Endocrinology Vol. 141, No. 6 2159-2165
Copyright © 2000 by The Endocrine Society
Npt2 Gene Disruption Confers Resistance to the Inhibitory Action of Parathyroid Hormone on Renal Sodium-Phosphate Cotransport1
Ningxia Zhao2 and
Harriet S. Tenenhouse
Departments of Pediatrics (N.Z., H.S.T.) and Human Genetics
(H.S.T.), McGill University-Montréal Children’s Hospital
Research Institute, Montréal, Québec, Canada H3H 1P3
Address all correspondence and requests for reprints to: Harriet S. Tenenhouse, Ph.D., Montréal Children’s Hospital, 2300 Tupper Street, Montréal, Québec, Canada H3H 1P3. E-mail:
mhdt{at}www.debelle.mcgill.ca
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Abstract
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PTH inhibition of renal sodium-phosphate (Na-Pi) cotransport is
associated with the endocytic retrieval of the type II Na-Pi
cotransporter, Npt2, from the renal brush border membrane into the late
endosomal/lysosomal compartment. The aim of the present study was to
determine whether mice homozygous for the disrupted Npt2
gene (Npt2-/-) exhibit decreased renal Pi
reabsorption in response to PTH. We demonstrate that PTH has no effect
on the serum Pi concentration, fractional excretion of Pi, or
Na-dependent Pi transport in renal brush border membrane vesicles in
Npt2-/- mice. In contrast, PTH elicits a
fall in the serum Pi concentration, an increase in urinary Pi
excretion, a decrease in brush border membrane Na-Pi cotransport, and a
corresponding reduction in the relative abundance of Npt2 protein in
wild-type mice (Npt2+/+). Both
Npt2-/- and
Npt2+/+ mice exhibit a significant rise in
the urinary cAMP/creatinine ratio in response to PTH, indicating that
generalized resistance to PTH cannot account for the absence of the PTH
response in Npt2-/- mice. In addition, we
demonstrate that Pi-depleted normal mice respond to PTH with a decrease
in renal brush border membrane Na-Pi cotransport and Npt2 protein,
indicating that Pi deficiency per se does not account
for PTH resistance in Npt2-/- mice. Taken
together, our data provide compelling evidence that Npt2
gene expression is crucial for PTH effects on renal Pi handling.
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Introduction
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THE KIDNEY IS a major arbiter of phosphate
(Pi) homeostasis by virtue of its ability to increase or decrease
its Pi reabsorptive capacity to accommodate Pi need. The bulk
of filtered Pi is reabsorbed in the proximal tubule, with approximately
60% of the filtered load reclaimed in the proximal convoluted tubule
and 15–20% in the proximal straight tubule (1). Physiological studies
have demonstrated that sodium (Na)-Pi cotransporters in the renal brush
border membrane of proximal tubular cells mediate the rate-limiting
step in the overall Pi reabsorptive process and are subject to
regulation by PTH and dietary Pi, key regulators of renal Pi
reabsorption (1, 2, 3, 4, 5).
Complementary DNAs encoding two distinct renal Na-Pi
cotransporters were identified by expression and homology cloning and
have been designated type I (Npt1) (6, 7, 8) and type II (Npt2) (9, 10, 11, 12, 13, 14).
Both Npt1 and Npt2 immunoreactive proteins are localized to the brush
border membrane of proximal tubular cells (15, 16, 17). In mouse kidney,
Npt2 is approximately 5 times more abundant than Npt1 at the messenger
RNA (mRNA) level (18). In addition, Npt2 is a target for regulation by
PTH and dietary Pi. PTH decreases Na-Pi cotransport through the
endocytic retrieval of Npt2 protein from the brush border membrane to
the endosomal/lysosomal compartment (19) and its subsequent lysosomal
degradation (20). In contrast, dietary Pi restriction elicits an
adaptive increase in Na-Pi cotransport that can be ascribed to
microtubule-dependent recruitment of Npt2 protein to the apical surface
from an intracellular pool (21, 22).
The importance of Npt2 in the overall maintenance of Pi homeostasis was
recently demonstrated in mice in whom the Npt2 gene was
disrupted by targeted mutagenesis (23). Mice homozygous for the
disrupted gene (Npt2-/-) exhibit
decreased renal Pi reabsorption, an approximately 85% loss in renal
brush border membrane Na-Pi cotransport, hypophosphatemia, and skeletal
abnormalities (23). We recently demonstrated that other known Na-Pi
cotransporters, namely Npt1, Glvr-1, and Ram-1, do not compensate for
the loss of Npt2 function in Npt2-/-
mice (24). In addition, we showed that
Npt2-/- mice fail to exhibit an
increase in Na-Pi cotransport in response to Pi restriction (24). On
the basis of these data we concluded that Npt2 is essential for the
adaptive renal response to a low Pi diet.
