The xenotropic and polytropic retrovirus receptor 1 (XPR1) has long been considered as a candidate component of the inorganic phosphate (Pi) efflux mechanism because of its high degree of homology with PHO1 protein in plants, which has been shown to mediate Pi transport from roots to shoots.1,2 However, evidence has only recently emerged supporting a role of XPR1 in Pi transport. Battini and colleagues have shown in vitro that XPR1 depletion or inhibition results in a marked decrease in Pi efflux.3 They also demonstrated that XBRD, a XPR1 ligand derived from the X-MLV envelope glycoprotein, could efficiently inhibit Pi efflux, thereby providing evidence on the direct role of XPR1 in Pi transport. Wege and Poirier have demonstrated that ectopically expressed mouse XPR1 mediates Pi efflux in tobacco leaves.4 Most recently, Legati et al. have shown an association between genetic polymorphisms in Xpr1 and primary familial brain calcification disorder.5 However, the role of XPR1 in the maintenance of Pi homeostasis remains unknown. Here, we addressed this issue in mice deficient for Xpr1 in the nephron.
Because Xpr1-null mice exhibit embryonic lethality (viable pups: wild type, 254; heterozygous, 384; null, 0), we generated mice with a doxycycline (DOX)-inducible, Pax8-rtTA–driven,6 conditional deletion of Xpr1 in the renal tubule (Xpr1lox/lox/Pax8-rtTA/LC1 mice, hereafter referred to as conditional knockout [cKO] mice). Littermate Xpr1lox/lox mice treated with DOX were used as controls. Males and females were investigated separately to assess possible sex differences. As shown in Supplemental Figure 1, DOX treatment resulted in a significant reduction in Xpr1 mRNA and protein levels in whole kidneys and in microdissected proximal tubules of cKO mice. The decrease in renal XPR1 expression was accompanied by a progressively increasing difference in body weight between control and cKO mice that reached −20.4% (cKO males) and −12.1% (cKO females) 28 days after the end of DOX treatment (Figure 1A). Assessment of renal Pi handling revealed that cKO mice exhibited hypophosphatemia (Figure 1B), phosphaturia (transient in males, Supplemental Figure 2), inappropriately low maximal tubular reabsorption of Pi per volume of glomerular filtrate (TmPi/GFR) (Figure 1C), and significantly increased fractional excretion of Pi (Figure 1D). Furthermore, we assessed the role of XPR1 in Pi efflux in primary cultures of proximal tubular cells isolated from kidneys of DOX-untreated control and cKO mice. Xpr1 deficiency was induced ex vivo by 24 hours of DOX exposure. Twenty four hours after the end of DOX treatment, the Xpr1 mRNA expression was significantly decreased in the proximal tubular cells isolated from cKO mice, as assessed by quantitative PCR (Xpr1 mRNA expression in cKO versus control cells: 18.9±7.3%; n=3; P=0.01, t test). As shown in Figure 1E, proximal tubular cells from cKO mice had a nonsignificant trend toward lower [33Pi]phosphate uptake. In contrast, [33P]phosphate efflux was strongly affected by XPR1 deficiency (Figure 1F). The latter correlated with higher percentage of [33P]phosphate remaining in the proximal tubular cells from cKO mice after 60 minutes of efflux (Figure 1G). Importantly, efflux of [14C]glucose was not different between proximal tubular cells isolated from kidneys of control or cKO mice (Figure 1H), indicating that the short-term ex-vivo Xpr1 deficiency did not result in the overall depression of efflux transport activity. The Pi efflux was also evaluated in renal tubules freshly isolated from kidneys of control or cKO mice treated with DOX for 5 days. As shown in Figure 1I, the 30-minute [33P]phosphate uptake was similar in both genotypes, and was significantly reduced in the presence of phosphonoformic acid (PFA), a low potency competitive inhibitor of apical Na+/Pi cotransporters. The persistent Pi uptake in the presence of PFA likely results from partial inhibition of apical Pi transport, but importantly, the fraction of PFA-sensitive Pi uptake was not different between genotypes. At the end of the 30-minute uptake period, the [33P]phosphate was removed from the bath and Pi efflux was measured. In tubules isolated from kidneys of cKO mice, the Pi efflux was significantly slower compared with control mice, providing further evidence for an XPR1-mediated Pi efflux. Pi efflux is generally considered to occur through the basolateral membrane; indeed, an apical Pi efflux is very unlikely because intracellular Pi concentration remains far below the thermodynamic equilibrium for Na+-dependent Pi transporters.7 Collectively, these experiments demonstrate a critical role of XPR1 in Pi efflux from renal tubular cells, and suggest Xpr1 deficiency as the primary cause of phosphaturia in cKO mice.
