Differential effects of Npt2a gene ablation and X-linked Hyp mutation on renal e - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:48 |只看该作者 |倒序浏览 |打印

作者：Harriet S. Tenenhouse, Josée Martel, Claude Gauthier, Hiroko Segawa,  Ken-ichi Miyamoto作者单位：Departments of Pediatrics and Human Genetics, McGill University, and Montreal Children‘s Hospital Research Institute, Montreal, Quebec, H3Z 2Z3 Canada; and Department of Nutrition, School of Medicine, Tokushima University, Tokushima City, 770-8503 Japan
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      【摘要】
   The present study was undertaken to define the mechanisms governing the regulation of the novel renal brush-border membrane (BBM) Na-phosphate (P i ) cotransporter designated type IIc (Npt2c). To address this issue, the renal expression of Npt2c was compared in two hypophosphatemic mouse models with impaired renal BBM Na-P i cotransport. In mice homozygous for the disrupted Npt2a gene ( Npt2 - / - ), BBM Npt2c protein abundance, relative to actin, was increased 2.8-fold compared with Npt2  /  littermates, whereas a corresponding increase in renal Npt2c mRNA abundance, relative to -actin, was not evident. In contrast, in X-linked Hyp mice, which harbor a large deletion in the Phex gene, the renal abundance of both Npt2c protein and mRNA was significantly decreased by 80 and 50%, respectively, relative to normal littermates. P i deprivation elicited a 2.5-fold increase in BBM Npt2c protein abundance in Npt2  /  mice but failed to elicit a further increase in Npt2c protein in Npt2 - / - mice. P i restriction led to an increase in BBM Npt2c protein abundance in both normal and Hyp mice without correcting its renal expression in the mutants. In summary, we report that BBM Npt2c protein expression is differentially regulated in Npt2 - / - mice and Hyp mice and that the Npt2c response to low-P i challenge differs in both hypophosphatemic mouse strains. We demonstrate that Npt2c protein is maximally upregulated in Npt2 - / - mice and suggest that Npt2c likely accounts for residual BBM Na-P i cotransport in the knockout model. Finally, our data indicate that loss of Phex function abrogates renal Npt2c protein expression. 3 Y) @, m5 ^( r# E. \
      【关键词】 hypophosphatemia mouse Phex NaP i cotransporter type I low phosphate! h0 n0 U$ |: |# o/ M  F
   THE KIDNEY PLAYS a critical role in the maintenance of phosphate (P i ) homeostasis ( 22 ). Evidence from physiological studies suggests that Na-dependent P i transporters in the brush-border membrane (BBM) of proximal tubular cells mediate the rate-limiting step in the overall P i reabsorptive process ( 21 ). Two distinct classes of Na-P i cotransporters, type I (Npt1; 36 ) and type II (Npt2; 19 ), identified by expression cloning, have been localized exclusively to the BBM of proximal tubular cells ( 6, 9 ) and thus are appropriately positioned to reabsorb filtered P i.

Npt1 mediates Na-P i cotransport when expressed in Xenopus laevis oocytes ( 36 ) and accounts for 15% of Na-P i cotransporter mRNAs in mouse kidney ( 33 ). However, the precise contribution of Npt1 to renal P i reabsorption remains unclear ( 22 ). Furthermore, electrophysiological studies demonstrated that Npt1 also operates as a channel for Cl - and mediates the transport of anionic xenobiotics ( 7, 39 ).5 P% c" a- ]4 m% U) |/ m2 a& \7 {
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Npt2 is the most abundant of the renal Na-P i cotransporters ( 33 ) and is a target for regulation by parathyroid hormone (PTH; 16 ) and dietary P i ( 18 ), the major regulators of renal P i handling. We demonstrated that disruption of the Npt2 gene in mice results in increased urinary P i excretion, an 80% loss in BBM Na-P i cotransport and significant hypophosphatemia ( 4 ). In addition, mice homozygous for the disrupted Npt2 gene ( Npt2 - / - ) fail to respond to P i deprivation with an adaptive increase in BBM Na-P i cotransport ( 14 ) and to PTH with a decrease in transport ( 42 ). These findings underscore the significant role of Npt2 in renal P i reabsorption and its regulation by dietary P i and PTH.

