INTRODUCTION

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INORGANIC PHOSPHATE (P_i) is essential for a variety of cellular processes and, to accommodate these needs, transport systems have evolved to permit the efficient translocation of P_i anion across the cell membrane. The mammalian kidney is a major arbiter of P_i homeostasis by virtue of its ability to increase or decrease P_i reabsorptive capacity, and thus considerable effort has been devoted to the characterization of renal P_i transporters (4, 30, 32). Recently, expression and homology cloning led to the molecular identification of two distinct classes of renal high-affinity, Na⁺-coupled P_i transporters, NPT1 (type I) (7, 8, 51) and NPT2 (type II) (9, 17, 18, 28, 38, 49), in several mammalian species. Both NPT1 and NPT2 are expressed in the brush-border membrane (BBM) of proximal tubular cells (5, 11, 12), where the bulk of filtered P_i is reabsorbed, and serve to translocate P_i from the lumen into the cell against an electrochemical gradient. NPT1 is also expressed in liver, but the level of expression is significantly lower than that in kidney (26, 51). In contrast, NPT2 expression appears to be exclusive to the kidney (28).

Dietary P_i and parathyroid hormone (PTH) are major regulators of renal P_i handling. The increase in P_i reabsorption in response to chronic P_i deprivation can be attributed to an increase in BBM Na⁺-dependent P_i transport that is associated with a corresponding increase in the renal abundance of NPT2 (24, 25, 50), but not NPT1 (49), mRNA and immunoreactive protein. The response to acute P_i restriction, however, does not require de novo RNA and protein synthesis and is mediated by microtubule-dependent translocation of presynthesized NPT2 protein to the BBM (27). Similarly, the decrease in renal P_i reabsorption following PTH administration is not associated with changes in NPT2 mRNA and depends upon the internalization of cell surface NPT2 protein (23).

Recent studies have demonstrated that renal expression of Npt2¹ is also subject to regulation by Pex, a P_i-regulating gene with homology to endopeptidases on the X chromosome (48). Large deletions in the 5' region of the Pex gene in Gy mice (39) and in the 3' region of the Pex gene in Hyp mice (3) are associated with a significant decrease in the renal abundance of Npt2 mRNA and immunoreactive protein and a corresponding reduction in BBM Na⁺-P_i cotransport (2, 47). In addition, we reported that the deficit in renal Npt2 mRNA in Hyp mice (36) is not corrected by the administration of growth hormone (GH) which, in normal rats, stimulates renal P_i conservation and Na⁺-P_i cotransport in renal BBM vesicles (6, 16, 43). To date, similar studies on the effects of the Hyp and Gy mutations and of GH on renal expression of Npt1 have not been performed.

A recent study demonstrated that cell surface receptors for gibbon ape leukemia virus (Glvr-1) and murine amphotropic virus (Ram-1) have multiple membrane-spanning domains and share ~25% identity with a putative P_i permease of Neurospora crassa (21, 22). When expressed in Xenopus oocytes, both Glvr-1 and Ram-1 mediate high-affinity Na⁺-dependent P_i transport (22). Moreover, in cultured cells Glvr-1 and Ram-1 mRNAs are upregulated by extracellular P_i depletion (22). Glvr-1 and Ram-1 share ~60% sequence identity, have no sequence similarity to NPT1 or NPT2 (22), and have been designated type III Na⁺-P_i cotransporters. Both Glvr-1 and Ram-1 appear to be ubiquitously expressed, with Glvr-1 expression highest in bone marrow and Ram-1 expression highest in the heart (22). To date little is known about the renal abundance of Glvr-1 and Ram-1, relative to Npt1 and Npt2. In addition, there are no data on the distribution, membrane localization, and regulation of Glvr-1 and Ram-1 gene expression in mammalian kidney.

The aim of the present study was to determine the tissue distribution of Npt1, Npt2, Glvr-1, and Ram-1 mRNAs, to quantitate their relative expression in the mouse kidney, to localize Npt2 and Glvr-1 mRNA in the kidney by in situ hybridization, and to examine the effects of low-P_i diet, the Hyp mutation, and GH on renal Npt1, Npt2, Glvr-1, and Ram-1 mRNA expression.

