Iron Chelation Resulting in Renal Phosphate Wasting

Accepted Manuscript Iron chelation resulting in renal phosphate wasting Lynda Cheddani, MD, Thierry Leblanc, MD, PhD, Caroline Silve, MD, Nahid Tabibzadeh, MD, Dominique Prié, MD, PhD, Jean-Philippe Haymann, MD, PhD, Marie-Noëlle Péraldi, MD, PhD, Michel Daudon, PhD, Paul Meria, MD, PhD, Emmanuel Letavernier, MD, PhD PII:

S2468-0249(17)30318-2

DOI:

10.1016/j.ekir.2017.07.011

Reference:

EKIR 203

To appear in:

Kidney International Reports

Received Date: 20 June 2017 Revised Date:

17 July 2017

Accepted Date: 24 July 2017

Please cite this article as: Cheddani L, Leblanc T, Silve C, Tabibzadeh N, Prié D, Haymann JP, Péraldi MN, Daudon M, Meria P, Letavernier E, Iron chelation resulting in renal phosphate wasting, Kidney International Reports (2017), doi: 10.1016/j.ekir.2017.07.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Iron chelation resulting in renal phosphate wasting

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Lynda Cheddani3, MD, Thierry Leblanc4, MD, PhD, Caroline Silve5,6,7, MD, Nahid Tabibzadeh1,2,3 ,MD,

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Dominique Prié

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Michel Daudon1,2,3 PhD, Paul Meria11 MD, PhD, Emmanuel Letavernier1,2,3 MD, PhD

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, MD, PhD, Jean-Philippe Haymann

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, MD, PhD, Marie-Noëlle Péraldi10 MD, PhD,

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Sorbonne université- UPMC Paris 06, UMR S 1155, F-75020, Paris, France

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INSERM, UMR S 1155, F-75020, Paris, France

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Explorations fonctionnelles multidisciplinaires, AP-HP, Hôpital Tenon, F-75020, Paris, France

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Service d’Hématologie, AP-HP, Hôpital Saint-Louis, F-75010, Paris, France

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Service de Biochimie et Génétique Moléculaire, AP-HP, Hôpital Cochin, Paris, France.

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INSERM U1169, Université Paris Sud, Hôpital Bicêtre, Le Kremlin Bicêtre, France.

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Centre de Référence des Maladies Rares du Métabolisme du Phosphore et du Calcium, Filière de Santé

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Maladies Rares OSCAR, AP-HP, Paris, France.

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Paris Descartes Université- Paris 05, F-75015, Paris, France

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Service des explorations fonctionnelles, AP-HP, Hôpital Necker, F-75015, Paris, France

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Service de Néphrologie, AP-HP, Hôpital Saint-Louis, F-75010, Paris, France

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Service d’Urologie, AP-HP, Hôpital Saint-Louis, F-75010, Paris, France

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Funding: This research did not receive any specific grant from any funding agency in the public, commercial

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or not-for-profit sector.

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Correspondence:

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Emmanuel LETAVERNIER,

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Service des Explorations Fonctionnelles Multidisciplinaires, Hôpital TENON,

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4 rue de la Chine,

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75020 Paris (France).

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Phone 33 1 56 01 67 73 ; Fax 33 1 56 01 70 03

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E-mail: [email protected]

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Abbreviated Title: Iron chelation resulting in phosphate wasting

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Key words: phosphate, FGF23, deferasirox, nephrolithiasis, rickets

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ACCEPTED MANUSCRIPT INTRODUCTION

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Acting on fibroblast growth factor receptor 1 (FGFR1), FGF23 reduces renal-phosphate reabsorption through

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downregulation-of the sodium phosphate cotransporters NPT2a and NPT2c at the apical membrane of the

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proximal renal tubule (1,2).

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Human disorders of phosphate handling and hypophosphatemic rickets have been related to increased FGF23

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serum levels resulting from mutations affecting FGF23 gene itself or genes regulating FGF23 secretion such

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as PHEX or DMP1. These mutations present as autosomal-dominant (ADHR), X-linked dominant (XLHR),

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and autosomal-recessive hypophosphatemic rickets (ARHR), respectively (3).

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Iron plays a role in FGF23 regulation in humans, and an inverse relationship between iron status and FGF23

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concentrations has been described in several populations (4–6) .

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Inflammation and iron deficiency increase fgf23 transcription in mice by activating Hif1α signalling but

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relationships between iron and FGF23 secretion in humans remain a matter of debate (7,8).

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Kapelari et al. reported an association between iron supplementation and a complete loss of biochemical

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ADHR phenotype, allowing withdrawal of rickets medication (6).