Studies in our laboratory also demonstrated that decreased renal expression of Npt2 mRNA and immunoreactive protein are responsible, at least in part, for decreased BBM Na-P i cotransport, renal P i wasting, and hypophosphatemia in X-linked Hyp mice ( 35 ). The latter harbor a large 3'-deletion in the Phex gene ( 5 ) and serve as a model for X-linked hypophosphatemia (XLH), the most prevalent form of inherited rickets in humans ( 10, 29 ). The findings in both Hyp mice and Npt2 knockout mice underscore the importance of Npt2 in the maintenance of P i homeostasis.2 y* j3 G' S, r  X7 E
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More recently, two additional type II Na-P i cotransporters, with homology to Npt2 (now designated Npt2a), have been identified. The type IIb transporter is expressed in mammalian small intestine, but not in kidney, and is a candidate for apical intestinal Na-Pi cotransport ( 13 ). The type IIc transporter (Npt2c) was identified in rat and human kidney and is expressed exclusively in the BBM of proximal tubular cells ( 25 ). Moreover, Npt2c is regulated by dietary P i, and the relative abundance of Npt2c protein is significantly higher in kidneys of 22-day-old rats than in those of 60-day-old rats, suggesting that Npt2c is a growth-related renal Na-P i cotransporter ( 25 ). In addition, hybrid depletion studies suggested that Npt2c accounts for 30% of Na-P i cotransport in kidneys of Pi-deprived adult mice ( 23 ).

The contribution of Npt2c to the overall renal P i reabsorptive process remains unclear. Moreover, the nature of the renal Na-P i cotransporter(s) responsible for residual P i reabsorption in Npt2 knockout mice has not yet been delineated. To clarify these issues, we examined the impact of Npt2a gene ablation on renal Npt2c expression. In addition, it is not clear whether the decrement in renal P i reabsorption in X-linked Hyp mice can be attributed entirely to downregulation of Npt2a. We thus sought to determine whether molecular events, secondary to loss of Phex function, also have an impact on renal Npt2c expression. We also examined the effect of P i deprivation on Npt2c protein abundance in Npt2 - / - and Hyp mice and the effects of Npt2a gene ablation and the Hyp mutation on renal Npt1 expression. We demonstrate that Npt2a gene ablation and the Hyp mutation differentially regulate renal Npt2c and Npt1 gene expression. Our data suggest that Npt2c may be responsible for residual BBM Na-P i cotransport in Npt2 - / - mice and that normal Phex function is essential for Npt2c regulation in mouse kidney.

MATERIALS AND METHODS2 w9 \# J6 O( S) w" d& d# t

Mice. Mice were bred and raised in the Animal Facility at the Montreal Children's Hospital Research Institute. Male Npt2 - / - mice and wild-type (WT) littermates ( Npt2  /  ) were derived by crossing Npt2 - / - males with Npt2 - / - females and Npt2  /  males with Npt2  /  females, and progeny were genotyped by PCR amplification of genomic DNA obtained from tail tissue, as described ( 4 ). Male Hyp mice and WT littermates were derived by breeding Hyp /  females with  /Y males as described ( 35 ). The original breeding pairs ( Hyp /  x  /Y on C57BL/6J background) were obtained from Jackson Laboratory, Bar Harbor, ME. Mice were weaned at 25 days of age and were killed between 60 and 70 days of age. Mice received water ad libidum and were fed a normal diet, containing 0.6% Pi (5001, Purina Lab Chow, Ralston Purina, St. Louis, MO), unless otherwise indicated. To examine the effect of dietary P i intake on renal BBM Npt2a and Npt2c protein abundance, Npt2 - / - and Hyp mice and their respective WT littermates were fed diets containing 0.02, 0.6, and 1.0% P i for 4 days [test diets (TD) 86128, TD 98243, and TD 86129, respectively, Harlan Teklad, Madison, WI]. All animal studies were carried out in compliance with, and were approved by, the Institutional Animal Care and Use Committee.

