Effects of Npt2 gene ablation and low-phosphate diet on renal Na⁺/phosphate cotransport and cotransporter gene expression

The renal Na⁺/phosphate (Pi) cotransporter Npt2 is expressed in the brush border membrane (BBM) of proximal tubular cells. We examined the effect of Npt2 gene knockout on age-dependent BBM Na⁺/Pi cotransport, expression of Na⁺/Pi cotransporter genes Npt1, Glvr-1, and Ram-1, and the adaptive response to chronic Pi deprivation. Na⁺/Pi cotransport declines with age in wild-type mice (Npt2^+/+), but not in mice homozygous for the disrupted Npt2 allele (Npt2^–/–). At all ages, Na⁺/Pi cotransport in Npt2^–/– mice is approximately 15% of that in Npt2^+/+ littermates. Only Npt1 mRNA abundance increases with age in Npt2^+/+ mice, whereas Npt1, Glvr-1, and Ram-1 mRNAs show an age-dependent increase in Npt2^–/– mice. Pi deprivation significantly increases Na⁺/Pi cotransport, Npt2 protein, and mRNA in Npt2^+/+ mice. In contrast, Pi-deprived Npt2^–/– mice fail to show the adaptive increase in transport despite exhibiting a fall in serum Pi. We conclude that (a) Npt2 is a major determinant of BBM Na⁺/Pi cotransport; (b) the age-dependent increase in Npt1, Glvr-1, and Ram-1 mRNAs in Npt2^–/– mice is insufficient to compensate for loss of Npt2; and (c) Npt2 is essential for the adaptive BBM Na⁺/Pi cotransport response to Pi deprivation.

Phosphate (Pi) is a major constituent of bone mineral and plays a crucial role in cellular function. The kidney is an important determinant of Pi homeostasis and functions to reabsorb Pi to accommodate Pi need. In mammals, approximately 60–70% of the filtered Pi load is reabsorbed in the proximal tubule by Na⁺-coupled Pi transport systems that mediate the translocation of Pi across the brush border membrane (BBM) from the lumen into the cell (1–3). The extent of Pi reabsorption in this segment of the nephron is subject to hormonal control and to regulation by dietary Pi content (4–8).

To date, 3 families of Na⁺/Pi cotransporters have been identified in mammalian kidney. The type I (Npt1) (9–11) and type II (Npt2) (12–17) cotransporters are expressed predominantly in the kidney and have been localized to the BBM of proximal tubular cells, where the bulk of filtered Pi is reabsorbed (18–20). The type III cotransporters (Glvr-1 and Ram-1) are retroviral receptors that are ubiquitously expressed and may serve as housekeeping Na⁺/Pi cotransporters (21).

We recently demonstrated that Npt1, Npt2, Glvr-1, and Ram-1 account for approximately 15%, 84%, 0.5%, and 0.5%, respectively, of total Na⁺/Pi cotransporter mRNA in mouse kidney (22). Not only is Npt2 the most abundant of the renal Na⁺/Pi cotransporters, it is also the major target for regulation by parathyroid hormone (PTH) and dietary Pi. PTH decreases Na⁺/Pi cotransport through the endocytic retrieval of Npt2 protein from the BBM (23) and its subsequent lysosomal degradation (24). In contrast, both acute and chronic Pi deprivation elicit an adaptive increase in BBM Na⁺/Pi cotransport that can be ascribed to microtubule-dependent recruitment of Npt2 protein to the apical surface (25). The increase in Npt2 protein in acute Pi restriction is not associated with an increase in Npt2 mRNA, whereas in chronic Pi deprivation, a corresponding augmentation in Npt2 transcript abundance might (26–28) or might not (22, 25) be apparent.

