher capability to form mineralization Biphasic effects of on osteoclast biology An expression of FGF-R1 Patient LOW An activation of Erk and Akt Effects driven through FGF-R Control NORMAL Masi CTI 2015 Inhibition of osteoclast differentiation A moderate albeit significant stimulation of resorption Allard, Calcified Tissue International

3 Klotho expression in long bones regulates production during renal failure New insights: physiology in the central nervous system Murine model: targeted deletion of Klotho in long bones Two main results Long bones from these mice did not increase expression after adenine diet or 5/6 th nephrectomy -treated bone cells required Klotho to increase expression and induce Erk phosphorylation Kaludjerovic FASEB 2017 Impact of on neuronal morphology and synaptic density In vitro model of primary murine hippocampal cultures Incubation with ± α-klotho Enhanced number of primary neurites And reduced arborization => Resulting in a less complex neuronal morphology New insights: and adipose tissue And the fat lady sings about phosphate and calcium Hensel J Neurochemist 2016 Wagner Kidney International 2017 Towards new endocrine regulations High adiponectin levels New insights: and (pediatric) CKD Suppressed Klotho renal expression Decreased levels Caused renal loss of calcium Rutkowski, Kidney International 2017; editorial comment by Wagner Kidney International

4 The paradigm: both and Klotho are deregulated early in CKD in pediatric CKD Cohort of 227 followed in a pediatric nephrology unit Normal renal function and CKD (pre-dialysis) GFR: inulin clearance Influence of GFR, age, solid organ transplantation and BMI on levels A likely influence of corticosteroids on loevels An association between and uric acid An association between and IGF1 Fliser, JASN 2007; Isakova JAMA 2011 Wolf, Kidney International 2012 Pereira, Bone 2010 Faul et al, J Clin Invest 2011 Gutierrez et al, N Eng J Med 2008 Bacchetta, J Clin Endocrinol Metab 2010 Bacchetta, Pediatr Nephrol 2012 Recombinant growth hormone increases levels Similar results of an open label prospective study in Pediatric Endocrinology, Birmingham, Alabama 23 children with GH deficiency Clinical trial: C terminal before and one year after rhgh Increased, TmP/GFR and phosphorus after rhgh The increase of remained significant after adjustment on age and phosphorus increases early in pediatric CKD Data from the CKID cohort N=464 children ages 1 16 years with predialysis CKD Gardner et al, J Pediatr Endocrinol Metab 2011 Portale, clin J Am Soc Nephrol 2014 as a risk factor of CKD progression New insights: and infections in CKD Data from the CKID cohort N=419 children ages 1 16 years with predialysis CKD Portale, clin J Am Soc Nephrol

5 as an inhibitor of extra-renal 1-alpha hydroxylase in monocytes High (and low 25-D) as risk factors for clinical infections in the HEMO-study Fold change in mrna relative to vehicle-treated cells * * * * CYP27B1 CYP24A1 VDR LL37 TNF : 100 ng/ml, 24 hours Controls = PBS 6 hours = black 24 hours = grey Data obtained in monocytes from healthy donors, but similar data obtained in PD cells from pediatric patients undergoing chronic PD * * 25OHD 25OHD 25OHD Vehicle 1,25(OH) 2D 1,25(OH)2D 1,25(OH)2D 1,25(OH) 2D IL-15 1,25(OH) 2D IL-15+ 1,25(OH) 2D Bacchetta, JBMR 2012 Chonchol, JASN 2015 and further confirmed JASN 2016 and left ventricular hypertrophy New insights: and cardiovascular disease in CKD Increased FGF 23 levels and left ventricular hypertrophy Epidemiological prospective CRIC cohort, 3070 CKD adults Increased FGF 23 levels at baseline and increased risk for onset of LVH RR= 2.4 Rat cardiomyocytes + Hypertrophy, dose-dependent Reactivation fetal genes Increased markers for LVH Faul et al, J Clin Invest 2011 and cardiomyocytes: a Klotho-independent effect Klotho is not expressed in CM, but FGF-R1 and 4 are Signaling pathways Through FGF-R signaling Through PLCγ and NFAT No activation of Akt Intramyocardial and intravenous injection of induces ventricular hypertrophy in mice Wild type Klotho deficient mice: high and LVH and LVH in pediatric CKD German autopsy study 17 dialysis, 17 KTx, 24 controls 67% LVH in patients Induction of cardiac -FGFR4 expression as well as a decreased Klotho expression is associated with LVH Faul et al, J Clin Invest 2011 Leifheit-Nestler, Nephrol Dial Transplant

6 LVH depends on but also on Klotho levels: loss of Klotho in CKD breaks one s heart! Anti antibodies: can they be relevant in CKD? Heterozygous Klotho deficient mice Klotho-deficient CKD mice display aggravated cardiac fibrosis as compared to WT-CKD mice Transgenic expression of soluble Klotho improves cardiomyopathy in Klotho-deficient CKD mice Xie JASN 2015 / Fu & Liu JASN 2015 Antibodies against improve hyperparathyroidism But they also increase mortality Early decreased PTH levels But increased phosphate, calcium and 1-25 D levels Shalhoub JCI 2012 Shalhoub JCI 2012 and cardiomyocytes: a potential therapeutic target FGFR inhibitors in a rat model of CKD (5/6 th nephrectomy) Decreased LVH No modification of blood pressure From prevention to treatment FGFR blockade can improve LVH in animal models Faul et al, J Clin Invest 2011 Di Marco NDT

7 and human diseases So should we trash anti antibodies? Wolf et al, JASN 2010 Hypophosphatemic rickets The revolution in 2014: KRN23 in patients, short-term Adults with XLH 38 patients receiving KRN23 or placebo, IV or sc Single dose Hypophosphatemic rickets The revolution in 2015: KRN23 in patients, long-term Adults with XLH 28 patients receiving KRN23: 0.05 to 0.6 mg/kg every 28 days (SC) Then extension study 22 patients receiving 0.1 to 1 mg/kg every 28 days (SC) Carpenter JCI 2014 Imel JCEM 2015 International pediatric trials are ongoing Back in the real life of a CKD patient Phosphate is a/the vascular toxin/silent killer Conclusion Vascular calcifications Scialla, Kidney International 2013 Shroff, Pediatric Nephrology

8 Phosphate and longevity Klotho -/- mice, Klotho and CKD in 2017 Both low Klotho and high levels are delerious in CKD for LVH Both low 25-D and high levels are deleterious in CKD for infections Conversion mg/dl: mmol/l Guinea pig Gerbil Dialysis patient Rhinoceros Elephant Centenarian human Kuro-O Mech Ageing Dev 2010 has become a therapeutic target in genetic hypophosphatemic diseases: the ongoing revolution! However antibodies have proved to be deleterious in CKD A potential role for drugs modulating FGF-R and for recombinant Klotho in the future? Clinical practice: target phosphate 8