BBM vesicle preparation and transport. Renal BBM vesicles were prepared from kidney cortex by the MgCl 2 preparation method as reported previously ( 35 ) and used for both transport studies and Western blot analysis. Uptake of 32 P i (100 µM) and [ 3 H]glucose (10 µM) was measured at 6 s in incubation medium containing either 100 mM NaCl or 100 mM KCl by rapid filtration as described ( 35 ). The Na -mediated component of transport was derived by subtracting uptake in KCl from that in NaCl.
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Crude membrane preparation. Mouse tissues were homogenized in 250 mM sucrose, 5 mM Tris·HCl, pH 7.4 using a Polytron homogenizer (Brinkman). After removal of debris by centrifugation at 1,000 g for 10 min, the supernatant was centrifuged at 50,000 g for 20 min to yield a pellet enriched in crude membranes. The pellet was resuspended in 50 mM mannitol, 20 mM HEPES/Tris, pH 7.5 and used immediately for Western blot analysis (40 µg protein/lane).% C2 R+ _2 q: f! B/ P. C
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Western blot analysis. Freshly prepared BBMs (10-50 µg protein, as required) were suspended in gel buffer ( 17 ), heated at 55°C for 3 min, fractionated by PAGE in the presence of 10% SDS, transferred to nitrocellulose membranes, and probed with rabbit polyclonal antibodies generated against Npt2a ( 35 ), Npt2c ( 25 ), actin (Sigma), and Npt1. The Npt1 polyclonal antibody was generated in rabbits against a mouse Npt1 COOH-terminal peptide (KGEIQDWAKEIKTTRL) ( 8 ) with a cysteine residue added to the NH 2 terminal for conjugation to keyhole limpet haemocyanin. Immune complexes on Western blots were visualized by chemiluminescence using an ECL kit for Npt2a and actin and an ECL Plus kit for Npt1 and Npt2c (Amersham Biosciences, Montreal, Quebec). The abundance of each Na-P i cotransporter, relative to actin, was quantified using Fuji-Scan software as described ( 14 ).
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Validation of Npt1 antibody. HEK (293) cells (CRL-1753, ATCC, Manassas, VA) were transfected with a full-length mouse Npt1 cDNA, subcloned in pCDNA3, and with empty vector, using lipofectamine (Invitrogen, Burlington, Ontario), as described previously ( 28 ). Immunoblots revealed that the antibody detected a 55-kDa protein in cells transfected with the Npt1 cDNA but not in cells transfected with the empty vector ( Fig. 1 A ) and in crude membrane preparations from mouse kidney but not other tissues ( Fig. 1 B ).) c  N, C( X+ w: F) d0 J9 y8 s4 B
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Fig. 1. Validation of Na-P i cotransporter type I (Npt1) antibody. A : expression of Npt1 protein in transfected HEK(293) cells. HEK(293) cells were transiently transfected with full-length Npt1 cDNA subcloned in pCDNA3 or with vector alone. Whole cell lysates, prepared from 10 x 10 6 mock-transfected cells (Mock; lane 1 ) and from Npt1 cDNA-transfected cells (4 x 10 6 cells, lane 2; 6 x 10 6 cells, lane 3; and 10 x 10 6 cells, lane 4 ), were electrophoresed on 10% SDS-PAGE and analyzed by immunoblotting with the polyclonal rabbit anti-Npt1 antibody as described in MATERIALS AND METHODS. B : crude membrane preparations (40 µg protein loaded/lane) from mouse kidney ( lane 1 ), brain ( lane 2 ), lung ( lane 3 ), heart ( lane 4 ), and liver ( lane 5 ) were electrophoresed on 10% SDS-PAGE and analyzed as described above. Lane 6 depicts Npt1 and actin protein expression in purified BBM vesicles (40 µg BBM protein) prepared from mouse kidney.1 ?* d0 D1 F3 X