Npt2 protein also appears to be the target for ontogenic regulation of renal Na⁺/Pi cotransport. Recent studies have demonstrated that BBM Na⁺/Pi cotransport is significantly higher in 24-day-old weanling rats than in 12-day-old suckling rats, and that the increase in transport activity is accompanied by a corresponding increase in Npt2 protein and mRNA (29). In addition, studies in weanling and 3-month-old rats provided evidence for higher BBM Na⁺/Pi cotransport in the weanlings and suggested that a unique growth-related Na⁺/Pi cotransporter that is homologous to, but distinct from, Npt2 may be mediating this activity (30). Finally, Sorribas et al. (31) demonstrated that aged rats (12–16 months old) exhibit a 2-fold decrease in BBM Na⁺/Pi cotransport when compared with young adult rats (3–4 months old), and that the decrease in transport is associated with a comparable decrease in the renal abundance of Npt2 protein and mRNA. Although age-related differences in Pi requirement likely play an important role in the ontogenic changes in Na⁺/Pi cotransporter gene expression, the contribution of Npt1, Glvr-1, and Ram-1 to this process is not known.

The crucial role of Npt2 protein to renal Pi reabsorption and the overall maintenance of Pi homeostasis was recently demonstrated in mice in which the Npt2 gene was inactivated by targeted mutagenesis (32). Mice homozygous for the disrupted gene (Npt2^–/–) exhibit increased urinary excretion of Pi, decreased BBM Na⁺/Pi cotransport, hypophosphatemia, and an appropriate increase in the serum concentration of 1,25-dihydroxyvitamin D [1,25-(OH)₂D] with attendant hypercalcemia and hypercalciuria (32).

In the present study, we examined the effect of Npt2 gene ablation and age on renal BBM Na⁺/Pi cotransport and the relative renal expression of Npt1, Glvr-1, and Ram-1. In addition, we sought to determine whether disruption of the Npt2 gene had an impact on the adaptive increase in BBM Na⁺/Pi cotransport in response to Pi deprivation. We demonstrate that the age-dependent upregulation of Npt1, Glvr-1, and Ram-1 mRNAs is not associated with an increase in BBM Na⁺/Pi cotransport in Npt2^–/– mice and cannot compensate for the loss of Npt2 function in homozygous mutants. Furthermore, we show that Npt2 is essential for the BBM adaptive response to dietary Pi restriction.

Effect of Npt2 gene ablation on renal BBM Na⁺/Pi cotransport and renal expression of Na⁺/Pi cotransporter mRNAs. We first measured the initial rate of Na⁺/Pi cotransport in BBM vesicles derived from Npt2^+/+ and Npt2^–/– mice as a function of age. The data in Figure 1 demonstrate that at all ages examined, Na⁺/Pi cotransport is significantly reduced in BBM vesicles derived from Npt2^–/– mice when compared with Npt2^+/+ mice. Figure 1 also shows that Na⁺/Pi cotransport decreases with age in Npt2^+/+ mice (activity at 115 days: 77 ± 8% and 84 ± 7% of that at 21 and 45 days, respectively; P < 0.05), but is not age-dependent in homozygous mutant mice. In agreement with previous findings (32), Na⁺-dependent glucose transport is not significantly decreased in BBM from Npt2^–/– mice when compared with age-matched Npt2^+/+ mice (at 21 days: 63 ± 17 vs. 53 ± 4 pmol/mg protein per 6 seconds for Npt2^+/+ vs. Npt2^–/– mice; at 45 days: 59 ± 12 vs. 63 ± 11 pmol/mg protein per 6 seconds for Npt2^+/+ vs. Npt2^–/– mice; and at 115 days: 58 ± 21 vs. 53 ± 5 pmol/mg protein per 6 seconds for Npt2^+/+ vs. Npt2^–/– mice).