	MATERIALS AND METHODS

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Mice. C57Bl/6J normal (+/Y, +/+) and mutant (Hyp/Y, Hyp/+) mice raised in our laboratory were used between 4 and 8 mo of age and were age matched (±1 mo) within each experiment. The initial breeding pairs (+/Y males and Hyp/+ females) were obtained from the Jackson Laboratory (Bar Harbor, ME). The mice were maintained on a standard diet (Teklad Rodent Diet; Harlan Teklad, Madison, WI) containing 1.46% calcium, 0.99% phosphorus, and 4.96 IU vitamin D₃/g. Normal and Hyp mice were fed diets containing 0.03% (low) and 1% (control) P_i (test diets 86128 and 86129; Teklad) for 8 days. Alternatively, mice fed the control diet were injected twice per day, for 4 days, with recombinant GH (2 µg/g body wt sc; a gift of Eli Lilly, Indianapolis, IN) or vehicle (0.05% BSA in PBS) and killed 2 h after the last injection. All animal studies were conducted in accord with the principles and procedures of the Canadian Council on Animal Care.

Isolation of total RNA. Kidney, liver, bone (femur), and intestine (duodenum) were removed immediately after death, frozen in liquid nitrogen, and stored at 85°C until ready to be processed. The capsule and papilla were removed from kidneys prior to freezing. The tissues were ground into a fine powder with a mortar and pestle on dry ice, and total RNA was extracted using the TRIzol reagent (GIBCO-BRL; Life Technologies, Burlington, Ontario, Canada).

Preparation of riboprobes. Riboprobes for mouse Npt1, Npt2, Glvr-1, and Ram-1 were generated by subcloning cDNA fragments for the respective Na⁺-P_i cotransporter into pBluescript II KS (Stratagene, La Jolla, CA). For the size and position of the subcloned cDNA fragments, see Table 1. As an internal standard, we used a Hind III-Kpn I -actin cDNA fragment, subcloned in pGEM3 (Promega, Madison, WI) as described previously (3). The plasmids were linearized, and ³²P-labeled antisense riboprobes were synthesized with either T3 or T7 RNA polymerases, depending on the orientation of the subcloned cDNA fragment, and [-³²P]UTP (800 Ci/mmol; ICN, Mississauga, Ontario). Digoxigenin-labeled sense and antisense Npt2 and Glvr-1 riboprobes were prepared according to the directions of the digoxigenin RNA labeling kit (Boehringer Mannheim Biochemica, Indianapolis, IN), as described previously (37).

Ribonuclease protection analysis. The ribonuclease protection assay was performed as described previously (3) using the method of Gilman (14). Total cellular RNA (5-20 µg) was hybridized with the appropriate labeled riboprobes (5 × 10⁵ cpm) at 50°C for 18 h and treated with 2 µg/ml RNase T1 for 1 h at 30°C. The protected fragments were precipitated, heat denatured, and electrophoresed on 6% denaturing polyacrylamide gels. The gels were dried and exposed to a PhosphorImager screen for quantitation of radioactive signals under conditions where linearity is achieved and to Kodak Biomax MR1 film for photography.

