Aromatase deficiency causes altered expression of molecules critical for calcium reabsorption in the kidneys of female mice *.

Abstract

Kidney stones increase after menopause, suggesting a role for estrogen deficiency. ArKO mice have hypercalciuria and lower levels of calcium transport proteins, whereas levels of the klotho protein are elevated. Thus, estrogen deficiency is sufficient to cause altered renal calcium handling.
The incidence of renal stones increases in women after menopause, implicating a possible role for estrogen deficiency. We used the aromatase deficient (ArKO) mouse, a model of estrogen deficiency, to test the hypothesis that estrogen deficiency would increase urinary calcium excretion and alter the expression of molecular regulators of renal calcium reabsorption.
Adult female wildtype (WT), ArKO, and estradiol-treated ArKO mice (n = 5-12/group) were used to measure urinary calcium in the fed and fasting states, relative expression level of some genes involved in calcium reabsorption in the distal convoluted tubule by real-time PCR, and protein expression by Western blotting or immunohistochemistry. Plasma membrane calcium ATPase (PMCA) activity was measured in kidney membrane preparations. ANOVA was used to test for differences between groups followed by posthoc analysis with Dunnett's test.
Compared with WT, urinary Ca:Cr ratios were elevated in ArKO mice, renal mRNA levels of transient receptor potential cation channel vallinoid subfamily member 5 (TRPV5), TRPV6, calbindin-D28k, the Na+/Ca+ exchanger (NCX1), and the PMCA1b were significantly decreased, and klotho mRNA and protein levels were elevated. Estradiol treatment of ArKO mice normalized urinary calcium excretion, renal mRNA levels of TRPV5, calbindin-D(28k), PMCA1b, and klotho, as well as protein levels of calbindin-D28k and Klotho. ArKO mice treated with estradiol had significantly greater PMCA activity than either untreated ArKO mice or WT mice.
Estrogen deficiency caused by aromatase inactivation is sufficient for renal calcium loss. Changes in estradiol levels are associated with coordinated changes in expression of many proteins involved in distal tubule calcium reabsorption. Estradiol seems to act at the genomic level by increasing or decreasing (klotho) protein expression and nongenomically by increasing PMCA activity. PMCA, not NCX1, is likely responsible for extruding calcium in response to in vivo estradiol hormonal challenge. These data provide potential mechanisms for regulation of renal calcium handling in response to changes in serum estrogen levels.

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Aromatase Deficiency Causes Altered Expression of Molecules Critical
for Calcium Reabsorption in the Kidneys of Female Mice*
Orhan K Öz,1,2Asghar Hajibeigi,1,2Kevin Howard,1Carolyn L Cummins,3Monique van Abel,4Rene JM Bindels,4
R Ann Word,5Makoto Kuro-o,6Charles YC Pak,2,7and Joseph E Zerwekh2,7
ABSTRACT: Kidney stones increase after menopause, suggesting a role for estrogen deficiency. ArKO mice
have hypercalciuria and lower levels of calcium transport proteins, whereas levels of the klotho protein are
elevated. Thus, estrogen deficiency is sufficient to cause altered renal calcium handling.
Introduction: The incidence of renal stones increases in women after menopause, implicating a possible role
for estrogen deficiency. We used the aromatase deficient (ArKO) mouse, a model of estrogen deficiency, to
test the hypothesis that estrogen deficiency would increase urinary calcium excretion and alter the expression
of molecular regulators of renal calcium reabsorption.
Materials and Methods: Adult female wildtype (WT), ArKO, and estradiol-treated ArKO mice (n ? 5–12/
group) were used to measure urinary calcium in the fed and fasting states, relative expression level of some
genes involved in calcium reabsorption in the distal convoluted tubule by real-time PCR, and protein expres-
sion by Western blotting or immunohistochemistry. Plasma membrane calcium ATPase (PMCA) activity was
measured in kidney membrane preparations. ANOVA was used to test for differences between groups
followed by posthoc analysis with Dunnett’s test.
Results: Compared with WT, urinary Ca:Cr ratios were elevated in ArKO mice, renal mRNA levels of
transient receptor potential cation channel vallinoid subfamily member 5 (TRPV5), TRPV6, calbindin-D28k,
the Na+/Ca+ exchanger (NCX1), and the PMCA1b were significantly decreased, and klotho mRNA and
protein levels were elevated. Estradiol treatment of ArKO mice normalized urinary calcium excretion, renal
mRNA levels of TRPV5, calbindin-D28k, PMCA1b, and klotho, as well as protein levels of calbindin-D28kand
Klotho. ArKO mice treated with estradiol had significantly greater PMCA activity than either untreated
ArKO mice or WT mice.