Figure 1

Effect of Npt2 gene knockout and age on renal BBM Na⁺/Pi cotransport. Renal BBM vesicles were prepared from Npt2^+/+ (filled bars) and Npt2^–/– (open bars) mice at 21, 45, and 115 days of age. The BBM vesicle preparations were used for both transport studies and Western analysis (Figure 2) as described in Methods. One hundred percent activity represents the Na⁺-dependent component of Pi transport (Na⁺/K⁺) in BBMs from 21-day-old Npt^+/+ mice (590 ± 30 pmol/mg protein per 6 seconds). Values (mean ± SEM) were derived from 3 experiments, each assayed in quadruplicate. *Effect of genotype, P < 0.05. ^§Effect of age, P < 0.05.

The effect of age on the abundance of Npt2 protein in renal BBMs derived from Npt2^+/+ mice was estimated by Western analysis (Figure 2). Quantitation of proteins bands revealed that the 13% decrease in abundance of Npt2 protein (83 kDa), relative to meprin (65 kDa), in mice at 115 days, compared with mice at 21 days of age (n = 4), was not statistically significant. Similarly, renal Npt2 mRNA abundance, relative to that of β-actin, does not appear to decrease significantly with age in Npt2^+/+ mice (Figure 3a). As reported previously (32), neither Npt2 immunoreactive protein (Figure 2) nor Npt2 mRNA (Figure 3b) is expressed in Npt2^–/– mice.

Figure 2

Effect of Npt2 gene knockout and age on renal BBM Npt2 and meprin immunoreactive protein. BBM proteins, prepared from Npt2^+/+ mice at 21, 45, and 115 days of age and from 115-day-old Npt2^–/– mice, were fractionated on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with a rabbit polyclonal anti-rat Npt2 antibody and an mAb raised against the α subunit of rat meprin. Bands depicting Npt2 (83 kDa) and meprin (65 kDa) immunoreactive proteins are indicated by arrows. The figure depicts 1 of 3 BBM preparations per group.

Figure 3

Effect of age on renal abundance of Npt2 mRNA in Npt2^+/+ mice (a) and ribonuclease protection assay of renal RNA from Npt2^+/+ and Npt2^–/– mice (b). Renal RNA was prepared from Npt2^+/+ mice at 21, 45, and 115 days of age, and the abundance of Npt2 mRNA, relative to β-actin mRNA, was determined by ribonuclease protection assay as described in Methods. The values are mean ± SEM derived from 5–8 mice per group (a). Protected Npt2 and β-actin mRNA fragments are evident with renal RNA from 45-day-old Npt2^+/+ mice, whereas only a protected β-actin mRNA fragment is detected with RNA from age-matched Npt2^–/– mice (b).

We then compared the renal abundance of Npt1, Glvr-1, and Ram-1 mRNAs, relative to β-actin mRNA, in Npt2^+/+ and Npt2^–/– mice as a function of age. Npt1 mRNA increases with age in both mouse strains, and, at both 21 and 45 days of age, Npt1 mRNA expression is significantly lower in the Npt2^–/– mice relative to age-matched Npt2^+/+ mice (Figure 4a). Age has no effect on the renal abundance of Glvr-1 (Figure 4b) and Ram-1 (Figure 4c) mRNAs in Npt2^+/+ mice. However, in Npt2^–/– mice, a significant age-related increase in the renal expression of both Glvr-1 (Figure 4b) and Ram-1 (Figure 4c) mRNAs is evident. In addition, although the abundance of both Glvr-1 and Ram-1 mRNAs is lower in 21-day-old Npt2^–/– mice relative to Npt2^+/+ mice, it is not significantly different from Npt2^+/+ mice at 45 days of age (Figure 4, b and c). At 115 days of age, Glvr-1 mRNA abundance is greater in Npt2^–/– mice than in Npt2^+/+ mice (Figure 4b), whereas Ram-1 mRNA abundance is similar in both genotypes (Figure 4c).