In the present study we sought to characterize the effect of
Npt2 gene ablation and Pi deprivation on the renal Pi
transport response to PTH. We demonstrate that Npt2 gene
expression is critical for the PTH-mediated decrease in renal brush
border membrane Na-Pi cotransport. In addition, we show that normal
mice fed a low Pi diet exhibit a significant decrease in brush border
membrane Na-Pi cotransport and Npt2 protein abundance in response to
PTH, indicating that Pi deficiency per se cannot account for
the refractory PTH response in
Npt2-/- mice.
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Materials and Methods
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Mice
Npt2 knockout mice were established in our
laboratory by homologous recombination as previously described (23).
Wild-type (Npt2+/+) and homozygous
mutant (Npt2-/-) mice were generated
by crossing heterozygous (Npt2+/-)
male and female mice and were genotyped by PCR amplification of genomic
DNA obtained from tail tissue using Taq polymerase (BRL
Life Technologies, Inc., Burlington, Canada) and three
primers [sense primer 3F (5'-TGC CCA GGT TGG CAC GAA GC-3') in exon 4
of Npt2, antisense primer 4R (5'-AGT CCT GTC CCC TGC CTG
CA-3') in exon 6 of Npt2, and antisense primer PGKR (5'-TGC
TAC TTC CAT TTG TCA CGT CC-3') in the
neor gene cassette] as described
previously (23). The mice were 65 ± 5 days old and were
maintained on a 0.6% Pi diet (5001 lab chow, Ralston Purina Co., J. E. Mondou, Montréal, Canada). For Pi
deprivation experiments, 65-day-old C57BL/6 mice (Charles River Laboratories, Inc., St.-Constant, Canada) were fed either
a low Pi (0.02% Pi) or a control Pi (0.6% Pi) diet for 2 days (test
diets TD 86128 and TD 98243, Harlan Teklad, Madison, WI). The test
diets were otherwise identical.
PTH administration
Mice were anesthetized with a mixture of ketamine, xylazine, and
acepromazine (5, 0.5, and 0.06 mg/100 g BW, ip). Bovine PTH(1–84),
which was provided by Dr. P. A. Friedman (University of Pittsburgh
School of Medicine, Pittsburgh, PA), was diluted in saline containing
0.1% BSA and 1 mM sodium acetate and injected via the tail
vein, as was the vehicle. Unless otherwise indicated, the dose of PTH
was 10 µg/100 g BW, and the mice were killed 2 h after PTH
administration. All animal studies were conducted in accordance with
the guidelines of the Canadian Council on Animal Care.
Brush border membrane isolation, transport studies, and Western
blot analysis
Renal brush border membrane vesicles were prepared from kidney
cortex by the MgCl2 precipitation method
described previously (25) and were used for both transport studies and
Western blot analysis. Kidneys from each of three mice were used for
brush border membrane vesicle preparations. The uptake of Pi (100
µM) and glucose (10 µM), performed in
quadruplicate on at least four different brush border membrane
preparations per group, was measured at 6 sec (initial rate) in medium
containing either 100 mM NaCl or 100 mM KCl by
the rapid filtration technique (25). An aliquot of brush border
membrane proteins (20–80 µg) was fractionated on 10% SDS-PAGE gels
according to the method of Laemmli (26), transferred to supported
nitrocellulose membranes (Hybond-C Extra, Amersham Pharmacia Biotech, Baie d’Urfe, Canada), and probed sequentially with
rabbit polyclonal antibodies raised against an N-terminal peptide of
rat Npt2 (17) and a C-terminal peptide of rabbit Npt1 (16) (gifts from
Drs. H. Murer and J. Biber, University of Zurich, Zurich, Switzerland),
and a monoclonal antibody raised against the 
-subunit of rat renal
endopeptidase-24.18 (meprin; provided by Dr. P. Crine, Université
de Montréal, Montréal, Canada) as described previously
(27). Primary antibodies were visualized using an enhanced
chemiluminescence kit (Amersham Pharmacia Biotech) and
were exposed to Kodak Biomax MRI film (Eastman Kodak Co., Rochester, NY). The abundance of Npt2 and Npt1 protein,
relative to that of meprin, was estimated using phosphorimager analysis
of scanned images.
Ribonuclease protection analysis
Total RNA (5–20 µg), isolated from kidney with Trizol reagent
(BRL Life Technologies, Inc.), was hybridized with
32P-labeled Npt1, Npt2, and ß-actin riboprobes
(5 x 105 cpm) as described previously (18, 24). The protected fragments were precipitated, heat denatured, and
electrophoresed on 6% denaturing polyacrylamide gels. The gels were
dried, exposed to a phosphorimager screen for quantification of
radioactive signals under conditions where linearity is achieved, and
the abundance of Npt2 and Npt1 mRNAs, relative to that of ß-actin,
was estimated.
