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NDT Advance Access originally published online on November 23, 2007
Nephrology Dialysis Transplantation 2008 23(2):730-733; doi:10.1093/ndt/gfm535
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org


Matrix extracellular phosphoglycoprotein causes phosphaturia in rats by inhibiting tubular phosphate reabsorption

Hamish Dobbie, Robert J. Unwin, Nuno J. R. Faria and David G. Shirley

Department of Physiology and Centre for Nephrology, University College London, Hampstead Campus, Rowland Hill Street, London NW3 2PF, UK

Correspondence to: Dr David G. Shirley, Department of Physiology and Centre for Nephrology, University College London, Hampstead Campus, Rowland Hill Street, London NW3 2PF, UK. Email: david.shirley{at}ucl.ac.uk



   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. Matrix extracellular phosphoglycoprotein (MEPE), first isolated from tumour-derived tissue from a patient with oncogenic hypophosphataemia, is a putative phosphatonin that has received much less attention than fibroblast growth factor-23. To date, its effect on renal tubular phosphate reabsorption remains undefined.

Methods. A renal clearance study was performed in anaesthetized rats infused intravenously with a range of doses of MEPE.

Results. MEPE had no effect on glomerular filtration rate (inulin clearance) but caused rapid, dose-dependent increases in absolute and fractional phosphate excretion, wholly attributable to reduced phosphate reabsorption. At a maximal dose, MEPE increased fractional phosphate excretion more than 2-fold, whereas no change was observed in time controls.

Conclusion. The results lend support to the hypothesis that MEPE contributes to the phosphaturia of oncogenic hypophosphataemia and of hypophosphataemic rickets.

Keywords: kidney; phosphatonin; phosphaturia; renal clearance



   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Acknowledgements
 References
 
Matrix extracellular phosphoglycoprotein (MEPE) was first isolated from tumour-derived tissue obtained from a patient with oncogenic hypophosphataemic osteomalacia [1]. It was independently isolated from rat primary bone marrow cultures during osteoblast differentiation (where it was given the name osteoblast/osteocyte factor 45; OF45) [2]. MEPE, along with a number of other putative humoural regulators of phosphate balance (‘phosphatonins’), is up-regulated in oncogenic hypophosphataemia [3,4] and in the murine form of X-linked hypophosphataemic rickets [5], but the current and prevailing view is that MEPE is a regulator of bone mineralization rather than a contributor to phosphate wasting in these conditions [6–8].

Nevertheless, circulating levels of MEPE correlate with serum phosphate concentrations [9], and Rowe's group has reported that repetitive high-dose intraperitoneal injections of MEPE into mice caused increased phosphate excretion, when compared with saline-injected controls [10]. However, since phosphate excretion was factored for creatinine excretion {an unreliable marker of glomerular filtration rate (GFR) in rodents [11]}, it was not possible to determine whether the tubular reabsorption of phosphate was altered. Here, we report the results of a renal clearance study in rats, in which we have used a series of infusions of MEPE, delivered intravenously to control its bioavailability; GFR was determined using inulin.



   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 Acknowledgements
 References
 
Adult male Sprague-Dawley rats (weighing 220–260 g) were anaesthetized with sodium thiopentone (100 mg/kg; intraperitoneally; Link Pharmaceuticals, Horsham, Sussex) and prepared surgically for clearance experiments (jugular venous cannulae; tracheotomy; bladder catheter; femoral arterial catheter) as described previously [12]. Isotonic saline was infused intravenously throughout at 4 ml/h. Thirty minutes after the completion of surgery, [3H]inulin (Amersham Biosciences, Little Chalfont, Bucks) was included in the infusate (2 µCi primer; 2 µCi/h). After a further hour of equilibration, all rats underwent a 1 h clearance period, at the end of which the animals were divided into four groups: one group (n = 6; time controls) continued to receive the saline vehicle for a further 2 h; the second group (n = 6; low-dose MEPE) was infused with recombinant full-length human MEPE (raised in insect Spodoptera frugiperda cells; a gift from Acologix, Hayward, CA, USA) at a dose of 30 µg/h for 2 h; the third group (n = 6; medium-dose MEPE) was infused with MEPE at 100 µg/h; and the final group (n = 6; high-dose MEPE) at 300 µg/h. During the final hour of the infusions, designated the experimental period, clearance measurements were repeated. Small arterial blood samples (~100 µl) were taken at the start and end of each clearance period.

Analyses
Urine and plasma samples were analysed for phosphate concentration and [3H]inulin activity by capillary electrophoresis [13] and β-emission spectroscopy (Packard Tricarb, model 2900TR), respectively.

Calculations and statistics
Glomerular filtration rate (GFR) was measured as the renal clearance of [3H]inulin (CIn). Renal clearances of phosphate (CPi) and [3H]inulin were calculated using the standard formula. The fractional excretion of phosphate (FEPi) was calculated as CPi/CIn.