Conclusions: Estrogen deficiency caused by aromatase inactivation is sufficient for renal calcium loss. Changes
in estradiol levels are associated with coordinated changes in expression of many proteins involved in distal
tubule calcium reabsorption. Estradiol seems to act at the genomic level by increasing or decreasing (klotho)
protein expression and nongenomically by increasing PMCA activity. PMCA, not NCX1, is likely responsible
for extruding calcium in response to in vivo estradiol hormonal challenge. These data provide potential
mechanisms for regulation of renal calcium handling in response to changes in serum estrogen levels.
J Bone Miner Res 2007;22:1893–1902. Published online on August 20, 2007; doi: 10.1359/JBMR.070808
Key words: aromatase, calcium transport proteins, estrogen, kidney, klotho
INTRODUCTION
T
HE INCIDENCE OF kidney stones in women is lower than
in men throughout adult life. Premenopausal women
have one half the prevalence of calcium stone disease than
men of comparable age.(1,2)However, this sex difference
disappears rapidly after the onset of menopause.(3–5)By
55–60 yr of age, the prevalence of calcium stone disease is
equal in men and women and remains equal henceforth.(5)
Several studies have provided evidence that urinary cal-
cium excretion increases at menopause,(6–8)thereby in-
creasing the risk for calcium-containing stones. However,
additional changes in urinary composition (e.g., hypocitra-
turia, hyperoxaluria, hyperuricosuria) could also contribute
*Presented in part at the 24th Annual Meeting of the American
Society for Bone and Mineral Research September 20–24, 2002,
San Antonio, TX, and the 25th Annual Meeting of the American
Society for Bone and Mineral Research September 19–23, 2003,
Minneapolis, MN.
The authors state that they have no conflicts of interest.
1Department of Radiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA;2Center for Mineral Metabolism
and Clinical Research, University of Texas Southwestern Medical Center, Dallas, Texas, USA;3Howard Hughes Medical Institute,
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, USA;4Department of Physiology,
University Medical Centre Nijmegen, Nijmegen, The Netherlands;5Department of Obstetrics and Gynecology, University of Texas
Southwestern Medical Center, Dallas, Texas, USA;6Department of Pathology, University of Texas Southwestern Medical Center, Dallas,
Texas, USA;7Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
JOURNAL OF BONE AND MINERAL RESEARCH
Volume 22, Number 12, 2007
Published online on August 20, 2007; doi: 10.1359/JBMR.070808
© 2007 American Society for Bone and Mineral Research
1893

Page 2

to an increased risk for renal stones. We recently undertook
a retrospective analysis of the database from our large kid-
ney stone patient registry and identified a major role for the
increased urinary calcium excretion in postmenopausal
women with kidney stones.(6)
Nordin and Polley(7)compared urinary calcium excretion
in matched pairs of normal pre- and postmenopausal
women who had not received hormone replacement
therapy. Urine calcium/creatinine was significantly greater
in the postmenopausal women. This rise in calcium excre-
tion at the menopause could not be accounted for by the
associated rise in plasma calcium or its fractions because it
remained significant even when pre- and postmenopausal
women were matched for total, ultrafilterable, ionized, or
complexed calcium. The authors suggested that estrogens
promote tubular reabsorption of calcium, an observation
that was subsequently confirmed in a second study by the
same investigators.(8)McKane et al.(9)also examined the
effect of estrogen replacement therapy (ERT) on renal tu-
bular calcium reabsorption before and after 6 mo of
therapy in early postmenopausal women (median age, 51
yr). Their study is significant in that they directly measured
serum ultrafilterable calcium and tubular reabsorption of
calcium before and during PTH clamp. Before PTH admin-
istration, ERT had no effect on serum ultrafilterable cal-
cium. ERT increased the tubular reabsorption of calcium,
an effect ascribed to a commensurate increase in PTH and
consistent with an effect of ERT in reducing bone resorp-
tion. However, after PTH clamp, the tubular reabsorption
of calcium was greater after ERT than at baseline, suggest-
ing a direct effect of ERT in promoting increased tubular
reabsorption of calcium independent of PTH’s action.