Figure 4

Effect of Npt2 gene knockout and age on renal abundance of Npt1 (a), Glvr-1 (b), and Ram-1 (c) mRNAs. The abundance of each transcript, relative to β-actin mRNA, was determined in renal RNA samples derived from 21-, 45-, and 115-day-old Npt2^+/+ (filled bars) and Npt2^–/– (open bars) mice by ribonuclease protection assay as described in Methods. The values depict the mean ± SEM derived from 5–8 mice per group. *Effect of genotype, P < 0.05. ^§Effect of age, P < 0.05.

Effect of Npt2 gene ablation on the renal adaptive response to Pi deprivation. Low-Pi diet (0.02%) elicits a significant fall in serum Pi in Npt2^+/+ and Npt2^–/– mice when compared with counterparts fed the control diet (1% Pi) (Table 1). Moreover, although serum Pi levels are similar in Npt2^+/+ and Npt2^–/– mice on the low-Pi diet, Npt2^–/– mice are significantly hypophosphatemic, relative to Npt2^+/+ mice, on the control diet (1% Pi) (Table 1), in agreement with previous data reported in mice fed standard chow (0.6% Pi) (32). Pi deprivation also elicits a decrease in urine Pi/creatinine and fractional Pi excretion index (FEI_Pi) in Npt2^+/+ and Npt2^–/– mice (Table 1). Under both dietary conditions, however, urine Pi/creatinine and FEI_Pi are significantly elevated in Npt2^–/– mice when compared with Npt2^+/+ mice.

Table 1

Effect of Npt2 gene knockout and low-Pi diet on serum Pi, urine Pi/creatinine, and FEI_Pi

BBM Na⁺/Pi cotransport is increased 3.4-fold in Npt2^+/+ mice after Pi restriction (Figure 5a), consistent with earlier studies (28, 37). Moreover, as reported previously (28), the increase in Na⁺/Pi cotransport in Pi-deprived Npt2^+/+ mice is associated with a corresponding increase (∼3-fold) in Npt2 protein (Figure 5b) and a modest but significant increase in Npt2 mRNA (Figure 6a). In contrast, Npt2^–/– mice, which are devoid of Npt2 protein (Figure 2 and Figure 5b) and mRNA (Figure 3b), fail to show an adaptive increase in BBM Na⁺/Pi cotransport in response to the low-Pi diet (Figure 5a).

Figure 5

Effect of Npt2 gene knockout and low-Pi diet on renal BBM Na⁺/Pi cotransport (a) and Npt2 and meprin immunoreactive protein (b). Renal BBM vesicles were prepared from 45-day-old Npt2^+/+ (filled bars) and Npt2^–/– (open bars) mice fed the control (1%) and low-Pi (0.02%) diets for 5 days. The BBM vesicle preparations were used for both transport studies (a) and Western analysis (b) as described in Methods and legends to Figures 1 and 2. The Na⁺-dependent component of Pi transport (mean ± SEM of 2 typical experiments, each assayed in quadruplicate) is depicted. *Effect of genotype, P < 0.05. ^‡Effect of diet, P < 0.05.

Figure 6

Effect of Npt2 gene knockout and low-Pi diet on renal abundance of Npt2 (a), Npt1 (b), Glvr-1 (c), and Ram-1 (d) mRNAs. Renal RNA was prepared from 45-day-old Npt2^+/+ (filled bars) and Npt2^–/– (open bars) mice fed the control and low-Pi diets for 5 days. The abundance of Npt1, Glvr-1, and Ram-1 mRNAs, relative to β-actin mRNA, was determined by ribonuclease protection assay as described in Methods. The values depict the mean ± SEM derived from 5–8 mice per group. *Effect of genotype, P < 0.05. ^‡Effect of diet, P < 0.05.

We also examined the effect of low-Pi diet on the renal expression of Npt1, Glvr-1, and Ram-1 mRNAs in normal and Npt2^–/– mice. The abundance of Npt1 mRNA is significantly increased, by 1.3- and 1.4-fold, respectively, in both Npt2^+/+ and Npt2^–/– mice fed the low-Pi diet when compared with counterparts on the control diet (Figure 6b). In contrast, Pi restriction failed to elicit a significant alteration in the renal abundance of Glvr-1 (Figure 6c) and Ram-1 (Figure 6d) mRNAs in either Npt2^+/+ or Npt2^–/– mice.