Ribonuclease protection assay. Total RNA was extracted from kidney cortex by standard TRIzol extraction (Invitrogen) and the abundance of Npt2a, Npt2c, and Npt1 mRNAs, relative to -actin mRNA, was estimated by ribonuclease protection assay as described ( 14, 33 ). Riboprobes for Npt1 and Npt2a were synthesized from subcloned cDNA fragments corresponding to nucleotides 869-1299 and 335-686, respectively, of their corresponding cDNAs, as described ( 33 ). A riboprobe for Npt2c was generated from a subcloned Npt2c cDNA fragment, corresponding to nucleotides 1547-1892 of the mouse cDNA ( 23 ) (GenBank Accession no. AB054999  ); this region of the Npt2c cDNA bears little homology to Npt2a, thereby providing high specificity to the Npt2c ribonuclease protection assay. A subcloned Hind III-Kpn I -actin cDNA fragment was used to generate the corresponding riboprobe that served as an internal standard, as described previously ( 5, 33 ).3 V6 b/ s9 D" K- C. ]( x8 p2 K

RT/PCR. RT-PCR was also used to estimate the abundance of Npt2c mRNA, relative to -actin mRNA, in kidneys of Npt2  /  and Npt2 - / - mice. Total renal RNA was reverse transcribed and the primer sequences used to amplify Npt2c ( 23 ) and -actin ( 30 ) cDNAs were as described previously. Amplified products for each transcript were examined after 15, 20, 25, 30, and 35 PCR cycles.% _0 z% |2 x6 ~/ x. ^
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Statistical analysis. The number of samples examined per group is indicated for each experiment, and the means ± SE are depicted. Statistical analysis was performed using ANOVA and Student's t -test where appropriate. A probability of P 0.05 was considered to be significant.- N: ~+ _2 ?+ H7 D) c3 Y( r
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RESULTS

Effect of Npt2a gene ablation on renal BBM Na-P i cotransport and cotransporter gene expression. BBM Na-P i cotransport is significantly reduced in Npt2 - / - mice compared with Npt2  /  littermates ( Table 1 ), as reported previously ( 4 ). BBM Na-glucose transport is significantly increased and the P i /glucose transport ratio is decreased in Npt2 - / - mice compared with Npt2  /  littermates ( Table 1 ).
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Table 1. Effect of Npt2a gene ablation and Hyp mutation on Na-P i and Na-glucose cotransport in renal BBM vesicles( p9 q8 _3 S* `( Z

Consistent with Npt2 gene ablation, Npt2a-immunoreactive protein was not detectable in the renal BBM of Npt2 - / - mice ( Fig. 2 A ). However, the relative abundance of BBM Npt2c-immunoreactive protein was significantly increased in Npt2 - / - mice when compared with Npt2  /  littermates ( Fig. 2 A ). In contrast, BBM Npt1 protein abundance was not significantly different in WT and Npt2 knockout mice ( Fig. 2 A ). In all genotype comparisons, there was no effect of Npt2 gene ablation on the BBM abundance of actin ( Fig. 2 A ).

Fig. 2. Effect of Npt2a gene ablation ( A ) and X-linked Hyp mutation ( B ) on renal brush-border membrane (BBM) abundance of Npt2a-, Npt2c-, Npt1-, and actin-immunoreactive proteins. Renal BBM vesicles were prepared from Npt2  / , Npt2 - / -, wild-type (WT), and Hyp mice, subjected to 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Npt2a, Npt2c, Npt1, and actin as described in MATERIALS AND METHODS. Representative gels for each Na-P i cotransporter are shown. The histograms represent the abundance of Npt2a, Npt2c, and Npt1, relative to that of actin. Means ± SE derived from 11 BBM preparations per genotype are shown. *Effect of Npt2a gene ablation ( A ) or Hyp mutation ( B ), P