Taken together, these observations are consistent with the
hypothesis that impaired tubular reabsorption of calcium
and renal calcium loss from estrogen deficiency could be
one factor contributing to increased stone formation in
postmenopausal women.
The mechanism by which estrogens might regulate cal-
cium reabsorption is incompletely understood. The major
part of renal tubular reabsorption of calcium occurs along
the proximal tubule and thick ascending limb by a passive
paracellular flux. The remaining calcium reabsorption oc-
curs in the distal part of the nephron and is hormonally
regulated. This process can be envisaged as a three-step
process consisting of facilitated entry across the apical
membrane mediated by an epithelial calcium channel
(TRPV5), cytosolic diffusion of calcium bound to calcium
binding proteins such as calbindin-D28k, and active extru-
sion of calcium across the basolateral membrane mediated
by PMCA and NCX1.(10)Thus, decreased expression or
function of any of these components could impair calcium
reabsorption.
The aromatase-deficient mouse is a model of estrogen
deficiency caused by inactivation of the gene responsible
for biosynthesis of estrogens.(11)In this study, we used the
ArKO mouse model to determine whether estrogen defi-
ciency, as opposed to complete gonadal failure of ovariec-
tomy models or surgically induced menopause, is sufficient
to alter renal calcium reabsorption and investigate potential
underlying mechanisms.
MATERIALS AND METHODS
Animals
The inactivated aromatase gene was maintained on the
hybrid 129SvEV/C57Bl6. Female ArKO mice were pro-
duced by breeding heterozygotes. In some studies, 10-wk-
old animals were treated three times per week with 20 ?g
estradiol/mouse, or sesame oil only (placebo) for 3 wk. All
procedures were approved by the UT Southwestern IA-
CUC.
Analytical studies
Urine was collected from wildtype (WT) and ArKO age-
matched mice (10–13 wk old) as single spontaneous voids
between 1:00 p.m. and 3:00 p.m. or in metabolic cages dur-
ing an 18-h fast. Urinary calcium was determined by atomic
absorption spectroscopy. Urinary creatinine and phospho-
rous were determined on an automated clinical system, the
Roche Mira System. Blood was harvested by closed cardiac
puncture at the time of death. Serum 1,25-dihydroxy-
vitamin D was determined by radioimmunoassay (RIA;
Gamma-B 1,25-dihydroxyvitamin D assay; Immunodiag-
nostic Systems, Fountain Hills, AZ, USA). This assay mea-
sures both D3and D2forms of 1,25-dihydroxyvitamin D. It
has a sensitivity of 8 pM, and intra- and interassay CVs of
8% and 14%, respectively. Mouse serum intact PTH(1-84)
was measured by enzyme immunoassay (EIA; Intact
Mouse PTH; Alpco Diagnostics, Salem, NH, USA). The
assay has a sensitivity of 4 pg/ml and intra- and interassay
CVs of 3.5% and 8.5%, respectively.
RT and quantitative PCR
Kidneys were powdered under liquid nitrogen (WT, n ?
12; ArKO, n ? 9; ArKO + E, n ? 6; independent samples).
The powder was homogenized in TriReagent (Sigma-
Aldrich), according to the manufacturer’s instructions, to
obtain protein, DNA, and RNA in three different phases.
Total RNA was treated with DNase to prevent contamina-
tion of genomic DNA and finally resuspended in diethyl-
pyrocarbonate-treated milliQ water. Thereafter, 2 ?g of to-
tal RNA was subjected to reverse transcription using
Moloney murine leukemia virus RT (Gibco BRL) as de-
scribed previously.(12)Expression levels of TRPV5,
TRPV6, calbindin-D28k, calbindin-D9k, plasma membrane
calcium ATPase 1b (PMCA-1b), and NCX1 mRNA were
analyzed by quantitative real-time PCR, using the ABI
Prism 7700 Sequence Detection System (PE Biosystems).
With the use of standard curves, the amount of copy num-
bers of the target genes in each sample was calculated and
expressed as a ratio to the hypoxanthine-guanine phospho-
ribosyl transferase (HPRT) gene. Primers and probes tar-
geting the genes of interest were designed using Primer
Express software (Applied Biosystems) and are listed in
Table 1.(13,14)
Expression levels of ER? and ER?
cDNA was synthesized from 2 ?g total RNA isolated
from kidney using multiscribe RT enzyme and random
hexamer primers (Superscript II; Invitrogen) in a total vol-
ÖZ ET AL. 1894

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ume of 20 ?l. Negative controls included RNA without RT.