Values are presented as means ± SEM. Statistical assessment of the changes in renal variables between the control and experimental periods in the four groups of rats was made by analysis of variance (ANOVA) with repeated measures, followed, where appropriate, by Bonferroni's multiple comparisons test. Comparison of the {Delta}FEPi in each MEPE-treated group with that in the vehicle group was made by one-way ANOVA followed by Dunnett's multiple comparisons test. A P-value of <0.05 was taken to be statistically significant.



   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 Acknowledgements
 References
 
Figure 1 shows values for GFR, absolute phosphate excretion and FEPi. GFR was similar in the four groups of rats during the baseline period and tended to fall slightly (NS) during the course of the experiment in all groups (Figure 1A). Phosphate excretion also did not differ significantly between the four groups during the baseline period. Whilst absolute phosphate excretion remained fairly stable in the time-control group, it tended to increase (though not significantly) in the low-dose MEPE group and increased markedly in both medium-dose and high-dose MEPE groups (Figure 1B). Because neither GFR nor plasma phosphate concentration differed significantly among the groups during either the control period or the experimental period, FEPi followed a similar pattern to absolute phosphate excretion. Thus, FEPi remained stable in the time controls, increased somewhat (NS) in the low-dose MEPE group and increased markedly in the medium- and high-dose MEPE groups (Figure 1C).


Figure 1
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  		Fig. 1. Glomerular filtration rate and phosphate excretion. (A) Glomerular filtration rate (GFR), (B) absolute phosphate excretion and (C) fractional phosphate excretion (FEPi) in rats infused with vehicle alone or with one of three doses of MEPE. Values shown are means ± SE; there were six rats in each group.

 
Figure 2 shows the dose–response curve for the effect of MEPE on FEPi, showing the change in FEPi between the control and experimental periods. MEPE had a dose-dependent effect on FEPi, and the effect was already maximal ({Delta}FEPi ~20% of the filtered load) at the medium dose.


Figure 2
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  		Fig. 2. Dose–response curve showing the change in fractional phosphate excretion ({Delta}FEPi) between the control and experimental periods in rats infused with vehicle alone (zero dose) or with one of three doses of MEPE. Note the log scale on the x-axis. Values shown are means ± SE; there were six rats in each group. *P < 0.01 compared with the value in rats infused with vehicle alone.

 


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Acknowledgements
 References
 
This study showed that intravenous infusion of MEPE into normal rats caused a rapid, dose-dependent increase in phosphate excretion that was wholly attributable to a reduction in absolute and fractional phosphate reabsorption by the nephron. Unfortunately, since no reliable assay for MEPE is available to us, we were unable to relate this effect to a particular plasma concentration of MEPE. Nevertheless, the findings lend support to the hypothesis that MEPE (or a derivative) is at least partly responsible for the phosphaturia seen in oncogenic hypophosphataemia and in hypophosphataemic rickets. This action of MEPE does not diminish the claims of other candidate phosphatonins such as fibroblast growth factor-23 (FGF-23), which has been shown to inhibit phosphate transport in rabbit proximal tubules in vitro [14] and to increase fractional phosphate excretion in mice in vivo [15], and frizzled related protein-4 (FRP-4), which has been shown to increase fractional phosphate excretion in rats [16]. Indeed, a recent study of patients with oncogenic hypophosphataemia showed that most of the subjects had raised plasma concentrations of FGF-23 [17]. However, a significant proportion did not. There is some suggestion that both MEPE and FGF-23 are involved in the disordered phosphate balance of these two conditions [18], though the nature of any such interaction is complex and unclear [19].

The site of action of MEPE along the nephron could not be ascertained from the present study, but, given the magnitude of the response and the fact that the bulk of phosphate reabsorption occurs in the (early) proximal tubule, this nephron segment must be a likely candidate. In support of this, in vitro studies have indicated that phosphate uptake in cultured renal proximal tubule cells is reduced during incubation with MEPE [10], while a recent study has reported MEPE immunoreactivity and mRNA in the proximal tubule [20]. However, in vivo confirmation of the renal site(s) of action of MEPE awaits a systematic micropuncture investigation.