The effect of Npt2 gene ablation on renal Na-P i cotransporter mRNA expression is shown in Fig. 3 A. Npt2a mRNA was not detectable in kidneys of Npt2 - / - mice ( Fig. 3 A ). Unexpectedly, the abundance of Npt2c mRNA, relative to -actin mRNA, was reduced in Npt2 - / - mice when compared with WT littermates ( Fig. 3 A ) and these findings were confirmed by time course RT/PCR (data not shown). In addition, the relative renal abundance of Npt1 mRNA was reduced in Npt2 - / - mice, consistent with an earlier report ( 14 ).* B2 e+ Q6 D% n, `9 ~7 k8 c! J( g

Fig. 3. Effect of Npt2a gene ablation ( A ) and X-linked Hyp mutation ( B ) on renal abundance of Npt2a, Npt2c, Npt1, and -actin mRNAs. Total RNA was prepared from kidneys of Npt2  / , Npt2 - / -, WT, and Hyp mice and the abundance of Npt2a, Npt2c, and Npt1 mRNAs, relative to -actin mRNA, was estimated by ribonuclease protection assay as described in MATERIALS AND METHODS. Representative gels are shown for each Na-P i cotransporter transcript and -actin mRNA. Each histogram represents means ± SE derived from 5-10 mouse kidneys per group. *Effect of Npt2a gene ablation ( A ) or Hyp mutation ( B ), P6 {. X* p) E$ G( A

Effect of Hyp mutation on renal BBM Na-P i cotransport and cotransporter gene expression. The X-linked Hyp mutation elicits a significant decrease in BBM Na-P i cotransport, consistent with earlier reports ( 34 ), an increase BBM Na/glucose cotransport, and a decrease in the P i /glucose transport ratio ( Table 1 ).( G& D& L2 j  f  T

Western analysis of BBM proteins revealed that renal Npt2a protein abundance, relative to actin, is significantly decreased in Hyp mice ( Fig. 2 B ), as reported previously ( 35 ). In addition, the relative abundance of both Npt2c- and Npt1-immunoreactive proteins is significantly decreased in the BBM of Hyp mice compared with WT littermates ( Fig. 2 B ). Similarly, Hyp mice exhibit a significant decrease in the renal abundance of Npt2a, Npt2c, and Npt1 mRNAs, relative to -actin mRNA, compared with WT littermates ( Fig. 3 B ). Thus all three renal Na-P i cotransporters are downregulated by loss of Phex function.$ Z2 M0 {* J& c7 G" ^1 W- b0 K" a
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Effect of Pi deprivation on serum P i and Npt2c protein abundance in renal BBMs of Npt2 -/- and Hyp mice and their WT littermates. Table 2 summarizes the effects of dietary P i intake on serum P i concentration in WT and mutant mice. The 0.02% P i diet elicits a significant drop in serum Pi in all four genotypes ( Table 2 ). Moreover, with the exception of Hyp mice, serum P i is significantly lower on the 0.6% Pi diet compared with values on the 1% P i diet ( Table 2 ). Finally, serum P i is significantly lower in Npt2 - / - mice than in Npt2  /  littermates on the 1 and 0.6% P i diets and significantly lower in Hyp mice than in WT littermates on all three diets ( Table 2 ).

Table 2. Effect of dietary P i intake on serum P i concentration in Npt2  /  and wild-type mice and their Npt2 - / - and Hyp littermates

