Am J Physiol Renal Physiol 308: F522–F534, 2015.
First published January 21, 2015; doi:10.1152/ajprenal.00386.2014.
CALL FOR PAPERS
Sex and Gender Differences in Renal Physiology
Estrogen directly and specifically downregulates NaPi-IIa through the
activation of both estrogen receptor isoforms (ER␣ and ER␤) in rat kidney
proximal tubule
Dara Burris,1 Rose Webster,1 Sulaiman Sheriff,2 Rashma Faroqui,1 Moshe Levi,3 John R. Hawse,4
and Hassane Amlal1
1
Submitted 9 July 2014; accepted in final form 5 January 2015
Burris D, Webster R, Sheriff S, Faroqui R, Levi M, Hawse
JR, Amlal H. Estrogen directly and specifically downregulates
NaPi-IIa through the activation of both estrogen receptor isoforms
(ER␣ and ER␤) in rat kidney proximal tubule. Am J Physiol Renal
Physiol 308: F522–F534, 2015. First published January 21, 2015;
doi:10.1152/ajprenal.00386.2014.—We have previously demonstrated that estrogen (E2) downregulates phosphate transporter NaPiIIa and causes phosphaturia and hypophosphatemia in ovariectomized
rats. In the present study, we examined whether E2 directly targets
NaPi-IIa in the proximal tubule (PT) and studied the respective roles
of estrogen receptor isoforms (ER␣ and ER␤) in the downregulation
of NaPi-IIa using both in vivo and an in vitro expression systems. We
found that estrogen specifically downregulates NaPi-IIa but not NaPiIIc or Pit2 in the kidney cortex. Proximal tubules incubated in a
“shake” suspension with E2 for 24 h exhibited a dose-dependent
decrease in NaPi-IIa protein abundance. Results from OVX rats
treated with specific agonists for either ER␣ [4,4=,4⬙;-(4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol, PPT] or ER␤ [4,4=,4⬙-(4-propyl-[1H]pyrazole-1,3,5-triyl) trisphenol, DPN] or both (PPT ⫹ DPN), indicated that only the latter caused a sharp downregulation of NaPi-IIa,
along with significant phosphaturia and hypophosphatemia. Lastly,
heterologous expression studies demonstrated that estrogen downregulated NaPi-IIa only in U20S cells expressing both ER␣ and ER␤,
but not in cells expressing either receptor alone. In conclusion, these
studies demonstrate that rat PT cells express both ER␣ and ER␤ and
that E2 induces phosphaturia by directly and specifically targeting
NaPi-IIa in the PT cells. This effect is mediated via a mechanism
involving coactivation of both ER␣ and ER␤, which likely form a
functional heterodimer complex in the rat kidney proximal tubule.
inorganic phosphate; postmenopause; sex steroids; proximal tubule;
hypophosphatemia.
SEVERAL CLINICAL STUDIES HAVE demonstrated that estrogen depletion (menopause) or repletion (hormone replacement therapy) is associated with changes in serum inorganic phosphate
(Pi) levels in women. Indeed, clinical studies have shown that
women treated with estrogen exhibit hypophosphatemia (2, 15,
18, 57, 59), and in some studies, a reduction in proximal
tubular Pi reabsorption was observed (12, 17, 59). Conversely,
Address for reprint requests and other correspondence: H. Amlal, Div. of
Nephrology and Hypertension, 231 Albert Sabin Way, MSB Rm. G-259,
Cincinnati, OH 45267-0585 (e-mail: [email protected]).
F522
estrogen-depleted patients showed elevated plasma levels of Pi
and increased rate of proximal tubule Pi reabsorption (2).
Kidney and intestine play an important role in the control
and maintenance of Pi homeostasis (8, 18, 20, 40). Pi enters the
body through its absorption in the intestine via an apical
sodium-dependent Pi transporters NaPi-IIb encoded by
Slc34a2 (18, 20). The involvement of kidney is more efficient
in controlling Pi metabolism, as it can reabsorb or excrete Pi
depending on the circulating levels of Pi and the needs of the
body. Indeed, most of filtered Pi is immediately reabsorbed in
the proximal tubule via the activity of several Pi transporters.
These transporters include NaPi-IIa (Slc34a1), NaPi-IIc
(Slc34a3), and Pit-2 (Slc20a2), all of which are sodiumdependent (NaPi) and expressed in the brush-border membrane
of the proximal tubule (see Refs. 9 and 40 for review).
Recently, studies demonstrated that estrogen stimulates the
expression of NaPi-IIb in the intestine (63) and inhibits phosphate uptake in brush border membrane vesicles harvested
from kidneys of estrogen-treated rats (6). Subsequently, we
demonstrated that estrogen downregulates NaPi-IIa and causes
hypophosphatemia and renal Pi wasting in ovariectomized rats
(23). We further demonstrated that the effect of estrogen on
renal Pi handling results from the downregulation of NaPi-IIa
and is independent of parathyroid hormone (PTH) levels or
changes in food intake (23).
PTH and fibroblast growth factor 23 (FGF23) are known to
regulate Pi balance through the downregulation of Pi transporters
in the proximal tubule with subsequent phosphaturia in humans,
as well as in experimental animals (7, 34, 52). Recent studies
demonstrated that estrogen stimulates the synthesis and release of
FGF23 from bone osteocytes, while decreasing the synthesis and
secretion of PTH in the parathyroid gland of rat (11). The effect
of estrogen on PTH appears to be indirect, as estrogen receptors
ER␣ or ER␤ are not expressed in the parathyroid cells and rather
results from the increase in the circulating levels of FGF23 and its
action in the parathyroid gland (11).
The majority of estrogen response is mediated through two
different isoforms of estrogen receptors (ER), usually referred
to as ER␣ and ER␤, each encoded by a separate gene (33, 38,
39, 50, 58). Immunoblot studies have shown that ER␣ and
ER␤ are expressed in the kidney of rat (61) and mice (29).
1931-857X/15 Copyright © 2015 the American Physiological Society
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Division of Nephrology and Hypertension, Department of Medicine, University of Cincinnati, Cincinnati, Ohio; 2Department
of Surgery, University of Cincinnati, Cincinnati, Ohio; 3Division of Renal Diseases and Hypertension, Department of
Medicine, University of Colorado Health Sciences Center, Denver, Colorado; and 4Department of Biochemistry and
Molecular Biology, Mayo Clinic, Rochester, Minnesota
ESTROGEN DOWNREGULATES NAPI-IIA IN THE RAT KIDNEY
MATERIALS AND METHODS
The experiments performed in these studies were approved by the
Institutional Animal Care and Use Committee of the University of
Cincinnati. Ovariectomized Sprague-Dawley rats were purchased
from Harlan (Harlan, Indianapolis, IN), housed two rats per cage with
free access to rat chow and distilled water, and maintained in a
temperature-controlled room regulated on a 12:12-h light-dark cycle
for 1 wk before and during the following treatments.
Dose-Dependent Effects of Estrogen
OVX rats were placed in metabolic cages and allowed free access
to food and water, as previously described in our laboratory (23).
After 3 or 4 days of adjustment to metabolic cages, rats were
randomly divided into several groups (n ⫽ 4 or 5 rats in each) and
injected subcutaneously with different doses of 17␤-estradiol (0.5, 5,
15, and 90 ␮g/100 g body wt/day) or vehicle (sesame oil). The
animals were injected daily and euthanized after 3 days of treatment.