Newly synthesized cDNA (50 ng) was used for real-time
PCR reactions. To correct for sample-to-sample variation,
mRNA amounts were normalized to cyclophilin as an en-
dogenous reference. Primers for ER? and ER? were de-
signed using Primer Express software (Table 2). Primers
that span an exon and Taqman probes were designed to
avoid amplification of residual contaminating DNA. For
each primer–probe set, serial dilutions of template were
conducted to ensure equal efficiency of amplification over a
wide range of template concentrations and to ensure that
efficiency of amplification was equal to that of cyclophilin.
Each real-time reaction included negative and positive con-
trols: cDNA (pooled from four wildtype cycling animals)
from mouse uterus and mouse bladder for ER? and ER?,
respectively. Real-time PCR reactions were conducted in
triplicate using the TaqManTM PCR Reagent Kit. The
ABI PRISMTM Sequence Detection System was used to
monitor the increase of the reporter fluorescence (Perkin-
Elmer, Applied Biosystems). Relative gene expression was
calculated using the comparative Ct method as described in
the Applied Biosystems User Bulletin 2. Expression levels
of klotho and mouse PTH receptor 1 were determined by
real-time PCR using Sybr Green and quantitated with the
ddCTmethod.(15)The primer sequences for klotho were
based on accession NM 013823. The forward primer se-
quence was 5?-AATTATGTGAATGAGGCTCTGAA-
AG-3? and the reverse primer sequence was 5?-TACGCA-
AAGTAGCCACAAAGG-3?. The primer sequences for
mPTHR1 were based on NM 011199. The forward primer
sequence was 5?-CAGGGACACTGTGGCAGATC-3?,
and the reverse primer sequence was 5?-CAAAAAATC-
CCTGGAAGGAGTT-3?. All investigators were blinded
to genotypes of the animals during performance of this
study.
Western blotting
Renal proteins were obtained from the organic phase of
a TriReagent (Sigma) extract of the kidney powder. Protein
was harvested from the organic phase by precipitation fol-
lowed by solubilization in a buffered 1% SDS solution con-
taining protease inhibitors. Protein concentration was de-
termined using a detergent compatible assay (BioRad). The
homogenates were subjected to SDS-PAGE on 12.5%
polyacrylamide gels or Tris-Tricine gels 9.5% (for calbin-
din-D9konly) followed by blotting onto PVDF membrane.
The following antibodies were used in this study: mouse
monoclonal anti-bovine calbindin-D28k(Alpha Diagnostic
International); rabbit polyclonal anti-calbindin-D9k
(Swant); and rat monoclonal anti-klotho KM2119.(16)Puri-
fied porcine intestinal calbindin-D9k was used as a standard
for anti-calbindin-D9kWestern blot (Calbiochem). Recom-
binant rat calbindin-D28kwas used as a standard for anti-
calbindin-D28kWestern blot (Swant). Recombinant klotho
was used as a standard for the anti-klotho antibody. Mem-
branes were probed with primary antibody at the following
dilutions or concentration: anti-calbindin-D28kmonoclonal
antibody (1:3000), anti-calbindin-D9k(1:1000), and anti-
klotho (3 ?g/ml) overnight at 4°C. Subsequently, the mem-
branes were probed with an appropriate horseradish per-
oxidase (HRP)-conjugated secondary antibody, and bands
were detected by ECL system (Amersham).
Immunohistochemistry
Kidneys were bisected and embedded in paraffin by stan-
dard techniques. Seven-micrometer-thick sections were
mounted on glass slides.
Anti-klotho immunohistochemistry: After deparaffiniza-
tion and rehydration of kidney sections, endogenous per-
oxidase activity was neutralized by incubating the sections
for 30 min with 0.3% H2O2in methanol. The slides were
blocked with normal rabbit serum for 20 min at room tem-
perature. Subsequently, the slides were washed several
times. The sections were incubated overnight with a rat
monoclonal antibody against recombinant klotho pro-
tein.(16)The bound primary antibody was detected using an
HRP-conjugated rabbit anti-rat IgG and DAB as substrate
(Vector Laboratories). Sections were counterstained with
2% methyl green.
