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BRIEF COMMUNICATION www.jasn.org Renal Fanconi Syndrome and Hypophosphatemic Rickets in the Absence of Xenotropic and Polytropic Retroviral Receptor in the Nephron Camille Ansermet,* Matthias B. Moor,* Gabriel Centeno,* Muriel Auberson,* † † ‡ Dorothy Zhang Hu, Roland Baron, Svetlana Nikolaeva,* Barbara Haenzi,* | Natalya Katanaeva,* Ivan Gautschi,* Vladimir Katanaev,*§ Samuel Rotman, Robert Koesters,¶ †† Laurent Schild,* Sylvain Pradervand,** Olivier Bonny,* and Dmitri Firsov* BRIEF COMMUNICATION *Department of Pharmacology and Toxicology and **Genomic Technologies Facility, University of Lausanne, Lausanne, Switzerland; †Department of Oral Medicine, Infection, and Immunity, Harvard School of Dental Medicine, Boston, Massachusetts; ‡Institute of Evolutionary Physiology and Biochemistry, St. Petersburg, Russia; §School of Biomedicine, Far Eastern Federal University, Vladivostok, Russia; |Services of Pathology and ††Nephrology, Department of Medicine, University Hospital of Lausanne, Lausanne, Switzerland; and ¶Université Pierre et Marie Curie, Paris, France ABSTRACT Tight control of extracellular and intracellular inorganic phosphate (Pi) levels is crit- leaves.4 Most recently, Legati et al. have ical to most biochemical and physiologic processes. Urinary Pi is freely filtered at the shown an association between genetic kidney glomerulus and is reabsorbed in the renal tubule by the action of the apical polymorphisms in Xpr1 and primary fa- sodium-dependent phosphate transporters, NaPi-IIa/NaPi-IIc/Pit2. However, the milial brain calcification disorder.5 How- molecular identity of the protein(s) participating in the basolateral Pi efflux remains ever, the role of XPR1 in the maintenance unknown. Evidence has suggested that xenotropic and polytropic retroviral recep- of Pi homeostasis remains unknown. Here, tor 1 (XPR1) might be involved in this process. Here, we show that conditional in- we addressed this issue in mice deficient for activation of Xpr1 in the renal tubule in mice resulted in impaired renal Pi Xpr1 in the nephron. reabsorption. Analysis of Pi transport in primary cultures of proximal tubular cells Because Xpr1-null mice exhibit em- or in freshly isolated renal tubules revealed that this Xpr1 deficiency significantly bryonic lethality (viable pups: wild affected Pi efflux. Further, mice with conditional inactivation of Xpr1 in the renal type,254;heterozygous,384;null,0), tubule exhibited generalized proximal tubular dysfunction indicative of Fanconi we generated mice with a doxycycline syndrome, characterized by glycosuria, aminoaciduria, calciuria, and albuminuria. (DOX)-inducible, Pax8-rtTA–driven,6 Dramatic alterations in the renal transcriptome, including a significant reduction in NaPi-IIa/NaPi-IIc expression, accompanied these functional changes. Additionally, Xpr1-deficient mice developed hypophosphatemic rickets secondary to renal dys- Received July 6, 2016. Accepted September 5, function. These results identify XPR1 as a major regulator of Pi homeostasis and as a 2016. potential therapeutic target in bone and kidney disorders. C.A., M.B.M., G.C., and M.A. contributed equally to this work. J Am Soc Nephrol 28: 1073–1078, 2017. doi: 10.1681/ASN.2016070726 Present address: Natalya Katanaeva, Swiss Federal Institute of Technology, Lausanne, Switzerland. Present address: Dr. Barbara Haenzi, Cambridge The xenotropic and polytropic retrovirus shown in vitro that XPR1 depletion or in- Centre for Brain Repair, University of Cambridge, receptor1(XPR1)haslongbeenconsidered hibition results in a marked decrease in Pi Cambridge, UK. as a candidate component of the inorganic efflux.3 They also demonstrated that Published online ahead of print. Publication date phosphate (Pi) efflux mechanism because XBRD, a XPR1 ligand derived from the available at www.jasn.org. of its high degree of homology with PHO1 X-MLV envelope glycoprotein, could effi- Correspondence: Dr. Dmitri Firsov or Dr. Olivier protein in plants, which has been shown ciently inhibit Pi efflux, thereby providing Bonny, Department of Pharmacology and Toxicol- ogy, University of Lausanne, 27 rue du Bugnon, to mediate Pi transport from roots to evidence on the direct role of XPR1 in Pi 1011 Lausanne, Switzerland. Email: dmitri.firsov@ shoots.1,2 However, evidence has only re- transport. Wege and Poirier have demon- unil.ch or [email protected] cently emerged supporting a role of XPR1 strated that ectopically expressed mouse Copyright © 2017 by the American Society of in Pi transport. Battini and colleagues have XPR1 mediates Pi efflux in tobacco Nephrology J Am Soc Nephrol 28: 1073–1078, 2017 ISSN : 1046-6673/2804-1073 1073 BRIEF COMMUNICATION www.jasn.org conditional deletion of Xpr1 in the renal knockout [cKO] mice). Littermate investigated separately to assess possible tubule (Xpr1lox/lox/Pax8-rtTA/LC1 mice, Xpr1lox/lox mice treated with DOX were sex differences. As shown in Supplemen- hereafter referred to as conditional used as