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Conversely, we report here the case of a man affected by a homozygous DMP1 rare variant, and presenting an

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hyperphosphaturia associated with high serum FGF23 levels. This exceptional case raises concerns about the

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relationships between iron chelation and renal phosphate leak, potentially promoting bone demineralization

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and urolithiasis in genetically predisposed patients.

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ACCEPTED MANUSCRIPT PATIENT AND METHODS

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Clinical case

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A 22 years-old man presented a medical history of Diamond Blackfan anemia. This rare disorder results from

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a mutation of RPS19 gene (RPS19: NM_001022.3; c.320T>G ; p.Leu107Arg) responsible for intrinsic

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progenitor cell defect. There was no consanguinity in the family. He was 150 cm tall, had moderately bowed

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legs, and his weight was 50 kg. Since birth, he received iterative transfusions inducing post-transfusional

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hemochromatosis. He had therefore been treated with iron chelators, mainly deferasirox. He progressively

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developed a mild renal failure (serum creatinine 108 µmol/L) and deferasirox was therefore replaced by

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deferiprone and deferoxamine with doses adapted to serum ferritin levels.

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When he was 21-years old, he developed massive kidney stones treated by reno-ureteroscopy. Stone analysis

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revealed a predominance of calcium phosphate (mildly carbonated apatite) and to a lesser extent the presence

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of calcium oxalate dihydrate (weddellite).

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Initial serum biochemistry findings [normal range] were as follows: calcium, 2.42 [2.16-2.52] mmol/L;

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phosphate, 0.56 [0.85-1.31] mmol/L; intact parathyroid hormone (PTH), 18 [8-76] pg/mL ; 25-hydroxy-

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Vitamin D (25(OH)D), 45 [30-100] ng/mL, and 1,25[OH]2D, 58 [17-67] pg/mL. Serum bicarbonate level was

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at the lower limit at 22.5 mmol/L, and potassium level was normal at 3.8 [3.7-5.1] mmol/L. Urine tests

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revealed a low tubular phosphate reabsorption rate (TPR), 75 % [85-97%] and decreased tubular maximum

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reabsorption of phosphate to glomerular filtration rate [TmP/GFR] at 0.44 [>0.67] mmol/L.GF. Urine calcium

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excretion was increased at 7.7 [<6] mmol/day.

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The patient presented therefore biological features associating low serum phosphate level caused by a massive

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renal loss and hypercalciuria, responsible for calcium phosphate nephrolithiasis. These features were

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consistent with an acquired proximal tubulopathy due to deferasirox. However, C-Terminal FGF23 serum

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level at 218 [33.7-96.5] RU/mL, suggesting that renal phosphate leak was rather due to increased FGF23

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secretion.

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A few months later, C-Terminal FGF23 decreased transiently to 89 RU/mL and both serum phosphate and

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1,25[OH]2D serum levels increased at the same time up to 0.72 mmol/L and 78 pg/mL, respectively.

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ACCEPTED MANUSCRIPT Hypotheses and methods

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These observations suggested that renal phosphate loss was due to excessive FGF23 secretion. Therefore, we

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conducted molecular genetic analysis to detect mutations affecting FGF23, PHEX and DMP1 genes. The

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PolyPhen-2 (Available on: http://genetics.bwh.harvard.edu/pph2/, 15/06/2017) and SIFT (Available on:

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http://sift.jcvi.org/, 15/06/2017) softwares were used to predict in silico whether an amino acid substitution

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affects protein function. Informed consent was obtained from the patient and both parents to perform DNA

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analyses and collect clinical and biochemical data.

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Moreover, the simultaneous fluctuations of FGF23 and phosphate levels supported the hypothesis that

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intermittent determinants, e.g. drugs, would be involved. Considering in vitro and animal studies suggesting

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that iron deficiency can increase FGF23 levels, the contribution of iron chelators in renal loss of phosphate

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has been hypothesized (7,8). Actually, this patient had intermittent iron chelators intake. To test this

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hypothesis, iron chelators were withdrawn for 72 hours. C-terminal FGF23 and phosphate levels were

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monitored during iron chelation, after discontinuation for 72 hours, and after treatment reintroduction several

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weeks later.

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Molecular genetic analysis

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DNA Sequencing of DMP1 gene (NM_004407) revealed a rare homozygous variant affecting exon 5

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(c.427C>A, p.Gln159Lys). No mutation has been identified in PHEX or FGF23 genes. The Gln159Lys

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variant is predicted to be deleterious. Both parents were heterozygous carriers of this rare variant.