Serum and urinary parameters
Serum Pi and calcium and urinary Pi and creatinine were assayed
using phosphorous, calcium, and creatinine kits (Stanbio Laboratories,
San Antonio, TX) as described previously (28). The fractional excretion
index for Pi (FEIPi) was calculated as follows:
urinary Pi/(urinary creatinine x serum Pi). Urinary cAMP was
determined by competitive binding assay using a commercial kit
(Amersham Pharmacia Biotech).
Statistical analysis
For serum and urine parameters, each group consisted of 11–18
mice. For brush border membrane parameters, each group consisted of 4
preparations. Statistical analysis was performed using two-way ANOVA.
P < 0.05 was considered statistically significant.
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Results
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PTH dose-response and time-course studies
To determine the experimental conditions necessary to elicit PTH
inhibition of renal brush border membrane Na-Pi cotransport in mice, we
examined the effect of PTH dose and the time course of the response to
PTH. The data in Fig. 1
, A and B,
demonstrate that approximately 50% inhibition of renal brush border
membrane Na-Pi cotransport was achieved at a PTH dose of 10 µg/100 g
BW 2 h after PTH administration. These conditions were used for
all subsequent experiments.
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Figure 1. Effects of PTH dose (A) and time (B) on Na-Pi
cotransport in renal brush border membrane vesicles prepared from
normal mice. Anesthetized mice were injected with vehicle or increasing
doses of PTH and killed 2 h later (A) or with vehicle or PTH (10
µg/100 g BW) and killed at various times thereafter (B). Brush border
membrane vesicles were prepared from kidney cortex, and transport of Pi
was measured under initial rate conditions in the presence of 100
mM KCl or NaCl as described in Materials and
Methods. The Na-mediated component of transport, derived by
subtracting uptake in KCl from that in NaCl, is depicted. The mean
± SEM derived from a representative experiment, each
assayed in quadruplicate, is shown. Similar results were obtained in
one other experiment.
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Effect of Npt2 gene disruption on PTH response
PTH elicited a significant reduction in serum Pi concentration in
wild-type mice compared with that in vehicle-treated controls (Fig. 2). In
Npt2-/- mice, the serum Pi
concentration was significantly lower than that in wild-type
littermates, as reported previously (23, 24), and was not further
decreased by the administration of PTH (Fig. 2). The fall in serum Pi
concentration in PTH-treated wild-type mice was associated with a
significant increase in FEIPi (Fig. 3). In contrast, PTH failed to elicit an
increase in FEIPi in Npt2-/- mice
(Fig. 3). The latter was significantly elevated in vehicle-treated
homozygous mutants relative to that in vehicle-treated wild-type mice,
in agreement with previous results (23, 24). Figure 4 demonstrates that both
Npt2+/+ and
Npt2-/- mice responded to PTH with a
robust increase in the urinary excretion of cAMP. The PTH-mediated
increase in urinary cAMP was modestly lower in
Npt2-/- mice than in
Npt2+/+ littermates (Fig. 4). PTH had
no effect on the serum calcium concentration under the conditions
studied (data not shown).
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Figure 2. Effect of PTH on serum Pi in
Npt2+/+ and
Npt2-/- mice. Anesthetized mice were
injected with vehicle or PTH (10 µg/100 g BW), and blood was
collected 2 h thereafter. The serum Pi concentration was
determined as described in Materials and Methods. The
mean ± SEM derived from 11–18 mice/group is shown.
#, Effect of PTH in Npt2+/+ mice,
P < 0.0003. *, Effect of genotype in
vehicle-treated mice, P < 0.0002.
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Figure 3. Effect of PTH on fractional excretion index for Pi
(FEIPi) in Npt2+/+ and
Npt2-/- mice. Anesthetized mice were
injected with vehicle or PTH (10 µg/100 g BW), and urine was
collected 2 h thereafter for the determination of Pi and
creatinine concentrations. FEIPi was calculated as
described in Materials and Methods. The mean ±
SEM derived from 11–18 mice/group is shown. #, Effect of
PTH in Npt2+/+ mice, P
< 0.0001. *, Effect of genotype in vehicle-treated mice,
P < 0.0021.
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Figure 4. Effect of PTH on urinary cAMP/creatinine ratio in
Npt2+/+ and
Npt2-/- mice. Anesthetized mice were
injected with vehicle or PTH (10 µg/100 g BW), and urine was
collected 2 h thereafter. Urinary cAMP and creatinine were
assayed, and the cAMP/creatinine ratio was determined as described in
Materials and Methods. The mean ± SEM
derived from 11–18 mice/group is shown. #, Effect of PTH in
Npt2+/+ mice, P <
0.0001; effect of PTH in Npt2-/- mice,
P < 0.0003. *, Effect of genotype in PTH-treated
mice, P < 0.0158.