Respective Roles of Estrogen Receptors ER␣ or ER␤ Using
Pharmacological Agonists
OVX rats were housed in metabolic cages and fed rodent chow and
distilled water and treated as follows: in a first set of the experiments,
rats were divided into three groups (n ⫽ 4 rats in each) with two
groups injected subcutaneously with either 150 or 500 ␮g/100 g body
wt of PPT, a specific agonist of ER␣ (26, 27, 50, 54, 57), and the third
group was injected with vehicle. Because it was expected that PPT
would cause a reduction in food intake, as previously shown by others
(49, 50), the access of vehicle group to food was restricted to the same
amount consumed by PPT-treated rats at 500 ␮g/100 g body wt
(pair-feeding). In a second set of experiments, rats were divided into
three groups (four rats in each) and injected subcutaneously with
either 150 or 500 ␮g/100 g body wt of DPN, a specific ER␤ agonist
(26, 27, 50, 54, 57), or vehicle (sesame oil). In the last experiment, a
group of rats (n ⫽ 4) was injected subcutaneously with a combination
of PPT ⫹ DPN at 150 ␮g/100 g body weight each. In all of these
experiments, rats were injected daily and killed after 3 days of
treatments. To confirm the findings of ER activation studies, the effect
of PPT or DPN at 150 ␮g/100 g body wt vs. vehicle was reevaluated
using another batch of four rats in each group, and NaPi-IIa protein
abundance was reexamined in the kidney cortex of these animals.
Our choice of 150 ␮g/100 g body wt of PPT or DPN is estimated
from studies that examined the dose-response effects of these compounds on food intake in OVX rats (26, 46, 50, 54, 57). Our doses are
slightly overestimated to account for the difference in the vehicle of
preference, i.e., sesame oil in our studies vs. DMSO in Santollo’s
work (50). To ascertain that PPT alone and DPN alone have no effect
on renal phosphate handling, we have also studied the effects of a
higher dose of 500 ␮g/100 g body wt of these compounds on food
intake and renal Pi handling.
During each of the above studies, food and water intake and urine
volume were measured daily. At the end of each experiment, rats were
euthanized and kidneys were removed. Slices of kidney cortex were
dissected and snap frozen in liquid nitrogen and stored at ⫺80°C for
total RNA and membrane protein isolation. Urine chloride concentration was measured using a chloridometer, and urine creatinine, Pi, and
calcium concentrations were determined using colorimetric assays
kits (BioAssay Systems, Hayward, CA; or BioVision, Milpitas, CA).
These parameters were measured in urine collected on the last day of
treatment. NH4⫹ concentration in culture media was measured as
previously described and used in our laboratory (1).
Effect of Estrogen on NaPi-IIa Expression In Vitro Using Proximal
Tubular Suspension
Isolation of renal proximal tubule suspension. Proximal tubule
suspensions (PTS) were prepared from 200- to 250-g ovariectomized
Sprague-Dawley rats using collagenase digestion protocol that has
been described and extensively used by others (14, 28, 31, 45, 46).
This method was used to study the regulation of Na⫹/H⫹ exchanger
(NHE3) activity in freshly isolated proximal tubule suspensions (14,
28, 31, 45, 46). We have also used PTS in our laboratory to study the
activity of basolateral Na⫹:HCO⫺
3 cotransporter in the kidney proximal tubule of control and K⫹-depleted rats (3). The passage of the
final suspension through a 70-␮m opening nylon mesh eliminates
small fragments of distal tubules and isolated cells, while enriching
the suspension with proximal tubule fragments, as shown in our
RT-PCR data depicted below (Fig. 3).
Expression of estrogen receptor isoforms in the proximal tubule
cells. Total RNA isolated from proximal tubule suspensions and
cortex slices was reverse transcribed and used as a DNA template to
examine the expression of ER␣ and ER␤ by PCR. Primers for cDNA
synthesis and PCR amplification of ER␣ are 5=-CCA TGACCATGACCCTTCACAC-3= (forward) and 5=-GAGCCTGGGAGTTCTCAG ATG G-3=(reverse), corresponding to nucleotides 208 to 2026 of
rat ER␣ (accession no. NM_012689). The primers for ER␤ are
5=-GGCTGAGCGACAACCAGTGGCTG-3= (forward) and 5=GCGTGTGAGCATTCAGCATCTC-3= (reverse) corresponding to
nucleotides 90 to 1779 of rat ER␤ (accession no. NM_012754). The
primers for sodium glucose cotransporter SGLT1 are 5=-ATGGTGTGGTGGCCGATTGG-3= (forward) and 5=-GTGTAGATGTCCATGGTGAAGAG-3= (reverse) corresponding to nucleotides 362 to
1379 of rat SGLT1 (accession no. NM_013033). The primers for
neutral amino acid transporter SNAT3 or SN1 are 5=-CTGAAGACGCCCAACACTG-3= (forward) and 5=-CAGAATGATGATGACGGATA-3= (reverse) corresponding to nucleotides 201 to 700 of
rat SNAT3 (accession no. 145776). The primers for epithelial calcium
channel (ECaC1 or TRPV5) are 5=-CCTCAAGTTCCGTGATGCC-3=
(forward) and 5=-CATTAGCCAGCAGAAGCG-3= (reverse) corresponding to nucleotides 773 to 1211 of rat ECaC1 (accession no.
XM_006236376.2). The primers for the Na⫹/Ca2⫹ exchanger (NCX)
are 5=-AATGAGCTTGGTGGCTTCACA-3= (forward) and 5=CCGCCGATACAGCAGCAC-3= (reverse) corresponding to nucleotides 2113 to 2958 of the rat NCX (accession no. XM_008764435.1).
Lastly, the primers used for GAPDH are 5=-CCCATCACCATCTTCCAGGACC-3= (forward) and 5=-CCAGTGAGCTTCCCGTTCAGC-3= (reverse) corresponding to nucleotides 286 to 758 of rat
GAPDH (accession no. NM_017008.4). PCR products were resolved
on 1.2% agarose gel and visualized with ethidium bromide staining
using Kodak Gel Logic 100 Imaging instrument and UV light transilluminator.
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Immunohistochemistry studies demonstrated that ER␣ is expressed in both proximal tubule and distal tubule of both rat
and mice kidney (29, 61). The segmental distribution of ER␤
in rat kidney was not examined in this study (61). In mice, ER␤
is also expressed in both proximal tubule and distal nephron
segments (29). However, the specificity of the antibodies used
in both studies was not demonstrated. Nevertheless, whether
estrogen receptors mediate directly or indirectly the effect of
estrogen on renal Pi transporters remains to be studied.
In the present studies, we examined the dose-response effect
of estrogen on renal Pi handling and NaPi-IIa expression and
determined the effects of estrogen on other apical Pi absorbing
transporters in the kidney proximal tubule. Further, we examined whether the mRNA of ER␣ and ER␤ is expressed in the
kidney proximal tubule cells and studied whether they mediate
a direct effect of estrogen on NaPi-IIa expression using proximal tubular suspensions. Lastly, we determined the respective
roles of ER␣ and ER␤ in estrogen-induced downregulation of
NaPi-IIa using both in vivo and in vitro experiments.
F523
F524
ESTROGEN DOWNREGULATES NAPI-IIA IN THE RAT KIDNEY
Chronic effect of estrogen on NaPi-IIa expression in vitro using
proximal tubule suspensions. The approach used for these studies was
adapted from the method of shake medullary thick ascending limb
(MTAL) suspension developed by Attmane-Elakeb et al. (4) and used
for the long-term regulation of MTAL NKCC2/BSC1 (5, 30) and
ROMK (24) in rat kidney. Briefly, Proximal tubule suspensions were
suspended in aliquot of equal parts (volume) in 60-mm cell culture
dishes containing phenol red- and serum-free DMEMF/12 medium
supplemented with 400 UI/ml penicillin, 200 mg/ml streptomycin, 11
mM NaHCO3, 5 mM leucine, 15 mM HEPES, pH adjusted to 7.40
with tris(hydroxymethyl)-aminomethane (Tris). Dishes were then
placed on the orbital shaker (Lab Line rotator orbital shaker, model
no. 1314), which is housed in a water-jacketed CO2 cell culture
incubator (model no. 2406; Shel Lab, Cornelius, OR) quipped with a
path allowing the connection of the shaker to the electrical outlet.