Analysis of urine samples revealed that 1 week after beginning DOX treatment, cKO mice developed generalized proximal tubule dysfunction, or renal Fanconi syndrome, characterized by aminoaciduria (Figure 2A), glycosuria (Figure 2B), albuminuria (Figure 2C), magnesuria (Supplemental Figure 3A), calciuria (Supplemental Figure 3B), lower urinary pH (Supplemental Figure 4A), polyuria (Supplemental Figure 4B), and decreased urine osmolality (Supplemental Figure 4C). Transcriptome analysis (GSE87450) of kidneys from control and cKO mice (males) revealed dramatic changes in expression levels of RNAs encoding proteins involved in apical Pi reabsorption (NaPi-IIa [Slc34a1]: −7.19-fold; NaPi-IIc [Slc34a3]: −25.37-fold), glucose reabsorption (SGLT2 [Slc5a2]: −2.88-fold; GLUT2 [Slc2a2]: −2.87-fold), amino acid transport (LAT2 [Slc7a8]: −4.59-fold; BAT1 [Slc7a9]: −4.24-fold; LAT1 [Slc7a7]: −3.36-fold; 4F2hc [Slc3a2]: −1.83-fold), and in endocytic receptors required for reuptake of filtered albumin in the proximal tubule (megalin [Lrp2]: −3.52-fold; cubilin [Cubn]: −3.40-fold) (Supplemental Table 1). The impairment in tubular albumin reabsorption was assessed functionally by confocal microscopy analysis of kidney slices prepared from kidneys of mice intravenously injected with fluorescent albumin (Texas Red albumin). As shown in Figure 2D, Texas Red albumin was abundantly present in the subapical region of the proximal tubular cells in kidneys of control mice, whereas the fluorescence intensity was significantly lower in kidneys of cKO mice.
The kidneys of cKO mice exhibited reduced expression of genes encoding mitochondrially located proteins (Supplemental Figure 5, A and B) despite normal mitochondrial biogenesis (Supplemental Figure 5C) and apparently normal mitochondria, as examined by electron microscopy (Supplemental Figure 5E). The NAD+/NADH ratio was significantly reduced in kidneys of cKO mice, suggesting a shift in the metabolic status resulting from the XPR1 deficiency (Supplemental Figure 5D).
The GFR was significantly decreased in male cKO mice, along with an increase in plasma creatinine levels in cKO mice of both sexes (Table 1). The cKO mice exhibited slightly higher calcemia, whereas plasma levels of glucose, sodium, and potassium, and plasma osmolality were not different from controls (Table 1). Plasma aldosterone levels were significantly increased, suggesting extracellular volume contraction in cKO mice (Table 1).
Table 1. -
Plasma chemistry and GFR in control and cKO mice euthanized on day 28 after DOX withdrawal
|Osmolality, mosm/kg H2O
|GFR (inulin), μl/min
|ALP activity, U/L
Data are means±SEM (n). P values calculated using unpaired t test. CTX1, C-terminal telopeptides of type I collagen; PTH, parathyroid hormone; ALP, alkaline phosphatase; TRAP, tartrate-resistant acid phosphatase.