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Na-Pi cotransport was significantly reduced in renal brush border
membrane vesicles prepared from PTH-treated wild-type mice compared
with that in vehicle-treated controls (Fig. 5). The decrease in transport was
associated with a 50 ± 11% decrease (n = 4;
P < 0.05) in the abundance of Npt2 protein relative to
meprin (see Fig. 6 for a representative
Western blot). However, a corresponding decrease in Npt2 mRNA was not
apparent in PTH-treated Npt2+/+ mice (data not
shown). In Npt2-/- mice, PTH had no
effect on Na-Pi cotransport across the renal brush border membrane
(Fig. 5). In agreement with previous studies (23, 24), mice homozygous
for the disrupted Npt2 gene had significantly reduced Na-Pi
cotransport (Fig. 5, vehicle-treated
Npt2-/- mice vs.
vehicle-treated Npt2+/+ mice) and did
not express Npt2 protein (Fig. 6) or Npt2 mRNA (data not shown) (23, 24).
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Figure 5. Effect of PTH on brush border membrane Na-Pi
cotransport in Npt2+/+ and
Npt2-/- mice. Anesthetized mice were
injected with vehicle or PTH (10 µg/100 g BW) and killed 2 h
thereafter. Renal brush border membrane vesicles were prepared from
kidney cortex, and transport of Pi was assayed under initial rate
conditions in the presence of 100 mM KCl or NaCl as
described in Materials and Methods. The Na-mediated
component of transport, derived by subtracting uptake in KCl from that
in NaCl, is depicted. The mean ± SEM derived from
four brush border membrane preparations per group, each assayed in
quadruplicate, is shown. #, Effect of PTH in
Npt2+/+ mice, P <
0.0015. *, Effect of genotype in vehicle-treated mice,
P < 0.0001; effect of genotype in PTH-treated
mice, P < 0.0041.
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Figure 6. Effect of PTH on brush border membrane Npt2, Npt1,
and meprin immunoreactive protein in Npt2+/+
and Npt2-/- mice. Anesthetized mice were
injected with vehicle or PTH (10 µg/100 g BW) and killed 2 h
thereafter. Renal brush border membrane vesicles were prepared from
kidney cortex, subjected to 10% SDS-PAGE, and immunoblotted as
described in Materials and Methods. Representative gels
from four brush border membrane vesicle preparations per group are
shown.
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Of interest, PTH had no effect on either renal brush border membrane
Na+-glucose cotransport (data not shown) or the
renal abundance of Npt1 protein (Fig. 6
) and Npt1 mRNA (data not shown)
in both Npt2+/+ and
Npt2-/- mice. Furthermore, these
parameters were not affected by Npt2 gene disruption
(24).
Effect of low Pi diet on PTH response in normal mice
To determine whether the failure of renal Pi transport in
Npt2-/- mice to respond to PTH could
be ascribed to Pi deficiency per se (29, 30), we also
examined the effect of PTH on the same parameters in Pi-deprived normal
mice. Both vehicle- and PTH-treated normal mice responded to the low Pi
diet with a marked decrease in the FEIPi (Fig. 7) and a significant adaptive increase in
renal brush border membrane Na-Pi cotransport (Fig. 8) and Npt2 protein abundance (Fig. 9) compared with counterparts fed the
control diet (
Figs. 7–9).
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Figure 7. Effect of PTH on fractional excretion index for Pi
(FEIPi) in control and Pi-deprived normal mice.
Anesthetized mice, fed control and low Pi diets for 2 days, were
injected with vehicle or PTH (10 µg/100 g BW), and blood and urine
were collected 2 h thereafter for the determination of Pi and
creatinine concentrations. FEIPi was calculated as
described in Materials and Methods. The mean ±
SEM derived from 11–18 mice/group is shown. #, Effect of
PTH in control mice, P < 0.0001. *, Effect of diet
in vehicle-treated mice, P < 0.0030; effect of
diet in PTH-treated mice, P < 0.0001.
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Figure 8. Effect of PTH on brush border membrane Na-Pi
cotransport in control and Pi-deprived normal mice. Anesthetized mice,
fed control and low Pi diets for 2 days, were injected with vehicle or
PTH (10 µg/100 g BW) and killed 2 h thereafter. Renal brush
border membrane vesicles were prepared from kidney cortex, and
transport of Pi was assayed under initial rate conditions in the
presence of 100 mM KCl or NaCl as described in
Materials and Methods. The Na-mediated component of
transport, derived by subtracting uptake in KCl from that in NaCl is
depicted. The mean ± SEM derived from five brush
border membrane preparations per group, each assayed in quadruplicate,
are shown. #, Effect of PTH in control mice, P <
0.0107; effect of PTH in low Pi mice, P < 0.0054.
*, Effect of diet in vehicle-treated mice, P <
0.0001; effect of diet in PTH-treated mice, P <
0.0003.
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Figure 9. Effect of PTH on brush border membrane Npt2 and
meprin immunoreactive protein in control and Pi-deprived normal mice.