In situ hybridization. Mice were anesthetized with Nembutal (5 mg/kg ip) and perfused with fixative (10% Formalin in Tris-buffered saline, pH 7.4) as described previously (37). The kidneys were harvested, cut, and embedded in paraffin, and 5-µm sections were prepared and fixed on Superfrost glass slides for in situ hybridization (20, 37). After dewaxing and rehydration, the tissue sections were deproteinated with proteinase K for 30-60 min at 37°C, postfixed in 4% paraformaldehyde for 5 min, and acetylated with 0.25% acetic acid in 0.1 M triethanolamine for 10 min. The sections were prehybridized in the hybridization solution (50% formamide, 2× SSC, 1× Denhardt's, 10% dextran sulfate, 0.5% sodium pyrophosphate, pH 7.2, 1 mM EDTA, 100 mM dithiothreitol, 0.5% SDS, 200 µg/ml transfer RNA, and 250 µg/ml denatured salmon sperm DNA) at 42°C for Npt2 and 48°C for Glvr-1, for 1 h in a humidified chamber. The slides were drained and hybridized with digoxigenin-labeled, heat-denatured sense or antisense riboprobes overnight in a humidified chamber. The slides were washed with 2× SSC for 5 min and with STE (500 mM NaCl, 1 mM EDTA, and 20 mM Tris · HCl, pH 7.5) for 1 min at room temperature. Hybridization and washing was performed at 42°C for Npt2 and at 48°C for Glvr-1. The slides were treated with RNase A (10 µg/ml) for 30 min at 37°C and washed in 2× SSC in 50% formamide and 1× SSC for 10 min each. This was followed by washes, at room temperature, in 0.1× SSC for 10 min and PBS for 5 min. The slides were then placed in buffer 1 (100 mM maleic acid, 100 mM NaCl, and 0.3% Triton X-100, pH 7.5) for 2 min and incubated in buffer 1 with 10% normal horse serum (NHS) for 1 h at room temperature. After blocking, the slides were incubated with anti-digoxigenin alkaline phosphatase-conjugated antibody, diluted 1:500 in buffer 1 with 10% NHS, in a humidified chamber at 4°C overnight. After rinsing for 5 min in buffer 1 and 5 min in buffer 3 (100 mM Tris, 100 mM NaCl, and 50 mM MgCl₂, pH 9.5), colorimetric detection of RNA:RNA hybrids was performed with nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl P_i for 24 h. The sections were mounted with Aquaperm (Lipshaw Immunon; Fisher), examined with a Leitz Aristoplan microscope, and photographed.

Analytical procedures. A commercial kit was used for the determination of the serum concentration of inorganic P_i (Stanbio Laboratories, San Antonio, TX), as described previously (36). The concentration of human GH was determined by radioimmunoassay (15).

Statistical analysis. Data are expressed as means ± SE of 4-8 animals per group. Statistical significance was determined by one-factor analysis of variance and Scheffé's F-test or Student's t-test, as appropriate. A probability of P < 0.05 was taken to be statistically significant.

	RESULTS

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Expression and relative abundance of Npt1, Npt2, Ram-1, and Glvr-1 mRNAs in mouse tissues. Using ribonuclease protection assay, we demonstrate that Npt2 is the predominant Na⁺-P_i cotransporter mRNA expressed in mouse kidney (Fig. 1A). We also show that protected fragments for Npt1, Glvr-1, and Ram-1 are detected in mouse kidney. However, for the latter two transcripts which are of low abundance, it was necessary to use a larger quantity of RNA in the hybridization reactions (Fig. 1A). Based on analysis of eight different renal RNA samples, we estimated that Npt1, Npt2, Glvr-1, and Ram-1 accounted for 15 ± 1.0, 84 ± 1.0, 0.5 ± 0.2, and 0.5 ± 0.3% of total Na⁺-P_i cotransporter mRNAs in the kidney, respectively (Fig. 2).

View larger version (54K): [in this window] [in a new window]   View larger version (33K): [in this window] [in a new window]   View larger version (31K): [in this window] [in a new window]   Fig. 1.   Ribonuclease protection assay of RNA from mouse kidney (A), liver and intestine (B), and bone and heart (C). Total RNA (two aliquots/tissue, 5-20 µg, as indicated) was hybridized with 32P-labeled Npt1, Npt2, Glvr-1, Ram-1, and -actin riboprobes and treated with RNase T1 as described in MATERIALS AND METHODS. The protected fragments were precipitated, electrophoresed on polyacrylamide gels, and visualized by autoradiography.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Relative abundance of Na+-Pi cotransporter mRNAs in normal mouse kidney. Total RNA (5 to 20 µg) was hybridized with 32P-labeled Npt1, Npt2, Glvr-1, Ram-1, and -actin riboprobes and treated with RNase T1 as described in MATERIALS AND METHODS. The protected fragments were precipitated and electrophoresed on polyacrylamide gels. The gels were dried and exposed to a PhosphorImager screen for quantitation of radioactive signals. Npt1, Npt2, Glvr-1, and Ram-1 phosphorimage signals were related to those of -actin under conditions in which we previously demonstrated a linear relationship between the quantity of RNA used and the resulting phosphorimage signal; 100% refers to the sum of Npt1/, Npt2/, Glvr-1/, and Ram-1/-actin ratios. Data represent the means ± SD derived from 8 different mouse renal RNA preparations.