controls. Males and females were tal Figure 1, DOX treatment resulted in a Figure 1. Altered renal handling of Pi in cKO mice. White circles/bars indicate control mice. Blue and red circles/bars indicate male or female cKO mice, respectively. (A) Body weight in control and cKO male (left panel) or female (right panel) mice. The body weight was measured during 5 days preceding DOX treatment (baseline), during the 2-week period of DOX treatment (days DOX), and during 28 days after DOX withdrawal (days post DOX). n=6 in each group; ANOVA. (B) Plasma Pi levels in control and cKO male (left panel) or female (right panel) mice. Plasma Pi concentration was measured on the day preceding the 2-week period of DOX treatment (baseline), on day 7 of DOX treatment (7 days DOX), and on days 3, 14, 21, and 28 after DOX † †† withdrawal (days post DOX). *P,0.05; **P,0.01; ***P,0.001; t test, statistical significance between control and cKO mice. P,0.05; P,0.01; ††† P,0.001; t test, statistical significance between plasma Pi levels measured at baseline and plasma Pi levels measured on day 7 of DOX treatment or after DOX withdrawal (days 3, 14, and 28). (C) TmPi/GFR in control and cKO male (left panel) or female (right panel) mice. The TmPi/GFR was de- termined on day 28 after DOX withdrawal. *P,0.05; **P,0.01; ttest. (D) FEPi in control and cKO male (left panel) or female (right panel) mice. The FEPi was determined on day 28 after DOX withdrawal. *P,0.05; t test. (E) [33Pi]phosphate uptake in primary cultures of proximal tubule cells isolated from DOX-untreated control or male cKO mice. Cells were exposed to DOX for 24 hours and the [33Pi]phosphate uptake was measured 24 hours after the end of DOX treatment (see Supplemental Material for details). n=4 in each group; ANOVA. (F) [33P]phosphate efflux from primary cultures of control or cKO proximal tubule cells. n=4 in each group; ANOVA. For (E), (F), and (H), white and blue circles indicate primary cultures of control or cKO proximal tubule cells, respectively. (G) [33P]phosphate remaining in primary cultures of control or cKO proximal tubule cells at the end of the efflux experiment (60 minutes of efflux); n=4 in each group. *P,0.05; t test. (H) [14C]glucose efflux from primary cultures of control or cKO proximal tubule cells. n=4 in each group; ANOVA. (I) [33P]phosphate uptake (30 minutes) and efflux (3 minutes, 8 minutes, and 40 minutes) from renal tubules freshly isolated from kidneys of control or cKO mice induced with DOX for 5 days (for efflux experiments, the 30-minute [33P]phosphate uptake was set as the zero time point). Pi uptake was determined in the presence or absence of 5 mmol PFA. Pi efflux was measured in the presence of 5 mmol PFA (see Supplemental Material for details). Background represents nonspecificbindingof[33P]phosphate to the renal tubules. n=4 in each group. The difference in the efflux kinetics was evaluated by ANOVA (genotype–time interaction). The differences in background, 30’ uptake + PFA, and 30’ uptake conditions was evaluated by t test. Numbers inside of bars represent the number of animals. Data are mean6SEM. *P,0.05. FEPi, fractional excretion of Pi. 1074 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1073–1078, 2017 www.jasn.org BRIEF COMMUNICATION significant reduction in Xpr1 mRNA and expression was accompanied by a progres- 212.1% (cKO females) 28 days after the protein levels in whole kidneys and in mi- sively increasing difference in body weight end of DOX treatment (Figure 1A). Assess- crodissected proximal tubules of cKO between control and cKO mice that ment of renal Pi handling revealed that mice. The decrease in renal XPR1 reached 220.4% (cKO males) and cKO mice exhibited hypophosphatemia Figure 2. XPR1 deficiency in the nephron is associated with aminoaciduria, glucosuria, albuminuria, and impaired albumin reabsorption in the proximal tubule. (A) Aminoaciduria in cKO mice. The urinary excretion rate of 19 of 20 proteinogenic amino acids (at the exception of cysteine) was measured by mass spectrometry on urine collected on day 28 after DOX withdrawal. White bars indicate the urinary excretion rates of amino acids in control mice. Blue and red bars indicate the urinary excretion rates of amino acids in male or female cKO mice, respectively; n=6 in each group. *P,0.05; **P,0.01; ***P,0.001; t test. (B) Glucosuria in cKO mice. The urinary excretion rate of glucose was measured on urine collected on the day preceding the 2-week period of DOX treatment (baseline), on days 7 and 14 of DOX treatment (days DOX), and on days 7, 14, 21, and 28 after DOX withdrawal (days post DOX). White bars indicate the urinary excretion rates of glucose in control mice (n=6 for males and n=4 for females). Blue and red bars indicate the urinary excretion rates of glucose in male or female cKO mice, respectively (n=6 for males and n=6 for females). *P,0.05; **P,0.01; ***P,0.001; t test, statistical significance between control and † †† ††† cKO mice.

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