   Acknowledgements
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
References
 
We thank Dr Peter Rowe for helpful discussion and Acologix for the gift of MEPE, and we gratefully acknowledge financial support from Kidney Research UK and St Peter's Trust for Kidney, Bladder & Prostate Research.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Rowe PS, de Zoysa PA, Dong R, et al. MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia. Genomics (2000) 67:54–68.[CrossRef][Web of Science][Medline]
  2. Petersen DN, Tkalcevic GT, Mansolf AL, Rivera-Gonzalez R, Brown TA. Identification of osteoblast/osteocyte factor 45 (OF45), a bone-specific cDNA encoding an RGD-containing protein that is highly expressed in osteoblasts and osteocytes. J Biol Chem (2000) 275:36172–36180.[Abstract/Free Full Text]
  3. Shimada T, Mizutani S, Muto T, et al. Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia. Proc Natl Acad Sci USA (2001) 98:6500–6505.[Abstract/Free Full Text]
  4. De Beur SM, Finnegan RB, Vassiliadis J, et al. Tumors associated with oncogenic osteomalacia express genes important in bone and mineral metabolism. J Bone Miner Res (2002) 17:1102–1110.[CrossRef][Web of Science][Medline]
  5. Argiro L, Desbarats M, Glorieux FH, Ecarot BMepe. the gene encoding a tumor-secreted protein in oncogenic hypophosphatemic osteomalacia, is expressed in bone. Genomics (2001) 74:342–351.[CrossRef][Web of Science][Medline]
  6. Blumsohn A. What have we learnt about the regulation of phosphate metabolism? Curr Opin Nephrol Hypertens (2004) 13:397–401.[CrossRef][Web of Science][Medline]
  7. Liu S, Brown TA, Zhou J, et al. Role of matrix extracellular phosphoglycoprotein in the pathogenesis of X-linked hypophosphatemia. J Am Soc Nephrol (2005) 16:1645–1653.[Abstract/Free Full Text]
  8. White KE, Larsson TE, Econs MJ. The roles of specific genes implicated as circulating factors involved in normal and disordered phosphate homeostasis: frizzled related protein-4, matrix extracellular phosphoglycoprotein, and fibroblast growth factor 23. Endocr Rev (2006) 27:221–241.[Abstract/Free Full Text]
  9. Jain A, Fedarko NS, Collins MT, et al. Serum levels of matrix extracellular phosphoglycoprotein (MEPE) in normal humans correlate with serum phosphorus, parathyroid hormone and bone mineral density. J Clin Endocrinol Metab (2004) 89:4158–4161.[Abstract/Free Full Text]
  10. Rowe PS, Kumagai Y, Gutierrez G, et al. MEPE has the properties of an osteoblastic phosphatonin and minhibin. Bone (2004) 34:303–319.[Medline]
  11. Namnum P, Insogna K, Baggish D, Hayslett JP. Evidence for bidirectional net movement of creatinine in the rat kidney. Am J Physiol (1983) 244:F719–F723.[Web of Science][Medline]
  12. Walter SJ, Zewde T, Shirley DG. The effect of anaesthesia and standard clearance procedures on renal function in the rat. Q J Exp Physiol (1989) 74:805–812.[Abstract/Free Full Text]
  13. Faria NJR, Dobbie H, Slater JM, Shirley DG, Stocking CJ, Unwin RJ. Simultaneous determination of anions in nanoliter volumes. Kidney Int (2005) 67:357–363.[CrossRef][Web of Science][Medline]
  14. Baum M, Schiavi S, Dwarakanath V, Quigley R. Effect of fibroblast growth factor-23 on phosphate transport in proximal tubules. Kidney Int (2005) 68:1148–1153.[CrossRef][Web of Science][Medline]
  15. Imel EA, Econs MJ. Fibroblast growth factor 23: roles in health and disease. J Am Soc Nephrol (2005) 16:2565–2575.[Free Full Text]
  16. Berndt T, Craig TA, Bowe AE, et al. Secreted frizzled-related protein 4 is a potent tumor-derived phosphaturic agent. J Clin Invest (2003) 112:785–794.[CrossRef][Web of Science][Medline]
  17. Imel EA, Peacock M, Pitukcheewanont P, et al. Sensitivity of fibroblast growth factor 23 measurements in tumor-induced osteomalacia. J Clin Endocrinol Metab (2006) 91:2055–2061.[Abstract/Free Full Text]
  18. Rowe PSN. The wrickkened pathways of FGF23, MEPE and PHEX. Crit Rev Oral Biol Med (2004) 15:264–281.[Abstract/Free Full Text]
  19. Liu S, Rowe PS, Vierthaler L, Zhou J, Quarles LD. Phosphorylated acidic serine-aspartate-rich MEPE-associated motif peptide from matrix extracellular phosphoglycoprotein inhibits phosphate regulating gene with homologies to endopeptidases on the X-chromosome enzyme activity. J Endocrinol (2007) 192:261–267.[Abstract/Free Full Text]
  20. Ogbureke KUE, Fisher LW. Renal expression of SIBLING proteins and their partner matrix metalloproteinases (MMPs). Kidney Int (2005) 68:155–166.[CrossRef][Web of Science][Medline]
Received for publication: 28. 2.07
Accepted in revised form: 10. 7.07


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