Tubule suspensions were, therefore, shaken at 37°C in a humid and
95% O2-5% CO2 atmosphere in the presence of different doses of
estrogen (60, 300, or 600 nM) or its vehicle (ethanol) for up to 24 h.
The viability of proximal tubule cells incubated in the above medium
was verified using trypan blue exclusion method and by measuring
their capacity to produce ammonium (NH4⫹) over a 24-h period (See
RESULTS below). At the end of treatment, PT suspensions were washed
twice with gentle centrifugation (50 g for 2 min) in the above Ringer
solution and the suspension was then frozen in liquid nitrogen until
used for membrane fractions isolation.
total protein lysates extraction. The protein concentrations were then
determined using BCA kit (Thermo Scientific, Rockford, IL).
Cloning and Generation of Rat Full-Length NaPi-IIa Plasmid and
Transfection Studies
U20S cells expressing both ER␣ and ER␤ were grown to confluence in four-well chamber slides in the above culture medium, as
previously described (43) with some modifications. Briefly, cells were
transfected with rat full-length NaPi-IIa and treated with doxycycline
in the presence of vehicle or 100 nM estrogen as described above.
After washing with TBS and fixation with 1% paraformaldehyde, cells
were blocked with TBS containing 10% normal goat serum (NGS) for
1 h. Cells were then incubated with anti-NaPi-IIa antiserum diluted at
1:50 in TBS containing 10% NGS overnight at 4°C. After washing
with TBS-containing Tween-20 (TBST), cells were incubated with
Alexa Fluor 594 goat anti-rabbit IgG in TBS containing 10% NGS for
1 h. Cells were then washed in TBST, and coverslips were mounted
using Fluoromount-G. The immunofluorescence was then revealed by
confocal microscopy at original magnification of ⫻20.
Effect of Estrogen on NaPi-IIa Expression In Vitro Using U20S
Cells Expressing ER␣ and/or ER␤
We used human female U20S osteosarcoma cells stably expressing
estrogen receptors ER␣ and/or ER␤ under the control of doxycycline
as previously described (37). U20S cells were cultured in DMEMF/12
medium containing 10% FBS, 1⫻ antibiotic/antimycotic, 5 mg/l
blasticidin, and 500 mg/l zeocin and maintained at 37°C in a 5%
CO2-95% O2 air incubator. For estrogen treatment, cells (70 – 80%
confluence) were switched to a phenol red-free medium supplemented
with 10% charcoal-stripped serum and the same antibiotics described
above and 24 h later, 100 ng/ml doxycycline was added to the cells.
After an additional 24 h, cells were transfected with rat full-length
NaPi-IIa plasmid, and treated in the same media with vehicle or 100
nM estrogen. After 24 h of treatment, cells were harvested in RIPA
buffer containing 1⫻ protease inhibitor cocktail and processed for
Total cellular RNA was extracted from renal cortex by the method
of Chomczynski and Sacchi (15), and as previously described in our
laboratory (1, 3, 23). Total RNA samples (30 ␮g/lane) were fractionated on a 1.2% agarose-formaldehyde gel and transferred to Magna
NT nylon membranes using 10⫻ sodium chloride-sodium phosphateEDTA as a transfer buffer. Hybridization was performed, according to
Church and Gilbert (16) and as previously used in our laboratory (1,
3, 23). Specific probes for various Pi transporters were generated by
RT-PCR using total RNA from rat kidney cortex.
Membrane Protein Isolation and Immunoblotting
Total cellular fraction containing plasma membrane and intracellular membrane proteins were prepared from the cortex, as previously
described (1, 3, 23). Semiquantitative immunoblotting experiments
were performed, as previously described and used in our laboratory
(1, 3, 23). Actin was used as a control constitutive protein for the
equity in protein loading in all gels. Rabbit polyclonal NaPi-IIa,
NaPi-IIc, and Pit2 antiserum were generated, characterized, and used
in Dr. Levi’s laboratory, as described before (10, 64).
Immunofluorescence
Materials
32
P-dCTP was purchased from Perkin Elmer (Boston, MA). Paper
blotting nitrocellulose membranes used for Northern hybridization
were purchased from Midwest Scientific (St. Louis, MO). 17␤Estradiol was purchased from Cayman Chemical. PPT and DPN were
obtained from Tocris (Tocris/R&D System, Minneapolis, MN). High
prime DNA-labeling kit was purchased from Roche (Roche Diagnostics, Indianapolis, IN). NaPi-IIa, NaPi-IIc, and Pit2 were provided by
Dr. Levi’s laboratory. Actin antibody was purchased from Santa Cruz
Biotechnology (Santa Cruz, Dallas, TX). Secondary antibodies donkey anti-rabbit and mouse anti-goat were purchased from Thermo
Scientific (Thermo Scientific, Rockford, IL). Progesterone and all
other chemicals were purchased from Sigma Chemical (St. Louis,
MO). Alexa Fluor 594 goat anti-rabbit IgG and Fluoromount were
purchased from Life Technologies (Grand Island, NY) and Southern
Biotech (Birmingham, AL). NGS was purchased from Vector Laboratories (Burlingame, CA).
Statistical Analysis
Semi-quantification of immunoblots and northern hybridization
band densities was determined by densitometry using a scanner
(ScanJet ADF; Hewlett Packard, Palo Alto, CA) and UN-SCAN-IT
gel software (Silk Scientific, Orem, UT) and Image Quant software
(Molecular Dynamics, Sunnyvale, CA), respectively. Data were expressed as absolute values or % of control (vehicle). Results were
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The rat kidney full-length NaPi-IIa plasmid was generated using
commercial services (GeneCopoeia, Rockville, MD). Briefly, rat fulllength NaPi-IIa sequence was generated from kidney cDNA template
by PCR using the forward primer 5=-CGAGCTGGAGCTGAGCCATAGTCAAGGAC-3= and the reverse primer 5=-ATGATCAGGACAGATGCAACTTTTAAAACTTTTTC-3= of rat NaPi-IIa mRNA
sequence (GenBank accession no. NM_0.13030.1). The PCR product
was subsequently constructed into the recombination scaffold of
shuttle vector. The sequence in shuttle clone was verified through
full-length sequencing using the following primers: forward 5=CAGCCTCCGGACTCTAGC-3= and reverse 5=-TAATACGACTCACTATAGGG-3=, and then transferred into the expression vector
pEZ-M02 through recombination (GeneCopoeia). The plasmid containing full-length rat NaPi-IIa was amplified using Escherichia coli
(Life Technologies, Carlsbad, CA) and purified using a plasmid Midi
kit (Qiagen, Valencia, CA). For transient transfection studies, U20S
cells grown in 60-mm dishes were transfected with 2 ␮g of the
full-length rat NaPi-IIa cDNA construct using Lipofectamine (Life
Technologies) or LyoVec (InvivoGen, San Diego, CA), according the
manufacturer’s instructions. The transfected cells were incubated and
treated in appropriate media, as described below.