Npt1 mRNA expression was also evident in mouse liver (Fig. 1B), albeit at a much lower level than that in kidney. A direct comparison of Npt1 mRNA in both tissues indicated that its abundance in kidney is ~30-fold higher than that in the liver (data not shown). We could find no evidence for Npt1 mRNA expression in intestine (Fig. 1B), bone, or heart (Fig. 1C).

Although Npt2 mRNA expression was not detectable in liver (Fig. 1B), bone, or heart (Fig. 1C), a protected Npt2 fragment was apparent in the intestine (Fig. 1B). Of interest was the finding that Npt2 mRNA was not expressed in primary renal epithelial cell cultures derived from either mouse kidney cortex or purified mouse proximal tubules (data not shown).² In addition, we could find no evidence for Npt2 mRNA expression in immortalized S1, S2, or S3 proximal tubular cells derived from mice harboring the large SV40 T antigen (33, 52).²

Glvr-1 and Ram-1 mRNAs were expressed in all tissues examined (Fig. 1, A-C) as well as in renal proximal tubule cell cultures (data not shown). In agreement with previous findings in the bone marrow (22), Glvr-1 mRNA was more prominent than Ram-1 mRNA in mouse bone (marrow was not flushed out of bone used in the present study) (Fig. 1C). In addition, in the heart, Ram-1 transcripts were more abundant than Glvr-1 transcripts (Fig. 1C) as reported previously (22). These findings validate the specificity of the Glvr-1 and Ram-1 riboprobes used in the present study.

In situ hybridization of Npt2 and Glvr-1 in mouse kidney. The digoxigenin-labeled antisense and sense Npt2 and Glvr-1 riboprobes were first tested on slot blots. A signal was detected when the Npt2 antisense probe was hybridized with renal RNA, but not with liver or bone RNA. Conversely, the Glvr-1 antisense probe hybridized with RNA from kidney, liver, and heart. Hybridization signals were not detected with either the Npt2 or Glvr-1 sense probes.

Figure 3 depicts the in situ hybridization of Npt2 and Glvr-1 antisense and sense digoxigenin-labeled riboprobes to renal sections derived from normal mouse kidney. Npt2 transcripts were detected in proximal tubular cells (Fig. 3A) of the outer cortex with the Npt2 antisense riboprobe, whereas no signal was obtained with the Npt2 sense riboprobe under identical conditions (Fig. 3B). In contrast, Glvr-1 mRNA was observed throughout the kidney as determined by hybridization with the Glvr-1 antisense riboprobe (Fig. 3C); no signal was apparent with the Glvr-1 sense riboprobe (Fig. 3D).

View larger version (174K): [in this window] [in a new window]   Fig. 3.   Localization of Npt2 and Glvr-1 transcripts in mouse kidney by in situ hybridization. Renal sections (5 µm) were prepared from normal mice and hybridized with digoxigenin-labeled Npt2 antisense (A) and sense (B) and Glvr-2 antisense (C) and sense (D) riboprobes as described in MATERIALS AND METHODS. Magnification was ×120 for all sections (bar = 8 µm).

Effect of low-P_i diet, GH, and the Hyp mutation on renal Npt1, Npt2, Glvr-1, and Ram-1 mRNA expression. As reported previously (36, 42), serum P_i was significantly lower in Hyp mice than in normal littermates and was decreased by the low-P_i diet in both genotypes (Table 2). Administration of human GH led to an increase in the serum P_i concentration in normal but not in Hyp mice (Table 2). The serum concentration of human GH at death was 629 ± 50 ng/ml (n = 14) in GH-treated mice but was undetectable in vehicle-treated controls.

View larger version (25K): [in this window] [in a new window]   View larger version (24K): [in this window] [in a new window]   View larger version (25K): [in this window] [in a new window]   View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effect of low-Pi diet, growth hormone (GH), and the Hyp mutation on renal Npt1 (A), Npt2 (B), Glvr-1 (C), and Ram-1 (D) mRNA abundance. Normal and Hyp mice were fed control and low-Pi diets. Mice fed the control diet were also treated with vehicle or GH (2 µg/g sc) for 4 days as described in MATERIALS AND METHODS. Total renal RNA was prepared, and abundance of Npt1 (A), Npt2 (B), Glvr-1 (C), and Ram-1 (D) mRNAs, relative to -actin mRNA, was determined by ribonuclease protection assay as described in MATERIALS AND METHODS. Quantitation of the mRNA signals was performed by phosphorimage analysis as described in the legend to Fig. 2; 100% refers to the Npt1/, Npt2/, Glvr-1/, and Ram-1/-actin mRNA ratios in normal, vehicle-treated mice fed the control diet. Data are means ± SE of 4-8 animals per group. C, control. * Effect of GH, P < 0.05. # Effect of genotype, P < 0.05.