Effect of Npt2 gene ablation and low-Pi diet on renal BBM Npt1 protein abundance. Western analysis revealed the presence of a 64-kDa Npt1 immunoreactive protein in renal BBMs derived from Npt2^+/+ and Npt2^–/– mice fed control and low-Pi diets (Figure 7). The abundance of Npt1 protein, relative to meprin, was not increased by either Npt2 gene disruption or dietary Pi (Figure 7). The size of the detected Npt1 protein and the absence of regulation by dietary Pi are consistent with previous reports (14, 19).

Figure 7

Effect of Npt2 gene knockout and low-Pi diet on renal BBM Npt1 and meprin immunoreactive protein. Renal BBM vesicles were prepared from 55-day-old Npt2^+/+ and Npt2^–/– mice fed the control (1%) and low-Pi (0.02%) diets for 5 days. The BBM proteins were fractionated on 10% SDS-PAGE gels, transferred to nitrocellulose membranes, and probed with a rabbit polyclonal anti-rabbit Npt1 antibody and subsequently with an mAb raised against the α subunit of rat meprin. Bands depicting Npt1 (64 kDa) and meprin (65 kDa) immunoreactive proteins are indicated by arrows. The figure depicts 1 of 3 BBM preparations per group.

In the present study, we examined the impact of Npt2 gene ablation on age-dependent renal BBM Na⁺/Pi cotransport; the expression of Na⁺/Pi cotransporter genes Npt1, Glvr-1, and Ram-1; and the adaptive BBM Na⁺/Pi cotransport response to Pi deprivation. We demonstrate that Na⁺/Pi cotransport is not age-dependent and is significantly compromised (∼15% of Npt2^+/+) in Npt2^–/– mice. Moreover, we show that although the abundance of Npt1, Glvr-1, and Ram-1 mRNAs increases with age in Npt2^–/– mice, the augmentation in Na⁺/Pi cotransporter gene expression is not sufficient to compensate for the loss of Npt2. We also show that Npt2^–/– mice fail to exhibit an increase in BBM Na⁺/Pi cotransport in response to Pi deprivation, thereby documenting the crucial role of Npt2 in the renal adaptive response to low Pi intake.

Previous studies have provided evidence for higher Na⁺/Pi cotransport activity in BBMs from younger animals when compared with older animals, consistent with the notion that the requirement for Pi is increased during growth (30, 31, 38). Although it was suggested that the higher transport activity observed in weanling rats, relative to 3-month-old rats, is mediated by a Na⁺/Pi cotransporter that is distinct from, but related to, Npt2 (30), this growth-related Na⁺/Pi cotransporter has not yet been identified at the molecular level. In the present study, we also provide evidence for an age-dependent decrease in BBM Na⁺/Pi cotransport in Npt2^+/+ mice. Moreover, the demonstration that BBM Na⁺/Pi cotransport is not age-dependent in mice homozygous for the disrupted Npt2 gene supports the hypothesis that differences in the Npt2 protein abundance alone could account for the age-dependent decrease in transport in Npt2^+/+ mice.

In a previous study, it was reported that the decrease in BBM Na⁺/Pi cotransport in aged (12- to 16-month-old) rats, relative to young (3- to 4-month-old) rats, could be ascribed to a proportional decrease in both Npt2 protein and mRNA (31). In the present study, however, a significant age-dependent decrease in Npt2 protein and mRNA was not observed. The discrepancy between the 2 studies may be explained by the fact that the ages and the species investigated were different, with the age of the older mice in the present study comparable to the younger rats in the earlier report (31), or by the magnitude of the age-related decrease in transport activity (∼20% in the present study vs. a 2-fold decrease in ref. 31). In this regard, it is possible that the age-dependent reduction in BBM Na⁺/Pi cotransport observed in Npt2^+/+ mice (Figure 1) may not have been of sufficient magnitude to detect corresponding changes in Npt2 protein and mRNA.