RNA Isolation and Northern Hybridization
F525
ESTROGEN DOWNREGULATES NAPI-IIA IN THE RAT KIDNEY
B
20
18
16
14
12
10
8
6
4
2
0
*
**
30
Cl- /Creatinine x 1000
(mmol/mg/dl/day)
Food intake (g/24h)
A
25
20
15
*
10
**
5
0
0
0.5
5
15
90
0
E2, μg/100 gm BW
C
5
15
90
D
10
Pi /Creatinine
0.6
0.5
0.4
0.3
**
0.2
¶
0.1
0.0
8
NS
NS
6
4
2
0
0
0.5
5
15
90
0
E2, μg/100 gm BW
0.5
5
15
90
E2, μg/100 gm BW
presented as means ⫾ SE. Statistical significance between control and
experimental groups was determined by Student’s unpaired t-test or
one-way ANOVA, as needed using Sigma Plot software. P ⬍ 0.05
was considered significant.
RESULTS
Dose-Response Effects of Estrogen on Food Intake, Urinary
Chloride, and Pi Excretion, and on the Expression of NaPiIIa in the Kidney Cortex of OVX Rats
In our previous study, we demonstrated that a single dose
of estrogen 15 ␮g/100 g body wt administered once daily for
3 days to OVX rats caused a significant downregulation of
NaPi-IIa and phosphaturia (23). In the present work, we
examined these effects in a dose-response study as described
in METHODS. The results shown in Fig. 1A indicate that
estrogen treatment is associated with a significant reduction
in food intake at a lower dose of 0.5 ␮g/100 g body weight
(P ⬍ 0.001 vs. vehicle; Fig. 1A) and further decreased at 15
and 90 ␮g/100 g body wt (P ⬍ 0.0001 vs. vehicle, Fig. 1A).
A parallel dose-dependent decrease in chloride/creatinine
(Fig. 1B) and calcium/creatinine (Fig. 1C) excretion was
also observed in response to estrogen. However, Pi/creatinine excretion remained unchanged in response to all doses
of estrogen (Fig. 1D) despite a reduction Pi intake. Lastly,
estrogen decreased serum Pi concentration significantly
(P ⬍ 0.05, Table 2) but did not change the circulating levels
of calcium (Table 1).
The expression of NaPi-IIa was then examined in the kidney
cortex of vehicle or estrogen-treated OVX rats. The results
shown in Fig. 2A indicate that estrogen caused a significant
decrease in the mRNA expression of NaPi-IIa at 5 ␮g/100 g
body wt (⫺45%, P ⬍ 0.02; Fig. 2A) and further decreased by
63% (P ⬍ 0.001) and 52% (P ⬍ 0.001) at 15 and 90 ␮g/100
g body wt, respectively, compared with vehicle (Fig. 2A). Data
depicted in Fig. 2B indicate that NaPi-IIa protein abundance is
significantly reduced by ⬃22% (P ⬍ 0.003) at 5 ␮g/100 g
body wt by ⬃52% at 15 and 90 ␮g/100 g body wt (P ⬍ 0.001)
of estrogen, compared with vehicle (Fig. 2B).
Specificity of the Effects of Estrogen on Pi Transporters in
the Kidney
As described above, several transporters, including NaPi-IIa,
NaPi-IIc, and Pit2, are involved in Pi reabsorption in the
proximal tubule. The results shown in Table 2 indicate that the
mRNA expression levels and protein abundance of NaPi-IIc
and Pit2 in the kidney cortex of OVX rats were not altered in
response to 3 days of estrogen treatment (15 ␮g/100 g body
wt), compared with vehicle (P ⬎ 0.05; Table 2). In addition,
the mRNA expression level of Pit1 was also not affected by
estrogen (Table 2).
Table 1. Serum levels of inorganic phosphate and calcium
Pi, mM
Ca2⫹, mg/dl
Vehicle-PF
PPT, 150 ␮g/100 g
body wt
Vehicle
DPN, 150 ␮g/100 g
body wt
PPT ⫹ DPN
Estrogen, 15 ␮g/100 g
body wt
3.60 ⫾ 0.22
7.72 ⫾ 1.93
3.31 ⫾ 0.39
9.46 ⫾ 1.28
3.56 ⫾ 0.32
10.43 ⫾ 0.85
3.90 ⫾ 0.42
13.0 ⫾ 1.42
2.31 ⫾ 0.09*
10.40 ⫾ 0.80
2.63 ⫾ 0.08*
10.32 ⫾ 0.43
Data are expressed as means ⫾ SE. 4,4=,4⬙-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT) is compared to pair-fed vehicle (vehicle-PF) Vehicle group
combines vehicles for 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) and PPT⫹DPN and estrogen pooled in one vehicle group. Pi, inorganic phosphate. *P ⬍
0.05 compared with vehicle; n ⫽ 4 to 7 rats in each group.
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0.7
Ca++/Creatinine
0.5
E2, μg/100 gm BW
Fig. 1. Dose-dependent effects of estrogen on food intake,
chloride, and phosphate (Pi) excretion. Ovariectomized
(OVX) rats were placed in metabolic cages and injected
daily with vehicle (0) or indicated doses of estrogen for 3
days. Food intake (A), chloride/creatinine excretion (B),
calcium/creatinine excretion (C), and phosphate/creatinine
excretion (D). These parameters were measured on the third
day of treatment. *P ⬍ 0.001, **P ⬍ 0.0001 vs. vehicle (0).
¶P ⬍ 0.04 vs. E2 (0.5 ␮g/100 g body wt). n ⫽ 4 rats in each
group. NS, not significant vs. vehicle (0).
F526
ESTROGEN DOWNREGULATES NAPI-IIA IN THE RAT KIDNEY
A
B
E2, μg/100gm BW
E2, μg/100gm BW
0
5
15
0
90
Long
exposure
NaPi-IIa
mRNA
Short
exposure
5
15
90
NaPi-IIa
Protein
90 kDa
Actin
42 kDa
120
120
80
NaPi-IIa protein / Actin
(arbitrary unit)
100
P<0.02
60
P<0.001
P<0.001
40
20
0
0
5.0
15
90
NS
100
P<0.003
80
P<0.001
60
40
20
0
E2, μg/100 gm BW
0
0.5
5.0
15
90
E2, μg/100 gm BW
Fig. 2. Dose-response effect of estrogen on NaPi-IIa expression in the kidney. A, top: representative Northern blots showing long and short exposure of
NaPi-IIa mRNA (light bands) and GAPDH mRNA (dark band) in the renal cortex of rats injected daily for 3 days with indicated doses of estrogen vs.
vehicle (0). GAPDH was used as a constitutive gene for the control of the equity of RNA loading into Northern gels. Bottom: corresponding densitometric
analysis showing the mean of NaPi-IIa mRNA-to-GAPDH mRNA ratio (n ⫽ 4 rat in each group). B, top: representative immunoblots showing the
abundance of NaPi-IIa and actin proteins in membrane fractions isolated from renal cortex of vehicle- or estrogen-treated rats. B, bottom: average
densitometric analysis of NaPi-IIa/actin bands (n ⫽ 4 rat in each group). Each lane was loaded with 30 ␮g of total RNA or 40 ␮g of membrane proteins
from a different rat. n ⫽ 4 rats in each group.
Expression of ER Isoforms in Whole Cortex and Cortical
Tubule Suspensions of Female Rat Kidney
Effect of Long-Term Estrogen Treatment on NaPi-IIa
Expression in Cortical Tubular Suspensions In Vitro
The RT-PCR data depicted in Fig. 3 indicate the presence of
mRNA of both ER␣ and ER␤ in the kidney cortex of ovariectomized (OVX) rats (Fig. 3A). Both receptors are also
detected in a preparation of cortical tubule suspension harvested from cortex of OVX rat kidneys using collagenase
digestion, as described in METHODS. The cortical suspension is
enriched with PTS, as indicated by increased mRNA expression levels of SGLT1 and SNAT3 used as specific markers of
proximal tubule cells (Fig. 3B). As expected, distal nephron
segments are less abundant in PTS, as indicated by lower
expression levels of calcium transporters ECaC1 and NCX
(Fig. 3C), which are considered the specific makers of distal
tubule segments.