Anti-PTHR1 immunohistochemistry: Deparaffinized and
rehydrated sections of mouse kidney underwent antigen
retrieval using Vector Laboratories antigen retrieval solu-
tion and the manufacturer’s suggested procedure. After a
5-min wash in PBS, the slides were treated with 0.1% Triton
X-100 in PBS for 20 min, washed, and incubated with 5 mM
glycine for 10 min. The slides were further blocked by a
20-min incubation with 3% goat serum (Vector Laborato-
ries). The sections were exposed to a 1:100 dilution of rabbit
anti-PTHR1 antibody (Affinity Bioreagents) or to PBS
(negative control) overnight at 4°C. Slides were washed for
5 min in PBS and treated with a 1:200 dilution of biotinyl-
ated goat anti-rabbit IgG (Vector Laboratories) for 45 min.
After a 5-min wash in PBS, slides were further treated with
streptavidin-FITC (1:50; Molecular Probes) for 20 min.
Slides were rinsed in PBS for 5 min, briefly rinsed in dis-
tilled water, shaken, blotted dry, and immediately cover-
slipped with a xylene-based mounting medium. Sections
were examined by confocal microscopy with a Zeiss LSM-
510 confocal microscope at a wavelength of 488 nm and
×400 magnification. Human placenta, which does not ex-
press PTHR1, was used as a negative control and subjected
to the same procedure.
PMCA enzymatic activity
PMCA activity was measured according to a previously
published method.(17)Briefly, kidneys were harvested from
WT, ArKO, and estradiol-treated ArKO mice after perfu-
sion with cold PBS containing 1 mM EDTA + 1 mM PMSF.
The kidneys were minced on ice in a hypotonic buffer con-
taining protease inhibitors. After 15 min, the fragments
were homogenized in an equal volume of 0.5 M sucrose +
0.3 M KCl + 1.5 mM EDTA. The homogenate was spun at
5000g to remove unbroken cells and mitochondria. The su-
pernatant was centrifuged at 100,000g for 1 h. The pellet
was resuspended in a solution containing 0.25 M sucrose,
0.15 M KCl, 10 mM Tris-HCl, pH 7.5, 2 mM DTT, and 20
?M CaCl2. Protein concentration was determined using the
Bradford method (Bio-Rad). Membrane vesicles (50 ?g of
membrane protein) were added to 140 ?l of medium con-
ESTROGEN AND RENAL CALCIUM REABSORPTION 1895

Page 4

taining 100 ?M CaCl2and ouabain to inhibit Na-K ATPase,
thapsigargin to inhibit SERCA pumps, oligomycin and
NaN3to inhibit any remaining contaminating mitochondria,
and sufficient EGTA to obtain a free calcium concentration
of 100 nM.45CaCl2was added at an activity of 50 ?Ci/mol
of total Ca. The reaction was started by the addition of 6
mM ATP and allowed to proceed for 10 min. Radioactive
Ca2+accumulation in the membrane preparations was de-
termined by filtration through 0.45-?m filters presoaked in
1 mM CaCl2to reduce background. The membranes were
washed twice with 1 mM CaCl2before counting in a ?-
counter.
Statistical analysis
Data are expressed as mean ± SE. Comparison of mean
values for statistical significance was accomplished by one-
way ANOVA. Posthoc analysis was performed by the two-
tailed Dunnett’s test, where the ArKO and estrogen-
treated ArKO groups were compared with the WT group.
Differences were considered significant at the p < 0.05 level.
RESULTS
Hypercalciuria and normophosphaturia in
ArKO mice
Urinary calcium, expressed relative to creatinine, was sig-
nificantly higher in ArKO mice compared with WT mice
when examined in spot urine collections (Table 2). To bet-
ter define this hypercalciuria, urine was collected under
fasting conditions. In addition, a third group representing
estradiol-treated ArKO mice was included. One-way
ANOVA disclosed a significant interaction between the
three groups (p < 0.001). Posthoc analysis disclosed that
ArKO females again showed significant hypercalciuria
compared with WT mice. Estrogen-treatment reduced uri-
nary calcium excretion to a value not significantly different
from WT. Similar results were also observed when urinary
calcium was expressed relative to 100 ml glomerular filtra-
tion (Table 1). Urinary phosphate levels, expressed relative
to creatinine, were not different between the groups (WT,
TABLE 2. URINARY CALCIUM/CREATININE RATIOS IN ArKO AND
WT MICE
Group
Ca/Cr (mean ± SE)
Spontaneous
(mg/mg)
Fasting
(mg/mg)GF corrected
WT 0.123 ± 0.020
(n ? 10)
0.181 ± 0.020
(n ? 10)
—
0.108 ± 0.022
(n ? 5)
0.277 ± 0.050
(n ? 5)
0.078 ± 0.011
(n ? 5)
0.001*, 0.7†
0.011 ± 0.002
(n ? 5)
0.034 ± 0.008
(n ? 5)
0.012 ± 0.003
(n ? 5)
0.03*, 0.9†
ArKO
ArKO + E
p
0.001*
Urine was collected as an afternoon spontaneous void or during an 18-h
fast. ArKO + E represents 20 ?g of estradiol/mouse three times per week
for 3 wk. GF-corrected values were obtained by multiplying the fasting
Ca/Cr ratios by serum Cr.