The effect of dietary P i intake on BBM Npt2c- and Npt2a-immunoreactive protein in Npt2  /  and Npt2 - / - mice was examined. In Npt2  /  mice, the renal BBM abundance of Npt2c-immunoreactive protein, relative to actin, is significantly increased as dietary P i intake is decreased ( Fig. 4 A ), with values 1.5- and 2.5-fold greater in mice fed the low-P i diet (0.02%) compared with that in Npt2  /  mice fed the 0.6 and 1% P i diets, respectively ( Fig. 4 A ). Similarly, BBM abundance of Npt2a protein is increased in Npt2  /  with the reduction in dietary P i intake ( Fig. 4 A ), as described previously ( 14 ). In Npt2 - / - mice, the low-P i diet had no effect on BBM Npt2c protein abundance ( Fig. 4 A ), with similar values observed on all three test diets ( Fig. 4 A ). Moreover, the magnitude of the difference in Npt2c protein abundance in Npt2 - / - mice, relative to Npt2  /  mice, decreased with the reduction in dietary P i intake ( Fig. 4 A ). These data demonstrate that Npt2c protein is maximally upregulated in Npt2 - / - mice fed the 0.6 and 1% P i diets ( Fig. 4 A ). Furthermore, our findings suggest that Npt2c mediates residual BBM Na-Pi cotransport in Npt2 - / - mice. As reported previously ( 4, 14 ), Npt2a protein expression was not detected in Npt2 - / - mice (data not shown).

Fig. 4. Effect of dietary P i on Npt2c-, Npt2a-, and actin-immunoreactive protein abundance in the renal BBM of Npt2  /  and Npt2 - / - mice ( A ) and WT and Hyp mice ( B ). Mice were fed diets containing 0.02, 0.6, and 1.0% P i for 4 days. Renal BBM vesicles were prepared, subjected to 10% SDS-PAGE, and analyzed by immunoblotting with antibodies against Npt2c, Npt2a, and actin as described in MATERIALS AND METHODS. Representative gels of renal BBMs from Npt2  /  and Npt2 - / - mice ( A, top ) and WT and Hyp mice ( B, top ) are shown. The histograms represent the abundance of Npt2c, relative to that of actin, in BBMs from Npt2  /  and Npt2 - / - mice ( A, bottom ) and WT and Hyp mice ( B, bottom ). Means ± SE derived from at least 3 BBM preparations per genotype are shown. 100% Refers to the abundance of BBM Npt2c, relative to actin, in Npt2  /  mice ( A ) and WT mice ( B ) fed the 0.6% diet. *Effect of Npt2a gene ablation ( A ) or Hyp mutation ( B ), P P  n( ]5 r- I  M* b
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Both WT and Hyp mice responded to the decrease in dietary P i intake with an increase in the renal BBM abundance of Npt2c- and Npt2a-immunoreactive proteins ( Fig. 4 B ), with the observed changes in Npt2a protein in WT and Hyp mice consistent with earlier findings ( 31 ). Under all three dietary conditions, the BBM abundance of Npt2c protein, relative to actin, is significantly reduced in Hyp mice compared with WT littermates ( Fig. 4 B ).# N/ m5 b; e4 e. N3 W( c' J9 E

DISCUSSION; j) t( h7 \7 \5 e* r
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A novel type II Na-P i cotransporter, Npt2c, with homology to Npt2a, the major Na-Pi cotransporter in mammalian kidney, was recently identified in rat, human, and mouse kidney ( 23, 25 ). Npt2c colocalizes with Npt2a in the BBM of proximal tubular ( 25 ), suggesting that it plays an important role in the maintenance of P i homeostasis. In the present study, we examined renal Npt2c expression in two mutant mouse models that exhibit hypophosphatemia secondary to impaired renal BBM Na-P i cotransport. We report that Npt2c protein is significantly upregulated in mice homozygous for the disrupted Npt2a gene and downregulated in X-linked Hyp mice, which harbor a large deletion in the Phex gene. We also demonstrate that the Npt2c-adaptive response to P i restriction is markedly different in both mutant mouse strains. Our data suggest that Npt2c protein is maximally upregulated in Npt2 - / - mice and accounts for residual BBM Na-P i cotransport in this mutant model. Our data also indicate that hypophosphatemia is not sufficient for the adaptive Npt2c response to P i restriction and that normal Phex function is essential the regulation of Npt2c expression in mouse kidney.