Viability of proximal tubule suspensions. PTS incubated in
serum- and phenol red-free DMEMF/12 for 24 h are viable, as
indicated by the absence of dead cells, as determined by Trypan
blue exclusion method. Further, PTS are metabolically highly
active, as indicated by their ammoniagenic capacity assessed by
measuring NH4⫹ production and accumulation in cultured media.
Indeed, the results depicted in Fig. 4 indicate that NH4⫹ production increased from 0.35 ⫾ 0.008 mM at time 0 to 1.55 ⫾ 0.026
mM (P ⬍ 0.00001; Fig. 4A) after an 18-h incubation period. Time
0 is defined here as [NH4⫹] in the medium before the addition of
PTS. After 18 h, PTS were washed by centrifugation and resuspended in fresh serum-free medium and assayed again for NH4⫹
production after 3- and 6-h incubation periods. The results indicate an increase in NH4⫹ production by PTS from 0.33 ⫾ 0.008
mM at time zero to 0.56 ⫾ 0.015 mM (P ⬍ 0.0001 vs. time zero;
Fig. 4B) and to 0.70 ⫾ 0.008 mM (P ⬍ 0.002 vs. 3 h; Fig. 4B)
after 3- and 6-h incubation periods, respectively. These data
clearly demonstrate that PTS incubated for 24 h in a serum-free
medium are viable, and highly ammoniagenic, under baseline and
unstimulated condition.
Chronic treatment of PTS by estrogen. PTS were freshly
isolated and incubated in serum- and phenol red-free DMEMF/12 in
the presence of 60, 300, and 600 nM estrogen or its vehicle for 24
h. Membrane proteins were then extracted from these suspensions
and used for immunoblotting studies. The data depicted in Fig. 4
show a significant reduction in NaPi-IIa protein abundance in PTS
in response to 300 nM (⫺50%; P ⬍ 0.05) and 600 nM (⫺80%;
Table 2. Expression levels of NaPi-IIc, Pit2, and Pit1 in the
kidney cortex
mRNA
NaPi-IIc
Pit2
Pit1
Protein
Vehicle
EST
Vehicle
EST
100 ⫾ 3.0
100 ⫾ 4.5
100 ⫾ 5.0
92 ⫾ 4.0
106 ⫾ 4.0
98 ⫾ 5.0
100 ⫾ 13
100 ⫾ 6.0
ND
97 ⫾ 10
100 ⫾ 10
ND
Data are expressed as means ⫾ SE; n ⫽ 4 rats in each group. Ovariectomized (OVX) rats were treated with estrogen (EST; 15 ␮g/100 g body weight)
or vehicle for 3 days. The mRNA and protein expression of Pi transporters
were examined with Northern hybridization and immunoblotting, respectively.
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NaPi-IIa mRNA/ 28S rRNA
(arbitrary unit)
GAPDH
mRNA
0.5
F527
ESTROGEN DOWNREGULATES NAPI-IIA IN THE RAT KIDNEY
A
B
Cortex
+
RT
-
C
PTS
+
Cortex
-
L
L
+
PTS
-
+
PTS
-
L
RT
ERα
SNAT3
GAPDH
-
+
NCX
Sglt1
ERβ
+
Cortex
ECaC
GAPDH
Fig. 3. Expression of ER␣ and ER␤ in the kidney proximal tubule. Kidneys were harvested from OVX rats, and total RNA was isolated from the cortex or
proximal tubular suspension (PTS). RNA was reverse transcribed, and resulting cDNA was PCR amplified using specific primers for ER␣ (A, top) or ER␤ (A:
bottom). Specific markers of proximal tubule cells SGLT1 (B, top) and SNAT3 (B: bottom) and distal tubule cells NCX (C, top) and ECaC1 (C, bottom) were
used to evaluate the enrichment of proximal tubule fragments in the tubular suspensions. GAPDH is used as a control for cDNA loading. In negative samples,
the reverse transcriptase was omitted in the reverse transcription reaction (RT). L: 1 kb Plus DNA ladder. The ladders for GAPDH in A and for SNAT3 in B
were rearranged to maintain consistency in the position of the ladder in the entire graph.
P ⬍ 0.003) of E2, compared with vehicle (Fig. 4, C and D).
Estrogen did not alter NaPi-IIa protein abundance in PTS at 60
nM, compared with vehicle (P ⬎ 0.05; Fig. 4D).
intake and Pi/creatinine excretion at baseline or after PPT
treatment were not significantly different between the three
groups. Lastly, PPT did not alter the protein abundance of
NaPi-IIa in the kidney cortex at either dose used (P ⬎ 0.05,
Fig. 6, A and B), compared with the pair-fed vehicle group
(vehicle-PF). PPT did not alter the serum levels of Pi or
calcium compared with vehicle-PF group (Table 1).
Effects of specific activation of ER␣ on food intake, Pi
excretion, and the expression of NaPi-IIa
The results shown in Fig. 5 indicate that specific activation
of ER␣ using 150 or 500 ␮g/100 g body wt of the ER␣-specific
agonist PPT resulted in a significant reduction in food intake
(Fig. 5, A and B), which correlated with a significant reduction
in urinary Pi/creatinine excretion (Fig. 5C), compared with
baseline levels. Both of these effects are comparable to those
obtained in pair-fed and vehicle-treated animals. Note that food
A
The data depicted in Fig. 7 indicate that specific activation of
ER␤ using 150 ␮g/100 g body wt of DPN did not affect food
intake (Fig. 7, A and B), but caused a decrease, albeit not
B
P<0.00001
1.8
Effects of Specific Activation of ER␤ on Food Intake, Pi
Excretion, and the Expression of NaPi-IIa
P<0.002
0.8
1.6
[NH4+], mM
[NH4+], mM
1.4
1.2
1.0
0.8
0.6
0.4
P<0.0001
0.6
0.4
0.2
0.2
0.0
0.0
0h
0h
18h
C
Vehicle
NaPi-IIa
protein
Actin
E2, 600 nM
NaPi-IIa protein/Actin
D
3h
6h
Vehicle (0)
E2
120
100
80
P<0.05
60
P<0.003
40
20
0
0
60
0
300
0
600
Fig. 4. Long-term effect of estrogen on NaPi-IIa
protein expression in proximal tubular suspension in
vitro. PTS was prepared and incubated in phenol redand serum-free medium on a shaker in cell culture
incubator, as described in METHODS. Equal parts of
PTS were distributed in 100-mm culture dishes and
incubated for 18 h under normal conditions. A: after
18 h, NH4⫹ produced by PTS was measured in the
medium and compared with its level in the medium
at time 0 (0 h), before the addition of PTS (n ⫽ 4
dishes in each). B: after 18 h, PTS were washed by
centrifugation and resuspended in the same medium
for up to 6 h. NH4⫹ was measured in the media after
3 and 6 h and compared with its level at time 0 (n ⫽
4 dishes in each group). C and D: PTS were treated
with vehicle (0.016% ethanol) or 60, 300, or 600 nM
of E2 and incubated for 24 h. C: representative
immunoblots showing the abundance of NaPi-IIa and
actin proteins in membrane fractions isolated from
PTS treated with vehicle or 600 nM E2. D: average
densitometric analysis of NaPi-IIa/actin bands for
each E2 dose vs. vehicle. Each lane was loaded with
40-␮g membrane proteins from a different 100-mm
culture dish. n ⫽ 4 or 5 dishes for each group in two
separate experiments. P ⬍ 0.05 and P ⬍ 0.003 vs. no
estrogen (vehicle).