* p for ArKO vs. WT.
†p for ArKO + E vs. WT.
TABLE 1. SEQUENCES OF PRIMERS AND TAQMAN PROBES FOR QUANTITATIVE REAL-TIME PCR*
Gene
Forward primer
Reverse primer
Probe
TRPV5
5?-CGTTGGTTCTTACGGGTTGAAC-3?
5?-GTTTGGAGAACCACAGAGCCTCTA-3?
5?-TGTTTCTCAGATAGCTGCTCTTGTACTTCCTCTTT
GT-3?
TRPV6
5?-ATCCGCCGCTATGCACA-3?
5?-AGTTTTTCTCCTGAATCTTTTTCCAA-3?
5?-TTCCAGCAACAAGATGGCCTCTACTCTGA-3?
CaBP-D28K
5?-AACTGACAGAGATGGCCAGGTTA-3?
5?-TGAACTCTTTCCCACACATTTTGAT-3?
5?-ACCAGTGCAGGAAAATTTCCTTCTTAAATTCCA-3?
CaBP-D9K
5?-CCTGCAGAAATGAAGAGCATTTT-3?
5?-CTCCATCGCCATTCTTATCCA-3?
5?-CAAAAATATGCAGCCAAGGAAGGCGA-3?
PMCA1B
5?-CGCCATCTTCTGCACCATT-3?
5?-CAGCCATTGCTCTATTGAAAGTTC-3?
5?CAGCTGAAAGGCTTCCCGCCAAA-3?
NCX1
5?-TCCCTACAAAACTATTGAAGGCACA-3?
5?-TTTCTCATACTCCTCGTCATCGATT-3?
5?-ACCTTGACTGATATTGTTTTGACTATTTCATCATT
CT-3?
ER?
5?-TGCCTGGCTGGAGATTCTG-3?
5?-TGATTGGTCTCGTCTGGCGCTCC-3?
5?-GAGCTTCCCCGGGTGTTC-3?
ER?
5?-AAGCTGGCTGACAAGGAACTG-3?
5?-TGGCCCAGCCAATCATGTGCA-3?
5?-CCACAAAGCCAGGGATTTTC-3?
HPRT
5?-TTATCAGACTGAAGAGCTACTGTAAT
GATC-3?
5?-TTACCAGTGTCAATTATATCTTCAAC
AATC-3?
5?-TGAGAGATCATCTCCACCAATAACTTTTATGTCCC-3?
Cyclophilin
5?-GCTTTTCGCCGCTTGCT-3?
5?-CAACCCCACCGTGTTC-3?
5?-TCGTCATCGGCCGTGAT-3?
* PCR primers and fluorescent probes (5?FAM-3?TAMRA) were designed using the computer program Primer Express (Applied Biosystems) and purchased from Applied Biosystems or Biolegio (Malden, The
Netherlands). TRPV5 / TRPV6, epithelial Ca2+channel 1 and 2; CaBP-D28k, calbindin-D28k; CaBP-D9k, calbindin-D9k;PMCA1B, plasma membrane calcium ATPase; NCX1, sodium-calcium exchanger; ER? estrogen
receptor alpha; ER? estrogen receptor. HPRT, hypoxanthine-guanine phosphoribosyl transferase, C
ÖZ ET AL. 1896

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4.65 ± 0.73; ArKO, 4.62 ± 0.36; ArKO + E, 4.89 ± 0.66; n ?
9 per group; ANOVA, p ? 0.9). Total serum calcium was
also not significantly different between the experimental
groups (WT, 8.41 ± 0.18 mg/dl; ArKO, 8.45 ± 0.07 mg/dl;
ArKO + E, 8.68 ± 0.27 mg/dl; n ? 9 per group; ANOVA,
p ? 0.6).