Hypophosphatemia and decreased TmPi/GFR prompted us to study the bone phenotype in cKO mice. Analysis of vertebrae by microcomputed tomography revealed severely decreased bone mineral density, bone volume per total volume, trabecular thickness, and trabecular number in male cKO mice, and similar but milder features in female cKO mice (Figure 3A, Supplemental Table 2). The microcomputed tomography analysis of femora showed significantly decreased thickness and tissue mineral density in the distal diaphyseal and metaphyseal cortical bone in male cKO mice, with similar but nonsignificant changes in female cKO mice, and largely unaffected trabecular bone of the distal metaphysis (Supplemental Table 3).
Vertebral specimens of male control and cKO mice were further analyzed by nondecalcified bone histomorphometry. Although not clearly visible at low magnification (Figure 3B), high magnification analysis showed a striking increase in all unmineralized osteoid parameters in cKO mice (Supplemental Table 4). The excessive osteoid in vertebrae of cKO mice is distinctly visible on a representative image of Toluidine Blue staining (Figure 3C). Cellular osteoblast parameters (the number of osteoblasts and the osteoblast surface) were unchanged in cKO mice, whereas the number of osteoclasts was increased (Supplemental Table 4). Collectively, these data reveal a highly excessive fraction of unmineralized bone in cKO mice, consistent with rickets.
To gain further insight into the molecular mechanisms underlying the defective bone mineralization in cKO mice, we measured plasma levels of hormones involved in calcium/phosphate homeostasis and bone turnover biomarkers (Table 1). Most strikingly, fibroblast growth factor 23 (FGF23) levels were undetectable in cKO mice of both sexes, whereas 1,25-dihydroxyvitamin D3 [1,25(OH)2-D3, or calcitriol] and parathyroid hormone levels were unchanged. Collagen degradation product CTX1 was significantly increased in male cKO mice, and a nonsignificant trend in the same direction was found in female cKO mice, suggesting an increase in bone resorption consistent with the increased osteoclast numbers observed. However, alkaline phosphatase activity was unchanged. The levels of the osteoblast-produced hormone osteocalcin were increased in male cKO mice. To summarize, distinct signs of overall altered bone turnover were present in cKO mice.
To conclude, mice deficient for Xpr1 in the renal tubule develop complete Fanconi syndrome and hypophosphatemic rickets. The severity of renal dysfunction was similar in cKO mice of both sexes, whereas the bone phenotype was more prominent in males compared with females, an observation that has been made in human patients.8 Hypophosphatemic rickets represents a heterogeneous entity that can be further divided into conditions associated with high FGF23 levels and suppressed 1,25(OH)2-D3, such as X-linked hypophosphatemic rickets and autosomal recessive hypophosphatemic rickets, or with low FGF23 and high 1,25(OH)2-D3 levels, found when defects of renal phosphate transport are present. Indeed, mutations of NaPi-IIa and NaPi-IIc, the two sodium-phosphate cotransporters present in the brush border of the proximal tubule, lead to hereditary hypophosphatemic rickets with hypercalciuria.9,10 Here, we provide evidence for involvement of XPR1 in hypophosphatemic rickets associated with low FGF23 levels and normal 1,25(OH)2-D3 levels, reminiscent of hereditary hypophosphatemic rickets with hypercalciuria. Furthermore, we show that renal XPR1 is essential for phosphate homeostasis and bone physiology, and open new avenues for treatment options.
Detailed methods are described in the Supplemental Material.
The authors thank Drs. Jean-Luc Battini and Yves Poirier for helpful discussions, Dr. Florence Morgenthaler (Cellular Imaging Facility, University of Lausanne, Lausanne, Switzerland) for help with microcomputed tomography analysis, and the Lausanne Genomic Technologies Facility for transcriptome analysis.
This work was supported by the Swiss National Science Foundation Research grants 31003A-149440 (to D.F.) and 310030-163340 (to O.B.).
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