It was of interest to determine whether Npt2 gene ablation elicits the upregulation of other Na⁺/Pi cotransporter genes that are expressed in the kidney (22). We demonstrate that, with the exception of renal Glvr-1 mRNA expression in 115-day-old Npt2^–/– mice, there is no evidence for an increase in the abundance of Npt1, Glvr-1, or Ram-1 mRNAs in 21-, 45-, and 115-day-old Npt2^–/– mice, relative to age-matched Npt2^+/+ littermates. We also demonstrate that the abundance of Npt1, Glvr-1, and Ram-1 mRNAs is significantly increased with age in Npt2^–/– mice. However, because Na⁺/Pi cotransport continues to be markedly impaired and unchanged in Npt2^–/– mice at all ages examined, it is clear that the increases in Npt1, Glvr-1, and Ram-1 mRNA abundance are insufficient to compensate for the loss of Npt2.

The changes in renal Npt1, Glvr-1, and Ram-1 mRNA levels in Npt2^–/– mice may not reflect the actual abundance of functional cotransporter protein. We thus evaluated the effect of Npt2 gene ablation on BBM Npt1 and Glvr-1 protein abundance. We demonstrate that Npt1 protein is not upregulated in Npt2^–/– mice. However, the contribution of Npt1 to BBM Na⁺/Pi cotransport is not entirely clear. Electrophysiological studies in cRNA-injected oocytes suggest that Npt1 can function not only as a Na⁺/Pi cotransporter, but also as a channel for Cl^– and organic anions in the kidney (39, 40). Accordingly, the extent of the role of Npt1 in the maintenance of Pi homeostasis remains to be elucidated. To this end, we have cloned the murine Npt1 gene and initiated studies to construct a targeting vector to generate mice in which the Npt1 gene has been disrupted by homologous recombination (41).

We were unable to detect Glvr-1 protein expression in BBMs from either Npt2^+/+ or Npt2^–/– mice (data not shown). Using the same antibody, Boyer et al. provided evidence for an 85-kDa protein in crude membranes prepared from several mouse tissues (35), consistent with the ubiquitous expression of Glvr-1 (21). Our failure to detect Glvr-1 protein in mouse renal BBMs suggests that Glvr-1 may not be expressed at the apical surface. In this regard, Glvr-1 and Ram-1 have a higher affinity for Pi (10 μM) than does Npt2 (100 μM), and more closely resemble basolateral membrane–associated Na⁺-dependent Pi transporters (42). Thus, these transporters are postulated to subserve cellular metabolic requirements for Pi under conditions in which inadequate quantities of Pi are available in the glomerular filtrate (43). In any case, the age-dependent upregulation of Glvr-1 and Ram-1 mRNAs in Npt2^–/– mice may be a moot point. They only comprise about 1% of Na⁺/Pi cotransporter mRNAs expressed in mouse kidney (22) and, thus, are not likely to contribute significantly to the BBM transport process.

There are several reports documenting the importance of Npt2 in the adaptive regulation of BBM Na⁺/Pi cotransport by dietary Pi (25–28). Consistent with these data, we demonstrate a comparable adaptive increase in BBM Na⁺/Pi cotransport and Npt2 protein in Npt2^+/+ mice fed the low-Pi diet. In contrast, Pi-deprived Npt2^–/– mice fail to show the adaptive increase in BBM Na⁺/Pi cotransport. On the basis of these findings, we conclude that Npt2 gene expression is essential for the adaptive BBM Na⁺/Pi cotransport response to Pi deprivation.