Anesthetized mice, fed control and low Pi diets for 2 days, were
injected with vehicle or PTH (10 µg/100 g BW) and killed 2 h
thereafter. Renal brush border membrane vesicles were prepared from
kidney cortex, subjected to 10% SDS-PAGE, and immunoblotted as
described in Materials and Methods. Representative gels
from five brush border membrane vesicle preparations per group are
shown.
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PTH elicited a significant increase in FEIPi in
mice fed the control diet, whereas the hormone had no effect on
FEIPi in mice fed the low Pi diet (Fig. 7).
However, a significant increase in the urinary cAMP/creatinine ratio
was evident in mice fed both the control and low Pi diets in response
to PTH (Fig. 10). Urinary cAMP levels
were modestly lower in PTH-treated Pi-deprived mice than in PTH-treated
littermates receiving the control diet (Fig. 10).
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Figure 10. Effect of PTH on the urinary cAMP/creatinine
ratio in control and Pi-deprived normal mice. Anesthetized mice, fed
control and low Pi diets for 2 days, were injected with vehicle or PTH
(10 µg/100 g BW), and urine was collected 2 h thereafter.
Urinary cAMP and creatinine were assayed, and the cAMP/creatinine ratio
was determined as described in Materials and Methods.
The mean ± SEM derived from 11–18 mice/group is
shown. #, Effect of PTH in control mice, P <
0.0001; effect of PTH in low Pi mice, P < 0.0001.
*, Effect of diet in PTH-treated mice, P < 0.003.
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Mice fed both the control and low Pi diets responded to PTH
administration with a significant decrease in renal brush border
membrane Na-Pi cotransport (Fig. 8) that was accompanied by a
corresponding decrease in Npt2 protein abundance (Fig. 9). These
findings demonstrate that the Pi-deprived mice were not resistant to
the action of PTH under the conditions examined.
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Discussion
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PTH, a major regulator of renal Pi reabsorption, inhibits the
Na+-dependent flux of Pi across the brush border
membrane from the lumen into proximal tubule cells. In the present
study we demonstrate that mice homozygous for the disrupted
Npt2 gene are refractory to the action of PTH on renal brush
border membrane Na-Pi cotransport and renal Pi handling. We also show
that PTH can elicit a robust urinary cAMP response in
Npt2-/- mice, indicating that
generalized resistance to PTH cannot account for our findings in
Npt2-/- mice. Moreover, we
demonstrate that Pi-depleted normal mice respond to PTH with a decrease
in renal brush border membrane Na-Pi cotransport, indicating that Pi
deficiency per se does not account for the PTH resistance of
renal brush border membrane Na-Pi cotransport in
Npt2-/- mice. Taken together, our
data provide compelling evidence that Npt2 gene expression
is essential for PTH effects on renal Pi handling.
The present demonstration that mice devoid of Npt2 gene
expression in their proximal tubular cells are resistant to the action
of PTH on renal Pi transport suggests that PTH does not inhibit Pi
transport in more distal segments of the nephron. This idea is
consistent with previous studies in which we showed that PTH has no
effect on Na-Pi cotransport in mouse distal convoluted tubule (MDCT)
cells (31). The lack of a PTH effect on Pi transport in these cells
could not be explained by the absence of PTH receptors (32). Moreover,
forskolin and phorbol 12-myristate 13-acetate were without effect on Pi
transport in MDCT cells (31) despite their ability to stimulate the
protein kinase A and C signaling pathways (32) that mediate the effect
of PTH on Pi transport (33). We also demonstrated that MDCT cells do
not express Npt2 mRNA (31). On the basis of these findings and the
demonstration that PTH elicits the internalization of cell surface Npt2
protein in the proximal tubule (19), we suggested that the lack of Pi
transport inhibition by PTH in the distal tubule may be explained by
the absence of Npt2 gene expression in this segment of the
nephron (31).
Contrary to the idea that Npt2 is the only target for PTH is the recent
demonstration that Na-Pi cotransport in human embryonic kidney (HEK)
cells is inhibited by PTH, despite the absence of Npt2 gene
expression in these cells (34). However, the inhibition by PTH was
modest, transient, and only observed after transfection of HEK cells
with PTH/PTH-related peptide receptor complementary DNA (34).
Accordingly, the mechanism for PTH inhibition in HEK cells may differ
substantially from that elucidated under more physiological conditions
in PTH-treated intact animals (19) and in the proximal tubular,
Npt2-expressing OK cell line (20, 35, 36).
It is well known that PTH binding to its cognate receptor in the
basolateral membrane of renal proximal tubule cells stimulates cAMP
production by activation of adenyl cyclase (37) and that cAMP is a
second messenger in the PTH-mediated inhibition of renal Na-Pi
cotransport (38, 39). In the present study we demonstrate that
Npt2-/- mice, like their wild-type littermates,
are able to mount a significant increase in urinary cAMP excretion in
response to PTH. Although we did not examine the effect of
Npt2 gene knockout on renal protein kinase C signaling,
which also plays an important role in mediating the effects of PTH on
renal Pi transport (33), our cAMP data are consistent with the idea
that generalized resistance to PTH cannot account for the absence of
PTH-inhibitable Na-Pi cotransport in the homozygous mutants.