E2, nM
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GAPDH
F528
ESTROGEN DOWNREGULATES NAPI-IIA IN THE RAT KIDNEY
B
20
15
*
10
*
5
*P<0.003
0
-72
-48
-24
0
24
48
Baseline
After treatment
20
PPT, 500μg
Food intake (g/24h)
Food intake (g/day)
25
72
96
C
14
12
¶
15
Pi/Cr excretion
A
**
*
10
5
10
8
**
6
4
2
0
0
Vehicle-PF
PPT-150
Vehicle-PF
PPT-500
PPT-150
PPT-500
Time (hours)
Effects of Simultaneous Activation of ER␣ and ER␤ on Food
Intake, Pi Excretion and the Expression of NaPi-IIa
The results shown in Fig. 10 indicate that simultaneous
activation of ER␣ and ER␤ using daily injections of a cocktail
containing 150 ␮g/100 g body wt of each of PPT and DPN
(PPT ⫹ DPN) for 3 days resulted in a 42% reduction in food
intake (P ⬍ 0.001; Fig. 9A) but did not decrease Pi/creatinine
excretion (P ⬎ 0.05; Fig. 9B). The results further indicate that
the mRNA expression level of NaPi-IIa is significantly reduced
(⫺40%) in PPT ⫹ DPN vs. vehicle-treated rats (P ⬍ 0.0001,
Fig. 9, C and D). Unlike PPT or DPN alone, combined
A
Vehicle-PF
PPT, 150μg
NaPi-IIa
protein
Actin
Vehicle-PF
NaPi-IIa
protein
Actin
PPT, 500μg
treatment with PPT ⫹ DPN significantly reduced the protein
abundance of NaPi-IIa (⫺44%; P ⬍ 0.001), compared with the
vehicle-treated group (Fig. 9, E and F). Lastly, PPT ⫹ DPN
treatment caused a significant reduction in serum Pi concentration (P ⬍ 0.05; Table 1), but did not alter the serum levels
of calcium (Table 1).
Respective Roles of ER␣ and ER␤ in NaPi-IIa Regulation by
Estrogen Using an In Vitro Expression System
Rationale for using bone cells (U20S) for the following
studies. The above results (Fig. 9) clearly suggest that the
downregulation of NaPi-IIa by estrogen likely requires the activation of both receptor isoforms ER␣ and ER␤. Despite several
attempts, we could not find specific and sensitive antibodies
against rat ER␣ and ER␤ to further dissect the mechanism(s) by
which estrogen downregulates NaPi-IIa in rat kidney. Most of the
commercially available antibodies were nonspecific, as shown
by others (53). Moreover, the existing proximal tubule cells
from rat (NRK), opossum (OK), and HEK (human) either do
not express both ER isoforms or express ER␣ at a very low
level by RT-PCR (data not shown). To overcome these difficulties and determine the mechanism by which both ER isoforms mediate the downregulation of NaPi-IIa, we used a
B
Vehicle-PF (0)
NaPi-IIa protein/ Actin
significant, in Pi/creatinine excretion (Fig. 7C), compared with
vehicle-treated animals. A higher dose of DPN (500 ␮g/100 g
body wt) also did not alter food intake but significantly decreased Pi/creatinine excretion by 40% (P ⬍ 0.001; Fig. 7C),
compared with vehicle-treated animals. Lastly, Fig. 8 shows
that the protein abundance of NaPi-IIa was not altered in
response to DPN at the lower dose (150 ␮g/100 g body wt), but
was rather increased (⫹40%, P ⬍ 0.02) at the higher dose (500
␮g/100 g body wt), compared with the vehicle-treated group
(Fig. 8, A and B). DPN did not alter the serum levels of Pi or
calcium compared with vehicle group (Table 1).
PPT (μg/100 gm BW)
140
120
100
80
60
40
20
0
0
150
0
500
Fig. 6. Effect of ER␣ agonist (PPT) on NaPi-IIa protein expression in the kidney cortex. Total membrane proteins were isolated from vehicle and PPT-treated
OVX rats and used for immunoblotting experiments. A: immunoblots of NaPi-IIa and actin proteins in the cortex of rats treated with PPT at 150 ␮g/100 g body
wt (top) or 500 ␮g/100 g body wt (bottom) vs. pair-fed vehicle. B: corresponding densitometric analysis of NaPi-IIa/actin bands for each dose of PPT vs.
vehicle-PF (0). Each lane was loaded with 40 ␮g of proteins harvested from a different rat kidney cortex. The PPT experiment at 150 ␮g/100 g body wt was
repeated twice (n ⫽ 8 rats in each group). n ⫽ 4 rats in each group were used for the experiment of PPT at 500 ␮g/100 g body wt.
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Fig. 5. Effects of ER␣ agonist [4,4=,4==-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol, PPT] on food intake and Pi excretion. OVX rats were housed in metabolic
cages, and after 3 days of adjustment (baseline), they were injected daily with PPT (150 or 500 ␮g/100 g body wt) or vehicle for 3 days. Vehicle group was
pair-fed with PPT-treated animals. A: time course of food intake before (baseline) and after 500 ␮g/100 g body wt PPT injection. B and C: average of 3 days
of food intake and Pi excretion/creatinine at baseline (black bars) and 3 days after treatment (gray bars). n ⫽ 4 rats in each group. *P ⬍ 0.003. **P ⬍ 0.002.
¶P ⬍ 0.05 vs. average baseline.
F529
ESTROGEN DOWNREGULATES NAPI-IIA IN THE RAT KIDNEY
A
B
DPN
14
15
10
5
12
Pi/Cr excretion
20
15
10
5
-48
-24
0
24
48
72
96
10
NS
8
*
6
4
2
0
0
0
-72
C
20
Food intake (g/24h)
Food intake (g/day)
25
Baseline
After treatment
Vehicle
DPN-150
DPN-500
Vehicle
DPN-150
DPN-500
Time (hours)
human bone cell line (U20S), which stably expresses either
ER␣ alone, ER␤ alone, or both ER␣ and ER␤ under doxycycline control (37). Most importantly, ER␣ and ER␤ have been
shown to form a functional heterodimer complex, which mediates the regulation of several target genes by estrogen in
these cells (37). These cells were transiently transfected with
rat kidney full-length NaPi-IIa construct and used for the
following experiments.
Effects of estrogen on rat NaPi-IIa expression in U20S cells.
First, Northern blotting experiment demonstrated that doxycycline treatment induced the expression of both ER␣ and ER␤
in U20S cells (Fig. 10, A and B). The induction of ER␣
precedes that of ER␤, but their mRNA expression levels is
significantly higher after 48 h of doxycycline treatment (Fig.
10, A and B). Second, immunoblotting experiment shows that
NaPi-IIa protein is expressed only in cells transfected with the
rat NaPi-IIa expression plasmid (Fig. 10C). NaPi-IIa protein in
these cells is expressed as a ⬃160-kDa band, as previously
described by other investigators in rat and mouse bone cells
(35). Third, immunofluorescence studies depicted in Fig. 11
shows that rat NaPi-IIa is expressed in the plasma membrane of
transfected and vehicle-treated U20S cells expressing both
ER␣ and ER␤ (Fig. 11, top) and that the abundance of its
A
Vehicle
DPN, 150μg
expression is significantly reduced in the presence of 100 nM
estrogen (Fig. 11, bottom). Finally, immunoblotting experiments of U20S cells incubated with 100 nM estrogen or vehicle
for 24 h show that NaPi-IIa protein abundance was not altered
by E2 in U20S cells expressing ER␣ alone (P ⬎ 0.05; Fig. 12,
A and B) or ER␤ alone (P ⬎ 0.05; Fig. 12, C and D). However,
estrogen caused a sharp decrease in the abundance of NaPi-IIa
protein (⫺80%; P ⬍ 0.01) in cells expressing both ER␣ and
ER␤ (Fig. 12, E and F), compared with the vehicle-treated
group.