	DISCUSSION

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Physiological and molecular studies have provided evidence for heterogeneity of Na⁺-P_i cotransporter systems in mammalian kidney (31, 40). As a first step to define their contribution to the overall P_i reabsorptive process, we measured the relative abundance of Npt1, Npt2, Glvr-1, and Ram-1 mRNAs in mouse kidney. We demonstrate that Npt2 is the predominant renal Na⁺-P_i cotransporter and accounts for 84% of total cotransporter transcript in mouse kidney. Using in situ hybridization, we show that Npt2 mRNA is expressed exclusively in the proximal tubule, the nephron segment where the bulk of filtered P_i is reabsorbed. In addition, we demonstrate that Glvr-1 mRNA is expressed throughout the kidney, consistent with its role as a housekeeping Na⁺-P_i cotransporter gene (22).

The predominance of Npt2 mRNA expression in mouse kidney and its localization to the proximal tubule is consistent with the notion that Npt2 plays a crucial role in renal P_i reabsorption. This conclusion is supported by our recent studies in mice deficient in the Npt2 gene, generated by targeted mutagenesis (1). Indeed, mice homozygous for the disrupted Npt2 allele have lost ~80% of Na⁺-dependent BBM P_i transport compared with wild-type mice (1). Our data are also consistent with functional studies of renal mRNA in Xenopus oocytes, following RNase H-mediated hybrid depletion of Npt2 mRNA (29, 47). In both reports, Na⁺-P_i cotransport in oocytes injected with Npt2-depleted renal mRNA was significantly decreased compared with that in the absence of hybrid depletion and was similar to that in water-injected oocytes (29, 47). In contrast, Na⁺-P_i cotransport was not reduced in oocytes injected with Npt1- or Glvr-1-depleted renal mRNA (29). Although the oocyte data suggest that Npt2 is the only functional Na⁺-P_i cotransporter in mammalian kidney, the sensitivity and signal-to-noise ratio of the oocyte assay may not permit the detection of Npt1- and Glvr-1-mediated transport.

Although Npt2 is expressed exclusively in the proximal tubule in vivo, it is of interest that efforts to detect Npt2 mRNA expression in primary renal cell cultures derived from proximal tubules of normal adult mice or mice harboring the SV40 large T antigen (33, 52) have failed (data not shown, see Footnote 2). We did, however, find evidence for Npt1, Glvr-1, and Ram-1 mRNA expression in the primary renal cell cultures, a pattern that was similar to that recently described in mouse distal convoluted tubule cells (41). However, based on in situ hybridization in the present study, RT-PCR of isolated proximal tubules (11), and immunohistochemistry (11, 44), we would not expect to find Npt2 in the distal tubule cell cultures. The mechanism for the loss of Npt2 expression in renal proximal tubule cell cultures and in renal cell lines (19) is not understood, and studies dedicated to examining the regulation of Npt2 gene expression in vitro are restricted to opossum kidney (OK) cells, which continue to express a type II Na⁺-P_i cotransporter (NaPi-4) (19, 38).

In the present study, we also examined the effects of low-P_i diet, GH, and the Hyp mutation on the regulation of renal Npt1, Npt2, Glvr-1, and Ram-1 mRNA expression in mouse kidney. We show that P_i deprivation, which elicits an adaptive increase in BBM Na⁺-P_i cotransport (45), is not associated with an increase in the renal abundance of Npt1, Npt2, Glvr-1, or Ram-1 mRNA. However, GH, which also stimulates BBM Na⁺-P_i cotransport (16), does elicit a significant increase in renal expression of Glvr-1 mRNA in normal mice but has no effect on Npt1, Npt2, or Ram-1 mRNA expression. The murine Hyp mutation, which is characterized by renal P_i wasting (13), attributable to a specific defect in BBM Na⁺-P_i cotransport (46), is associated with a significant reduction in Npt1 (22%) and Npt2 (42%) mRNAs and an increase in Glvr-1 (45%) transcripts, with no change in Ram-1 mRNA abundance. Our data suggest that changes in Na⁺-P_i cotransporter mRNA abundance contribute to the effects of GH and the Hyp mutation on renal P_i handling. In contrast, the adaptive response to P_i restriction does not appear to be mediated by changes in renal Na⁺-P_i cotransporter mRNA abundance under the conditions studied.