Previous studies have documented renal resistance to PTH during Pi
deprivation. PTH failed to elicit a phosphaturic response (29, 30) or
an increase in urinary cAMP excretion (29) in thyroparathyroidectomized
rats fed a low Pi diet. In contrast, a robust increase in urinary Pi
(29, 30) and cAMP (29) excretion was evident in
thyroparathyroidectomized rats stabilized on control (30) or high Pi
(29) diets. In the present study we also noted the absence of an effect
of PTH on urinary Pi excretion in Pi-deprived mice. However, PTH did
elicit a large increase in urinary cAMP excretion as well as a
significant decrease in brush border membrane Na-Pi cotransport and
Npt2 protein abundance in Pi-deprived mice. Our results are in
agreement with the findings that PTH can evoke a comparable decrease in
Na-Pi cotransport in OK cells grown in either control or low Pi medium
(40). Taken together, the data suggest that the regulation of Na-Pi
cotransport by PTH and the adaptive response to low Pi diet involve
distinct regulatory control mechanisms that lead to changes in Npt2
protein abundance in the renal brush border membrane. Although the
intracellular signaling pathways mediating the response to low Pi have
not yet been elucidated, it is of interest that a Pi response element
in the promoter of the Npt2 gene has recently been
identified (41). However, the physiological role of the Pi response
element remains to be determined.
In the present study we demonstrate that PTH elicited a significant
decrease in renal brush border membrane Na-Pi cotransport and Npt2
protein abundance in wild-type mice, but was without effect on the
renal abundance of Npt2 mRNA. However, studies in parathyroidectomized
rats demonstrated that the PTH-mediated decrease in brush border
membrane Na-Pi cotransport and Npt2 protein was accompanied by a
decrease in Npt2 mRNA abundance (19). The basis for difference in the
Npt2 mRNA data is not clear, but may be ascribed to the PTH status
[parathyroidectomized (19) vs. intact] and species of the
animals used. In any case, because the PTH-mediated reduction in Npt2
protein was far more impressive than the reduction in Npt2 mRNA (19),
and because the PTH-mediated inhibition of Pi transport is evident in
the absence of a detectable change in Npt2 mRNA abundance, it is likely
that the endocytic retrieval of Npt2 protein is the more critical step
in the inhibitory action of PTH on renal Na-Pi cotransport. In this
regard, it was recently demonstrated that renal proximal tubular cells
become depleted of Npt2 protein 60 min after PTH treatment, suggesting
that internalization of the cotransporter is rapidly followed by its
degradation (42).
Although there is considerable evidence that Npt2 is a major target for
PTH regulation (19, 20, 36), the action of PTH on Npt1 gene expression
has not been examined directly heretofore. We demonstrate that PTH
decreases neither Npt1 protein nor Npt1 mRNA abundance in
Npt2+/+ or
Npt2-/- mice. In this regard, we
also showed that Npt1 gene expression is not affected by
either Npt2 gene knockout or dietary Pi restriction (24). On
the basis of these studies we conclude that Npt1 is not a target for
the regulation of renal Pi transport by either PTH or a low Pi
diet.
In summary, we demonstrate that mice homozygous for the disrupted
Npt2 gene are refractory to the inhibitory action of PTH on
renal brush border membrane Na-Pi cotransport and renal Pi
reabsorption. These data indicate that Npt2 is the major target for PTH
regulation of renal Pi handling and suggest that the hormone does not
play a significant role in the regulation of Pi transport in more
distal segments of the nephron.
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Acknowledgments
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We thank Danielle Boulais, Claude Gauthier, and Josée
Martel for their technical support, Dr. P. A. Friedman for bovine
PTH-(1–84), Drs. H. Murer and J. Biber for the Npt2 and Npt1
antibodies, and Dr. P. Crine for the meprin antibody.
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Footnotes
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1 This work was supported by a grant from the Medical Research Council
of Canada (GR-13297 to H.S.T.). 
2 Recipient of a McGill University-Montréal Children’s Hospital
Research Fellowship.
Received December 2, 1999.
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References
|
|---|
-
Berndt TJ, Knox FG 1992 Renal regulation of
phosphate excretion. In: Seldin DW, Giebisch G (eds) The Kidney:
Physiology and Pathophysiology. Raven Press, New York, pp
2511–2532
-
Levi M, Kempson SA, Lotscher M, Biber J, Murer H 1996 Molecular regulation of renal phosphate transport. J Membr Biol 154:1–9[CrossRef][Medline]
-
Tenenhouse HS 1997 Cellular and molecular
mechanisms of renal phosphate transport. J Bone Miner Res 12:159–164[CrossRef][Medline]
-
Murer H Biber J 1997 A molecular view of proximal
tubular inorganic phosphate (Pi) reabsorption and of its regulation.