The contribution of Npt1, Glvr-1, and Ram-1 to the adaptive increase in BBM Na⁺/Pi cotransport was also investigated in the present study. We demonstrate that Npt1 protein is not mRNA is increased in Pi-deprived Npt2^+/+ and Npt2^–/– mice. These data indicate that Npt1 does not contribute to the adaptive increase in BBM Na⁺/Pi cotransport in Npt2^+/+ mice, consistent with previous reports in Pi-deprived rabbits (14) and rats (44). Moreover, our findings demonstrate that Npt1 does not participate in an adaptive response in Npt2^–/– mice. We also show that Glvr-1 and Ram-1 mRNAs are not increased by Pi deprivation in either Npt2^+/+ or Npt2^–/– mice, in agreement with the report that the renal abundance of Glvr-1 and Ram-1 mRNAs is not increased in Pi-deprived normal and X-linked Hyp mice, relative to counterparts fed the control diet (22). In support of these findings is the recent demonstration that posttranslational modification accounts for the regulation of Ram-1–mediated Na⁺/Pi cotransport in response to low-Pi challenge (45).

It is of interest that serum Pi values are similar in Pi-deprived Npt2^+/+ and Pi-deprived Npt2^–/– mice. Although the underlying mechanism for these findings is not clear, our studies in X-linked Hyp mice, which harbor a large deletion in the Phex gene (36), suggest a possible explanation. In contrast to Pi-deprived Npt2^–/– mice, serum Pi in Pi-deprived Hyp mice is significantly lower than that in Pi-deprived normal mice (28, 37, 46). Moreover, Hyp mice exhibit abnormal regulation of both renal 1,25-(OH)₂D synthesis and catabolism by dietary Pi (46, 47), whereas Npt2^–/– mice show the expected increase in serum 1,25-(OH)₂D levels in the face of hypophosphatemia (32). On the basis of these findings, it is possible that normal Phex function and appropriate regulation of renal vitamin D metabolism account for the similar serum Pi concentrations in Pi-deprived Npt2^+/+ and Npt2^–/– mice. It is also of interest that Hyp mice that are significantly deficient in, but not devoid of, renal Npt2 mRNA and protein (33) retain the capacity to mount an adaptive increase in BBM Na⁺/Pi cotransport in response to low-Pi challenge (28, 37). While the mechanism whereby loss of Phex function elicits the decrease in renal Npt2 gene expression is not understood, it is clear that, in contrast to Npt2, Phex is not necessary for the adaptive Na⁺/Pi cotransport response at the renal BBM.

Mice homozygous for the disrupted Npt2 gene exhibit many of the biochemical features of patients with hereditary hypophosphatemic rickets with hypercalciuria, a rare Mendelian disorder of renal Pi reabsorption (48, 49). These include increased urinary excretion of Pi, hypophosphatemia, and an elevated serum concentration of 1,25-(OH)₂D with attendant hypercalciuria. To date, there is no clear association between Npt2 and hereditary hypophosphatemic rickets with hypercalciuria. Additionally, it is not known whether patients with this disorder exhibit an adaptive increase in renal Pi reabsorption in response to low-Pi challenge.

In summary, we demonstrate that Npt2 is responsible for approximately 85% of Na⁺/Pi cotransport at the renal BBM, and that homozygous disruption of the Npt2 gene cannot be compensated for by the age-dependent increase in renal expression of Npt1, Glvr-1, and Ram-1 mRNAs. In addition, we show that Npt2 is essential for the adaptive increase in renal BBM Na⁺/Pi cotransport in response to low-Pi challenge. Our results support the conclusion that Npt2 plays a fundamental role in the overall maintenance of Pi homeostasis.

We thank Danielle Boulais for technical assistance, H. Murer and J. Biber for the Npt2 and Npt1 antibodies, P. Crine for the meprin antibody, and R. Beliveau for the Glvr-1 antibody. This work was supported by a grant from the Medical Research Council of Canada (Medical Research Council Group Grant in Medical Genetics to H.S. Tenenhouse). H.M. Hoag is the recipient of a Medical Research Council Studentship Award.

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