The finding that Npt2 mRNA expression is decreased in mutant Hyp mice relative to normal littermates confirms that of a previous report (47). However, we also demonstrate for the first time that Npt1 mRNA expression is downregulated in the mutant strain. Although preliminary data by Chong et al. (7) failed to detect a difference in renal Npt1 mRNA abundance in normal and Hyp mice, the authors suggest that a sufficient number of normal and Hyp mice were not examined in their study. The precise mechanism for downregulation of renal Npt1 and Npt2 gene expression in Hyp mice is not clear. Nevertheless, it is likely that both events are coupled to the loss of Pex function in the mutant strain (3).

Our data show that chronic P_i deprivation fails to elicit a significant increase in the renal expression of Npt1, Npt2, Glvr-1, and Ram-1 mRNAs. It is of interest that previous studies have demonstrated that the increase in BBM Na⁺-P_i cotransport in response to chronic P_i restriction is associated with a corresponding increase in Npt2 mRNA and immunoreactive protein (24, 25, 50). However, it is apparent from more recent studies that an increase in Npt2 mRNA is not always observed (19, 35, 36), suggesting that regulation at the mRNA level may not be the only mechanism contributing to the adaptive response. Indeed, recent studies on the functional analysis of the Npt2 promoter showed that reporter gene activity in cells transfected with Npt2 promoter-reporter gene constructs was not responsive to changes in the extracellular P_i concentration (19). Moreover, we demonstrated that mice heterozygous for the disrupted Npt2 allele exhibit normal BBM Na⁺-P_i cotransport activity and normal Npt2 protein abundance, in the face of a 50% reduction in Npt2 mRNA, suggesting that the adaptive response to the loss of one copy of the Npt2 gene occurs at the Npt2 protein and not mRNA level (1).

In the present study, we detected an Npt2-protected fragment with intestinal RNA, whereas earlier studies failed to detect an Npt2 transcript by Northern analysis of intestinal RNA (28). This discrepancy may be explained by the increased sensitivity of the ribonuclease protection assay used in our study. Taken together, the data suggest that the intestine expresses a type II Na⁺-P_i cotransporter that is homologous to Npt2. The latter would likely play an important role in 1,25-dihydroxyvitamin D-regulated mucosal-to-serosal P_i transport since it is well established that increased 1,25-dihydroxyvitamin D levels secondary to P_i deprivation (42) are associated with increased intestinal P_i absorption (10). Although an intestinal Na⁺-P_i cotransporter has not yet been cloned, a novel protein that stimulates P_i transport in Xenopus oocytes was identified in rabbit intestine (34).

We demonstrate that Glvr-1 mRNA expression is significantly stimulated by the Hyp mutation and by GH administration to normal mice. Given that Glvr-1 mRNA comprises such a small proportion of total renal Na⁺-P_i cotransporter mRNAs (<1%), the physiological relevance of these findings is not clear. However, it is necessary to realize that the relative abundance of Npt1, Npt2, Glvr-1, and Ram-1 mRNAs in mouse kidney may not be an accurate reflection of the relative abundance of their corresponding proteins. Moreover, the relative efficiency with which each of these transporters mediates the translocation of P_i across membrane may differ significantly.

In summary, we demonstrate that Npt2 mRNA is the most abundant of the Na⁺-P_i cotransporter mRNAs in mouse kidney and is localized to proximal tubules in the renal outer cortex, whereas Glvr-1 is a low-abundance transcript that is expressed throughout the kidney. In addition, we show that the Hyp mutation elicits a significant reduction in renal Npt1 and Npt2 mRNAs, whereas neither low-P_i diet nor GH influence the renal abundance of Npt1 and Npt2 transcripts. We also demonstrate that renal Glvr-1 mRNA expression is significantly increased in Hyp mice and GH-treated mice whereas the renal abundance of Ram-1 mRNA is unaffected by either the Hyp mutation, low-P_i diet, or GH treatment.