We demonstrate that the increase in renal BBM Npt2c protein abundance in Npt2 - / - mice occurs in the absence of a corresponding increase in Npt2c mRNA. These findings, which were confirmed by two independent methods, suggest that the adaptive increase in Npt2c protein in Npt2 - / - mice cannot be explained by alterations in Npt2c gene transcription or mRNA stability. Rather, alternate mechanisms such as increased translation of Npt2c mRNA, enhanced translocation of presynthesized Npt2c protein from a subapical compartment to the BBM, or decreased Npt2c protein turnover may play a role in the observed response in Npt2 - / - mice. In contrast, previous studies reported that both renal Npt2c mRNA and protein abundance are increased in rats ( 25 ) and mice ( 23 ) fed a low-P i diet. A possible explanation for this discrepancy is that Npt2 - / - mice are fed a P i -sufficient diet, i.e., their dietary supply of P i is not limiting. Indeed, we demonstrated that serum P i in Npt2 - / - mice fed a P i -sufficient diet is significantly higher than that in P i -restricted WT mice ( Table 2 and Ref. 14 ). We thus speculate that either the set point necessary to turn on renal Npt2c mRNA production was not achieved in Npt2 - / - mice receiving a P i -sufficient diet or that different mechanisms account for the increase in Npt2c protein in Npt2 - / - mice and the P i -deprived animal models ( 23, 25 ).
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With the use of a novel antibody raised against a murine COOH-terminal Npt1 peptide, we show unequivocally that Npt1 protein is not upregulated in renal BBMs of Npt2 - / - mice. Although the precise role of Npt1 in BBM Na-P i cotransport is not yet clear ( 7 ), it is expressed exclusively in the BBM of proximal tubular cells ( 6 ). Moreover, Npt1 interacts with the Napi-Cap1, NaPi-Cap2, and NHERF-1, the same PDZ binding proteins that associate and colocalize with Npt2a in microvilli or the subapical compartment ( 11 ). Although these interactions of Npt1 are likely responsible for its BBM localization, future work is necessary to determine the contribution of Npt1 to renal P i reabsorption.
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We report that hypophosphatemia and renal P i wasting in Hyp mice are associated with the downregulation of all three renal Na-P i cotransporters, Npt2a, Npt2c, and Npt1, at both the mRNA and protein levels. These findings are in sharp contrast to those in Npt2 - / - mice and may be explained by loss of Phex function in Hyp mice, leading to the accumulation of a circulating phosphaturic factor(s) that is(are) normally degraded by Phex-mediated endopeptidase activity ( 29 ). We found that Phex mRNA expression in calvaria, tibia, and incisor is identical in Npt2  /  and Npt2 - / - mice (data not shown). These results suggest that Phex function is normal in Npt2 - / - mice and are consistent with the well-documented differences in skeletal manifestations ( 4, 12, 20 ) and the regulation of renal 1,25-dihydroxyvitamin D synthesis by P i ( 2, 32 ) in Npt2 - / - mice ( 4, 12, 32 ) and Hyp mice ( 2, 20 ). Furthermore, the present study clearly demonstrates significant differences in renal Na-P i cotransporter expression in both mutant mouse strains./ Z2 v% Y9 f" t/ ~7 a; u: g
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A likely candidate for the circulating phosphaturic factor in Hyp mice is FGF-23, a novel secreted peptide that is elevated in the serum of most patients with the homologous disorder XLH as well as in the serum of patients with the phenotypically similar acquired disorder oncogenic hypophosphatemic osteomalacia ( 15, 40 ). Moreover, recent studies demonstrated that Hyp mice 10-fold higher serum FGF-23 levels than WT littermates ( 41 ). In addition, missense mutations in the FGF23 gene, which replace Arg residues in the protein's consensus furin cleavage site and prevent its proteolytic processing, are responsible for autosomal dominant hypophosphatemic rickets, a disorder with phenotypic features similar to those in XLH and oncogenic hypophosphatemic osteomalacia ( 1 ).
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Relevant to the increased serum FGF-23 concentrations in the human hypophosphatemic disorders and in Hyp mice is the finding that the administration of FGF-23 to normal mice elicits hypophosphatemia associated with renal P i wasting ( 27 ), a reduction in renal BBM Na-P i cotransport ( 24 ), and decreased renal Npt2a expression ( 3 ). In addition, rats receiving intrahepatic injection of FGF-23 cDNA developed hypophosphatemia and a significant decrease in both renal BBM Na-P i cotransport and Npt2c protein abundance ( 26 ). Further work is necessary to establish whether FGF-23 also downregulates renal Npt1 expression and whether phosphaturic factors other than FGF-23 contribute to renal P i wasting in Hyp mice.: c% u/ I. f1 x; a& }5 [
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We also report that the relationship between renal BBM Npt2c protein abundance and dietary P i intake is different in both mutant hypophosphatemic mouse strains. In Npt2 - / - mice, Npt2c protein abundance, which is significantly higher than that in Npt2  /  mice, is not further increased by P i restriction. These findings are consistent with maximum upregulation of Npt2c protein in Npt2 - / - mice fed P i -replete diets (1 and 0.6%) and previous results demonstrating that Npt2 - / - mice fail to respond to dietary P i restriction with an adaptive increase in BBM Na-P i cotransport ( 14 ). In WT littermates, however, BBM Npt2c protein is significantly increased with P i deprivation, in agreement with previous studies that showed that this increase is associated with an increase in BBM Na-P i cotransport ( 23, 25 ). In contrast in Hyp mice, the adaptive increase in Npt2c protein is only apparent on the low-P i diet and the abundance of Npt2c protein is significantly lower than that in WT littermates under all three dietary conditions, which is clearly not the case in Npt2 - / - mice. Taken together, our data suggest that Npt2c protein is already maximally upregulated in Npt2 - / - mice fed the P i -replete diets and that loss of Phex function interferes with Npt2c protein adaptation in response to hypophosphatemia and changes in dietary P i.! p. t5 H0 m/ ^4 R