Pflugers Arch 433:379–389[CrossRef][Medline]
-
Murer H, Forster I, Hilfiker H, Pfister M, Kaissling B,
Lotscher M, Biber J 1998 Cellular/molecular control of renal
Na/Pi-cotransport. Kidney Int [Suppl] 65:S2–S10
-
Werner A, Moore ML, Mantei N, Biber J, Semenza G, Murer
H 1991 Cloning and expression of cDNA for a Na/Pi cotransport
system of kidney cortex. Proc Natl Acad Sci USA 88:9608–9612[Abstract/Free Full Text]
-
Chong SS, Kristjansson K, Zoghbi HY, Hughes MR 1993 Molecular cloning of the cDNA encoding a human renal sodium
phosphate transport protein and its assignment to chromosome
6p21.3-p23. Genomics 18:355–359[CrossRef][Medline]
-
Chong SS, Kozak CA, Liu L, Kristjansson K, Dunn ST,
Bourdeau JE, Hughes MR 1995 Cloning, genetic mapping, and
expression analysis of a mouse renal sodium-dependent phosphate
cotransporter. Am J Physiol 268:F1038–F1045
-
Magagnin S, Werner A, Markovich D, Sorribas V, Stange
G, Biber J, Murer H 1993 Expression cloning of human and rat renal
cortex Na/Pi cotransport. Proc Natl Acad Sci USA 90:5979–5983[Abstract/Free Full Text]
-
Sorribas V, Markovich D, Hayes G, Stange G, Forgo J,
Biber J, Murer H 1994 Cloning of a Na/Pi cotransporter from
opossum kidney cells. J Biol Chem 269:6615–6621[Abstract/Free Full Text]
-
Verri T, Markovich D, Perego C, Norbis F, Stange G,
Sorribas V, Biber J, Murer H 1995 Cloning of a rabbit renal Na-Pi
cotransporter, which is regulated by dietary phosphate. Am J
Physiol 268:F626–F633
-
Collins JF Ghishan FK 1994 Molecular cloning,
functional expression, tissue distribution, and in situ hybridization
of the renal sodium phosphate (Na+/Pi)
transporter in the control and hypophosphatemic mouse. FASEB J 8:862–868[Abstract]
-
Hartmann CM, Wagner CA, Busch AE, Markovich D, Biber J,
Lang F, Murer H 1995 Transport characteristics of a murine renal
Na/Pi-cotransporter. Pflugers Arch 430:830–836[CrossRef][Medline]
-
Helps C, Murer H, McGivan J 1995 Cloning, sequence
analysis and expression of the cDNA encoding a sodium-dependent
phosphate transporter from the bovine renal epithelial cell line NBL-1.
Eur J Biochem 228:927–930[Medline]
-
Custer M, Meier F, Schlatter E, Greger R, Garcia-Perez
A, Biber J, Murer H 1993 Localization of NaPi-1, a
Na-Pi-cotransporter, in rabbit kidney proximal tubules. I.
mRNA-localization by RT-PCR. Pflugers Arch 424:203–209[CrossRef][Medline]
-
Biber J, Custer M, Werner A, Kaissling B, Murer H 1993 Localization of NaPi-1, a Na/Pi cotransporter, in rabbit kidney
proximal tubules. II. Localization by immunohistochemistry. Pflugers
Arch 424:210–215[CrossRef][Medline]
-
Custer M, Lötscher M, Biber J, Murer H, Kaissling
B 1994 Expression of Na-Pi cotransport in rat kidney: localization
by RT-PCR and immunohistochemistry. Am J Physiol
266:F767–F774
-
Tenenhouse HS, Roy S, Martel J, Gauthier C 1998 Differential expression, abundance and regulation of
Na+ -phosphate cotransporter genes in murine
kidney. Am J Physiol 44:F527–F534
-
Kempson SA, Lotscher M, Kaissling B, Biber J, Murer H,
Levi M 1995 Parathyroid hormone action on phosphate transporter
mRNA and protein in rat renal proximal tubules. Am J Physiol
268:F784–F791
-
Pfister MK, Ruf I, Stange G, Ziegler U, Lederer E, Biber
J 1998 Parathyroid hormone leads to the lysosomal degradation of
the renal type II Na/Pi cotransporter. Proc Natl Acad Sci USA 95:1909–1914[Abstract/Free Full Text]
-
Pfister MF, Hilfiker H, Forgo J, Lederer E, Biber J,
Murer H 1998 Cellular mechanisms involved in the acute adaptation
of OK cell Na/Pi-cotransport to high- or low-Pi medium. Pflugers Arch 435:713–719[CrossRef][Medline]
-
Lotscher M, Kaissling B, Biber J, Murer H, Levi M 1997 Role of microtubules in the rapid regulation of renal phosphate
transport in response to acute alterations in dietary phosphate
content. J Clin Invest 99:1302–1312[Medline]
-
Beck L, Karaplis AC, Amizuka N, Hewson AS, Ozawa H,
Tenenhouse HS 1998 Targeted inactivation of Npt2 in
mice leads to severe renal phosphate wasting, hypercalciuria and
skeletal abnormalities. Proc Natl Acad Sci USA 95:5372–5377[Abstract/Free Full Text]
-
Hoag HM, Martel J, Gauthier C, Tenenhouse HS 1999 Effects of Npt2 gene ablation and low-phosphate diet on
renal Na/phosphate cotransport and cotransporter gene expression.