DISCUSSION
We have previously demonstrated that estrogen causes phosphaturia and hypophosphatemia in ovariectomized rats (23).
This effect is mediated through the downregulation of the
brush border sodium-dependent phosphate cotransporter NaPiIIa at both the mRNA and protein levels (23). The present
studies confirm the phosphaturic and hypophosphatemic effects of estrogen and its suppression of NaPi-IIa expression in
a dose-dependent manner (Figs. 1 and 2). Increased electrolyte
wasting despite decreased food intake is likely specific to Pi, as
chloride excretion and calcium excretion correlated with re-
B
Vehicle (0)
NaPi-IIa
protein
Actin
Vehicle
NaPi-IIa
protein
Actin
DPN, 500μg
NaPi-IIa protein/ Actin
160
DPN (μg/100 gm BW)
P<0.02
140
120
100
80
60
40
20
0
0
150
0
500
Fig. 8. Effect of ER␤ agonist (DPN) on NaPi-IIa expression in the kidney cortex. Total membrane proteins were isolated from vehicle and DPN-treated OVX
rats and used for immunoblotting experiments. A: immunoblots of NaPi-IIa and actin proteins in the cortex of rats treated with DPN at 150 ␮g/100 g body wt
(top) or 500 ␮g/100 g body wt (bottom) vs. vehicle. B: corresponding densitometric analysis of NaPi-IIa/actin bands for each dose of DPN vs. vehicle (0). Each
lane was loaded with 40 ␮g proteins harvested from a kidney cortex of a different rat. The DPN experiment at 150 ␮g/100 g body wt was repeated twice (n ⫽
8 rats in each group). n ⫽ 4 rats in each group were used for the experiment of DPN at 500 ␮g/100 g body wt. P ⬍ 0.02 vs. vehicle (0).
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Fig. 7. Effects of ER␤ agonist [2,3-bis(4-hydroxyphenyl)-propionitrile, DPN] on food intake and Pi excretion. OVX rats were housed in metabolic cages and
after 3 days of adjustment (baseline), they were injected daily with DPN (150 or 500 ␮g/100 g body wt) or vehicle for 3 days. A: time course of food intake
before and after 150 ␮g/100 g body wt DPN injection. B: average of 3 days of food intake before (baseline, dark bars) or food consumed on the third day after
vehicle or DPN injection (gray bars). C: average of 3 days Pi excretion/creatinine excretion before (baseline, dark bars) and Pi excretion/creatinine excretion of
the third day after vehicle or DPN injection (gray bars). n ⫽ 4 rats in each group. *P ⬍ 0.001 vs. baseline of DPN group.
F530
ESTROGEN DOWNREGULATES NAPI-IIA IN THE RAT KIDNEY
6
Urine Pi/creatinine
18
16
14
P<0.002
12
10
8
6
4
2
5
3
2
1
0
0
Vehicle
PPT + DPN
Vehicle
D
C
100
NaPi-IIa
mRNA
GAPDH
mRNA
E
40
20
PPT + DPN
F
120
NaPi-IIa/Actin
PPT + DPN
A
80
P<0.0001
60
40
20
Vehicle
± Dox
C
-
+ +
PPT + DPN
been evident if targeted by estrogen in a chronic setting (i.e.,
3-day treatment).
Our results demonstrate the presence of both estrogen receptor isoforms (ER␣ and ER␤) in the kidney cortex and in the
proximal tubule suspension (Fig. 3). The level of expression of
these receptors is identical between cortex and PTS preparations (Fig. 3), as the expression of ER␣ and ER␤ in the whole
cortex also includes the distal tubule where estrogen has been
reported to upregulate calcium-absorbing transporters, at least,
in mice kidney (60). These data confirm the presence of ER␣
B
ERα
mRNA
100
0
duction in food intake in response to estrogen (Fig. 1). Further,
the present data clearly show that the phosphaturic effect of
estrogen is specific to the downregulation of NaPi-IIa, as the
expression of the other apical Pi-absorbing transporters in the
proximal tubules (i.e., NaPi-IIc and Pit2) was not affected by
estrogen (Table 2). We have used cortical total membrane
fractions to examine the expression of NaPi-IIa and NaPi-IIc in
these studies. Because both proteins are known to be downregulated by membrane retrieval and lysosomal degradation
(reviewed in Ref. 9), a downregulation of NaPi-IIc would have
+
60
Vehicle
Actin
+ +
P<0.0001
80
0
NaPi-IIa
protein
-
PPT + DPN
120
NaPi-IIa/GAPDH
mRNA
PPT + DPN
Vehicle
Vehicle
± Dox
NS
4
+
NaPi-IIa
Sham
ERβ
mRNA
160 kDa
NaPi-IIa
protein
42 kDa
Actin
28S rRNA
18S rRNA
Fig. 10. Expression of ER␣ and ER␤ mRNA and NaPi-IIa protein in U20S cells. Northern hybridization of ER␣ (A), ER␤ (B), and ribosomal 28S-18S RNA
in U20S cells treated with doxycycline or vehicle for indicated time. Each lane was loaded with 30 ␮g of total RNA isolated from three culture dishes (100 mm)
pooled together in one sample. C: immunoblot showing NaPi-IIa protein (⬃160 kDa) and actin bands in lysates harvested from U20S cells transfected with rat
NaPi-IIa expression construct or the empty vector (Sham). Each lane was loaded with 30 ␮g of proteins harvested from different culture dishes (60 mm) in each
group.
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Fig. 9. Effects of combined ER␣ and ER␤
agonists (PPT ⫹ DPN) on food intake, Pi excretion, and NaPi-IIa expression. OVX rats
were placed in metabolic cages and injected
with PPT ⫹ DPN (150 ␮g/100 g body wt each)
or vehicle. Averages of food intake (A) and
Pi/creatinine excretion (B) of the last day of
treatment with Vehicle or PPT ⫹ DPN.
C: Northern hybridization of NaPi-IIa and
GAPDH mRNA. E: immunoblots of NaPi-IIa
and actin proteins in the cortex of rats treated
with PPT ⫹ DPN vs. vehicle. Corresponding
densitometric analysis of NaPi-IIa mRNA/
GAPDH mRNA (D) and NaPi-IIa protein/actin
(F). Each lane was loaded with 30 ␮g of total
RNA or 40 ␮g proteins harvested from a different rat. n ⫽ 3 or 4 rats in each group. NS, not
significant.
B
Food intake (g/day)
A
ESTROGEN DOWNREGULATES NAPI-IIA IN THE RAT KIDNEY
F531
and ER␤ proteins in the proximal tubule of both mice and rat
kidneys (29, 61). Estrogen receptors are ligand-regulated transcription factors that mediate the genomic effects of different
estrogenic molecules, including 17␤-estradiol (22). Our data
(Fig. 3) are also in agreement with several ligand-binding
studies, which reported the presence of estrogen receptor in
both the cytosol and the nucleus of rat kidney cells (19, 25, 41).
These receptors are expressed mainly in S1 and S2 segments of
rat proximal tubule (19). All together, these results would
suggest that estrogen likely acts directly through these receptors and downregulates NaPi-IIa in the proximal tubule cells.