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Metabolic response to iron chelation withdrawal and reintroduction

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After discontinuation of iron chelators for 72 hours, C-terminal FGF23 levels values halved and serum

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phosphate levels normalized from 0.56 to 0.89 mmol/L (Figure 1). Stopping iron chelation during 72 hours

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induced resolution of renal phosphate loss. Treatment reintroduction had the opposite effect and a decline of

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serum phosphate level was observed (back to 0.56 mmol/L), due to an increase in C-terminal FGF23 levels

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(116.6 to 276.4 RU/mL). These results provided evidence that iron chelation promoted renal loss of

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phosphate, through an increase of FGF23 levels (Figure 1).

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ACCEPTED MANUSCRIPT DISCUSSION:

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To our knowledge, this is the first report of an association between iron chelation in a human and

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“hypophosphatemic rickets”. Farrow et al. previously demonstrated in vitro that iron chelation with

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deferoxamin increases FGF23 mRNA expression in osteoblasts by twenty fold (7). David et al. evidenced that

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functional iron deficiency, following hepcidin injection to wild-type mice, stimulates FGF23 production and

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preferentially increases circulating concentrations of C-terminal FGF23 (8).

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Regarding the patient, we first hypothesized that renal phosphate loss resulted from an acquired proximal

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tubulopathy induced by deferasirox (9). Actually, nephrotoxicity is the most frequent adverse effect of

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deferasirox treatment. Nephrotoxicity can present as an acute or chronic decrease in glomerular filtration rate

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and features of proximal tubular dysfunction are also frequent (10). High levels of FGF23 rapidly ruled out

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this assumption. Genetic analysis identified a rare homozygous DMP1 variant but the pathogenicity of this

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variant is unknown to date. It seems likely that the presence of both minor alleles may predispose to renal

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phosphate leak through an increase in FGF23 but, in the absence of iron chelation, biological expression was

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minimal. It appears that DMP1 variant was a predisposing condition requiring a second hit to promote renal

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phosphate leak. The exact role of DMP1 remains unknown, but recent murine experimental models have

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evidenced that mutations in the DMP1 gene create a lower set point for extracellular phosphate and maintain

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it through the regulation of FGF23 cleavage and expression (11).

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In this case, chronic medical iron chelation played a major role in the ARHR biochemical phenotype by

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simulating a transitory iron deficiency. This hypothesis was confirmed by serum phosphate levels and C-

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terminal FGF23 levels evolution associated with iron chelators withdrawal and reintroduction.

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Previous studies reported an association between iron status and FGF23 levels. Imel and al. observed an

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inverse correlation between iron status and (i) C-Terminal FGF23 levels in healthy individuals and (ii) both

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intact and C-terminal FGF23 levels in patients presenting ADHR (FGF23 mutation) or XLH (PHEX

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mutation) (4). Iron substitution has been associated with a C-Terminal FGF23 levels reduction, and with

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phosphate renal loss reduction in ADHR patients (4–6). In this case, C-terminal FGF23 was dramatically

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increased, a further argument in favour of relative iron “deficiency”.

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Beyond this case, another issue of interest concerns nephrolithiasis composition (calcium phosphate).

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Recently, Wong et al. reported that deferasirox was associated with a high prevalence of nephrolithiasis and

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ACCEPTED MANUSCRIPT bone demineralization in patients affected by thalassemia (12). In this study, among patients receiving

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deferasirox, those affected by nephrolithiasis had significantly lower serum phosphate and ferritin levels.

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FGF23 was not assessed but it might be hypothesized that deferasirox would promote renal phosphate leak

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and calcium phosphate stones in genetically predisposed individuals. More recently, Wong et al. observed

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hypercalciuria in 92% of subjects on deferasirox, with a significant dose-dependent relationship, but also in

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83.4% of the smaller group of subjects on deferoxamine(13). In the present report, the patient was also

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affected by hypercalciuria but the role of iron chelators in tubular defects leading to hypercalciuria remains

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unknown.

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In conclusion, this case supports the hypothesis that the iron status would be involved in C-terminal FGF23

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and serum phosphate levels regulation. Iron chelation was associated with increased C-terminal FGF23 and

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decreased serum phosphate levels, rapidly reversible after stopping treatment, in a genetically predisposed

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individual. Further studies are required to identify the mechanisms involved, in particular the sequence of

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events modifying FGF23 secretion and cleavage.

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Beyond this case, the role of iron chelators in kidney stone formation, osteomalacia and bone demineralization

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is an open question which must be addressed.

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Disclosure Statement: The authors have nothing to disclose

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Figure 1:

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Evolution of serum phosphate and C-terminal FGF23 levels under deferiprone and deferoxamine, 72h after

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stopping iron chelation, and after iron chelators reintroduction.

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