J Clin Invest 104:679–686[Medline]
-
Tenenhouse HS, Werner A, Biber J, Ma S, Martel J, Roy S,
Murer H 1994 Renal Na+-phosphate cotransport
in murine X-linked hypophosphatemic rickets: molecular
characterization. J Clin Invest 93:671–676
-
Laemmli UK 1970 Cleavage of structural proteins
during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
-
Tenenhouse HS, Martel J, Biber J, Murer H 1995 Effect of Pi restriction on renal Na+-Pi
cotransporter mRNA and immunoreactive protein in X-linked
Hyp mice. Am J Physiol 268:F1062–F1069
-
Tenenhouse HS Scriver CR 1979 Renal brush border
membrane adaptation to phosphorus deprivation in the Hyp/Y
mouse. Nature 281:225–227[CrossRef][Medline]
-
Steele TH 1976 Renal resistance to parathyroid
hormone during phosphorus deprivation. J Clin Invest 58:1461–1464
-
Berndt T, Onsgard MJ, Knox FG 1988 Effect of cAMP
analogue infusion on phosphate reabsorption in phosphate-deprived rats.
Am J Physiol 255:F96–F99
-
Tenenhouse HS, Gauthier C, Martel J, Gesek FA,
Coutermarsh BA, Friedman PA 1998 Na+-phosphate cotransport in mouse distal
convoluted tubule cells: evidence for Glvr-1 and Ram-1 gene
expression. J Bone Miner Res 13:590–597[CrossRef][Medline]
-
Friedman PA, Coutermarsh BA, Kennedy SM, Gesek FA 1996 Parathyroid hormone stimulation of calcium transport is mediated
by dual signaling mechanisms involving protein kinase A and protein
kinase C. Endocrinology 137:13–20[Abstract]
-
Cole JA, Eber AL, Poelling RE, Thorne PK, Forte LR 1987 A dual mechanism for regulation of kidney phosphate transport by
parathyroid hormone. Am J Physiol 253:E221–E227
-
Fernandes I, Beliveau R, Friedlander G, Silve C 1999 NaPO4 cotransport type III (PiT1) expression
in human embryonic kidney cells and regulation by PTH. Am J
Physiol 277:F543–F551
-
Malstrom K Murer H 1987 Parathyroid hormone
regulates phosphate transport in OK cells via an irreversible
inactivation of a membrane protein. FEBS Lett 216:257–260[CrossRef][Medline]
-
Pfister MF, Lederer E, Forgo J, Ziegler U, Lotscher M,
Quabius ES, Biber J, Murer H 1997 Parathyroid hormone-dependent
degradation of type II Na/Pi cotransporters. J Biol Chem 272:20125–20130[Abstract/Free Full Text]
-
Potts Jr JT, Kronenberg HM, Rosenblatt M 1982 Parathyroid hormone: chemistry, biosynthesis, and mode of action. Adv
Protein Chem 35: 323–396
-
Martin KJ, McConkey CL, Garcia JC, Montani D, Betts
CR 1989 Protein kinase A and the effects of parathyroid hormone on
phosphate uptake in opossum kidney cells. Endocrinology 125:295–301[Abstract/Free Full Text]
-
Martin KJ, McConkey CL, Jacob AK, Gonzalez EA, Khan M,
Baldassare JJ 1994 Effect of U-73,122, an inhibitor of
phospholipase C, on actions of parathyroid hormone in opossum kidney
cells. Am J Physiol 266:F254–F258
-
Quamme G, Biber J, Murer H 1989 Sodium-phosphate
cotransport in OK cells: inhibition by PTH and "adaptation" to low
phosphate. Am J Physiol 257:F967–F973
-
Kido S, Miyamoto K, Mizobuchi H, Taketani Y, Ohkido I,
Ogawa N, Kaneko, Harashima S, Takeda E 1999 Identification of
regulatory sequences and binding proteins in the type II
sodium/phosphate cotransporter NPT2 gene responsive to dietary
phosphate. J Biol Chem 274:28256–28263[Abstract/Free Full Text]
-
Lotscher M, Scarpetta Y, Levi M, Halaihel N, Wang H,
Zajicek HK, Biber J, Murer H, Kaissling B 1999 Rapid
downregulation of rat renal Na/P(i) cotransporter in response to
parathyroid hormone involves microtubule rearrangement. J Clin
Invest 104:483–494[Medline]
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Abstract
Introduction