Our previous studies demonstrated that the downregulation of
NaPi-IIa and phosphaturia by estrogen are also produced in
parathyroidectomized rats, indicating that the estrogen effect
on NaPi-IIa is independent of the circulating levels of PTH
(23). In addition to PTH, FGF23 is a potent phosphaturic
hormone, as it downregulates both NaPi-IIa and NaPi-IIc
proteins in the proximal tubule (7, 34, 40, 52). Recent studies
demonstrated that estrogen treatment increased FGF23 levels
in rats (11). However, our studies clearly demonstrate that
estrogen downregulates NaPi-IIa but does not alter NaPi-IIc or
Pit2 expression levels (Table 2) in OVX rats. Further, the in
vitro studies depicted in Figs. 4 and 11 clearly demonstrate that
estrogen directly downregulates NaPi-IIa in proximal tubule
suspensions harvested from OVX rat kidneys (Fig. 4), as well
as in U20S cells expressing both ER␣ and ER␤ and transfected
with a rat NaPi-IIa expression construct (Figs. 11 and 12).
Taken all together, these data indicate that FGF23 could not
fully account for the downregulation of NaPi-IIa and subsequent phosphaturia in response to estrogen.
Altogether, this and previously published studies (6, 23)
clearly define estrogen as a new phosphaturic hormone, which
specifically targets NaPi-IIa in the rat kidney proximal tubule.
This implies that estrogen is an important regulatory hormone
of Pi metabolism. This new role of estrogen also exists in
humans and is supported by abundant clinical data in the
literature. Indeed, it has been shown that estrogen replacement
therapy in postmenopausal women reduces bone resorption and
prevents bone loss and osteoporosis (48). Such treatment is
associated with a reduction in the circulating levels of inorganic phosphate in postmenopausal women (12, 55, 56). The
phosphaturic effect of estrogen is not specific to female subjects, as a significant decrease in serum Pi levels was also
reported in male patients with metastatic prostate cancer
treated with estrogen (17). On the basis of our studies, it is
likely that the hypophosphatemic effect of estrogen observed in
these patients results from the downregulation of NaPi-IIa in
the kidney with subsequent uncompensated phosphaturia.
Interestingly, the physiological significance of the phosphaturic effect of estrogen is not clear at this time. This effect
appears to contradict the general view that this hormone is
essential for bone health. Estrogen stimulates absorption of
calcium from both the intestine and kidney and stimulates
absorption of Pi from the intestine. Hence, it is possible that
estrogen promotes bone mineralization, but also increases renal
Pi wasting to offset the increase in its intestinal absorption and
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Fig. 11. Immunofluorescence of rat NaPi-IIa in
U20S cells. U20S cells were grown to confluence
and incubated in serum- and phenol red-free medium containing doxycycline. Cells were transfected with rat NaPi-IIa full-length construct and
then treated with vehicle (top) or 100 nM E2
(bottom) for 24 h. The immunofluorescence was
then revealed by confocal microscopy at original
magnification of ⫻20 with the same settings (gain,
brightness, zoom) between groups.
F532
A
B
Vehicle
ERα
NaPi-IIa
protein
Actin
D
Vehicle
ERβ
NaPi-IIa
protein
Actin
E
F
Vehicle
U20S cells
NaPi-IIa
protein
ERα/β
Actin
E2
140
120
100
80
60
40
20
0
Vehicle
E2
Vehicle
E2
120
100
80
60
40
20
0
140
120
100
80
60
40
P<0.01
20
0
Vehicle
E2
Fig. 12. Effects of estrogen on NaPi-IIa protein abundance in U20S cells expressing ER␣, ER␤, or ER␣ and ER␤ and transfected with rat NaPi-IIa construct.
U20S cells were pretreated with doxycycline and transfected with rat NaPi-IIa full-length construct and then treated with E2 (100 nM) or vehicle for 24 h. The
Immunoblots show the abundance of NaPi-IIa and actin proteins (A, C, E) and the corresponding densitometric analysis of NaPi-IIa/actin (B, D, F) in cells
expressing ER␣ alone (A, B) or ER␤ alone (C, D) or both ER␣ and ER␤ (E, F). Each lane is loaded with 30 ␮g of protein harvested from a different cell culture
dish. Five different dishes were used for each group and in each experiment.
maintain the blood level of Pi at a very narrow physiologic
range. With this coordinated action, estrogen will minimize
Ca-PO4 precipitation in soft tissues and prevent vascular calcification and cardiovascular disease. This would suggest that
women are more prone to vascular calcification in postmenopausal life in the absence of HRT. Indeed, studies have shown
that there is an association between arterial calcification and
osteoporosis in postmenopausal women (reviewed in Refs. 21
and 32). Subsequent studies demonstrated that postmenopausal
women treated with estrogen exhibited a decrease in the levels
of coronary artery calcification (36). However, the role of Pi
excretion in this protective effect of estrogen remains to be
demonstrated.
Lastly, we examined the respective roles of ER isoforms in
estrogen-induced downregulation of NaPi-IIa and phosphaturia
in OVX rats. Our results indicate that the specific activation of
ER␣ with PPT caused a significant reduction in food intake
(Fig. 5, A and B), as previously shown by others (57). However, unlike estrogen, the decrease in food intake by PPT
correlates with a significant reduction in urinary Pi excretion in
(Fig. 5C). PPT treatment did not alter NaPi-IIa protein abundance (Fig. 6, A and B), indicating that its activity is maintained during specific activation of ER␣, which explains the
absence of phosphaturia in PPT vs. pair-fed vehicle animals
(Fig. 5C). When ER␤ was specifically activated using DPN,
food intake was not altered at any dose, whereas urinary Pi
excretion was rather reduced at a higher dose (Fig. 7). The
abundance of NaPi-IIa protein was unchanged (lower dose)
and significantly increased at a higher dose of DPN (Fig. 8, A
and B). The increase in NaPi-IIa protein abundance/activity
could account for the reduction in urinary Pi excretion observed at a higher dose of DPN (Fig. 7C). However, when the
animals were treated with a combination of PPT ⫹ DPN
(activation of both ER␣ and ER␤, respectively), a significant
reduction in food intake (Fig. 9A), phosphaturia (Fig. 9B), and
significant downregulation of NaPi-IIa at both the mRNA (Fig.
9, C and D) and protein levels (Fig. 9, E and F) were observed.
These effects of PPT ⫹ DPN reproduce the exact effects of
estrogen on feeding, renal Pi handling, and NaPi-IIa expression. The effects of estrogen on NaPi-IIa expression and Pi
handling are more pronounced than that of PPT ⫹ DPN,
indicating that estrogen is also likely acting through other
pathways, including membrane-bound receptor, such as
GPR30, in addition to nuclear receptors ER␣ and ER␤. The in
vivo findings were confirmed by in vitro experiments showing
that estrogen decreased the expression of rat NaPi-IIa protein
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U20S cells
E2
160
140
NaPi-IIa protein/ Actin
C
NaPi-IIa protein/ Actin
U20S cells
E2
NaPi-IIa protein/ Actin
ESTROGEN DOWNREGULATES NAPI-IIA IN THE RAT KIDNEY
ESTROGEN DOWNREGULATES NAPI-IIA IN THE RAT KIDNEY
ACKNOWLEDGMENTS
Parts of these studies were published in abstract form and presented at the
annual meeting of the American Society of Nephrology in Atlanta, GA, in
November 2013.
GRANTS
These studies were supported by National Institutes of Health Grant
DK-083582 to H. Amlal.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: D.B., R.W., S.S., R.F., M.L., J.R.H., and H.A.
performed experiments; D.B., R.W., S.S., R.F., and H.A. analyzed data; D.B.,
R.W., S.S., R.F., and H.A. prepared figures; D.B., R.W., S.S., R.F., M.L.,
J.R.H., and H.A. edited and revised manuscript; D.B., R.W., S.S., R.F., M.L.,
J.R.H., and H.A. approved final version of manuscript; H.A. conception and
design of research; H.A. interpreted results of experiments; H.A. drafted
manuscript.
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