In the present study, we demonstrate that renal BBM Na/glucose cotransport is significantly higher in both Npt2 - / - and Hyp mice compared with their normal counterparts. However, significant genotype differences in renal BBM Na/glucose cotransport were not evident in previous reports of both mutant mouse strains ( 14, 35 ). A possible explanation for this discrepancy is the difference in dietary P i content in both studies. In the present study, the mice were maintained on a 0.6% P i diet, whereas, in our earlier work, the mice were raised on a 1% P i diet. We suggest that BBM Na/glucose cotransport increased in the hypophosphatemic mutants in response to the reduction in P i supply. Consistent with this hypothesis is the demonstration that glucose-6-phosphatase, an enzyme that increases glucose production and glycemia and is abundantly expressed in kidney, is also upregulated in the liver of Hyp mice ( 38 ) and of P i -deprived rats ( 37 ).. T9 P! m- ~/ w" R9 p  H
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In summary, we provide evidence for differential regulation of renal Npt2c gene expression in Npt2 - / - and Hyp mice. Our data indicate that hypophosphatemia per se is not sufficient for upregulation of Npt2c gene expression and that normal Phex function is required for Npt2c regulation in mouse kidney. Although our data suggest that Npt2c is responsible for residual renal Na-P i cotransport in Npt2 - / - mice, additional studies are necessary to determine the precise contribution of Npt2c to overall renal P i reabsorption." ~" K: S' t% R; B. k8 X" w* ?% B  E

DISCLOSURES

This work was supported by grants from the Canadian Institutes of Health Research (FRN 44345 to H. S. Tenenhouse) and the Ministry of Education, Science, Sports and Culture of Japan (Grant 11557202 to K. Miyamoto).

ACKNOWLEDGMENTS

We thank Y. Soumounou for technical support and S. Aubin for maintaining the mutant mouse colonies.* F, R, h/ G+ |- k, E
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