PTH levels were unchanged between groups (WT, 75 ±
11; ArKO, 67 ± 10; ArKO + E, 61 ± 20 pg/ml; n ? 10 per
group; ANOVA, p ? 0.753) as were 1,25OH2D3levels
(WT, 196 ± 30 pM; ArKO, 186 ± 35 pM; ArKO + E, 123 ±
14 pM; n ? 10 per group; ANOVA, p ? 0.09).
ArKO mice express lower levels of some proteins
involved in transepithelial calcium reabsorption in
the DCT that is normalized with estradiol therapy
To determine whether the hypercalciuria was associated
with abnormal expression of proteins that mediate trans-
cellular calcium reabsorption, we performed real-time PCR
analysis of the expression of TRPV5 and 6, calbindins D28k
and D9k, PMCA1b, NCX1, and the PTH receptor isoform
PTHR1.
For the real-time PCR analysis of expression of each
protein analyzed, one-way ANOVA disclosed a significant
interaction between the three study groups (p ? 0.029 or
less), with the exception of PTHR1 (WT, 0.82 ± 0.08;
ArKO, 1.08 ± 0.11; ArKO + E, 0.99 ± 0.11; ANOVA, p ?
0.140). As expected, TRPV5 was more highly expressed
than TRPV6 for both genotypes. ArKO mRNA levels were
significantly decreased to 50–55% of WT levels for both
proteins (Fig. 1). Calbindin-D28kmRNA level was signifi-
cantly decreased in ArKO mice to ∼50% of the WT level.
The mRNA level of calbindin-D9kin ArKO mice was re-
duced to ∼70% of WT, but the difference was not signifi-
cant (p ? 0.054, WT versus ArKO). In ArKO kidneys, the
mRNA levels of the basolateral membrane proteins, NCX1
and PMCA1b, were decreased. ArKO mRNA level of
FIG. 1.
estradiol supplementation on mRNA levels
of genes encoding proteins that regulate cal-
cium transport in the kidney. RNA was pre-
pared from whole kidneys and DNase
treated to prevent contamination of genomic
DNA. cDNA was prepared, and the copy
number of the target gene was determined.
The data are expressed as the ratio (mean ±
SE) of copy number of target gene to the
copy number of HPRT or cyclophilin
(CPH). WT, wildtype (n ? 12); ArKO, aro-
matase-deficient mice (n ? 9); ArKO + E,
ArKO mice treated with estradiol (n ? 6),
20 ?g/mouse three times per week for 3 wk.
aArKO vs. WT andbArKO + E vs. WT.
Effect of aromatase deficiency and
ESTROGEN AND RENAL CALCIUM REABSORPTION 1897

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NCX1 was significantly reduced to ∼55% of WT (Fig. 1).
PMCA1b mRNA level was significantly lower in ArKO
animals, being ∼68% of WT (Fig. 1). After treatment with
estradiol, there was no significant difference in the mRNA
levels of TRPV5, calbindin-D28k, and PMCA1b between
treated ArKO mice and WT mice (Fig. 1). Whereas calbin-
din-D28kand D9kmRNA levels were increased by estradiol
therapy such that neither was significantly different from
WT, both were significantly higher than untreated ArKO
mice (ArKO versus ArKO + E, p < 0.04 by Tukey’s posthoc
analysis). NCX1 and TRPV6 mRNA levels in kidneys of
estradiol treated ArKO mice remained significantly lower
than those of the WT mice (Fig. 1).
Because estradiol acts through receptors ER? and ER?,
mRNA levels of both receptors were assessed. Normalized
mRNA level for ER? was higher in ArKO kidney than WT,
whereas normalized ER? level was lower (Fig. 1). After
estradiol treatment, there was no significant difference be-
tween ER? mRNA levels of WT and treated ArKO mice
(Fig. 1). Expression of ER? in ArKO kidney did not sig-
nificantly change with estradiol therapy (Fig. 1).
Protein levels of the calbindins in whole kidney extracts
were also assessed by Western blot analysis. PTHR1 recep-
tor protein levels and distribution were evaluated by immu-
nohistochemistry. At the protein level, calbindin-D28kwas
significantly decreased in ArKO mice (Fig. 2). Estradiol
therapy raised the levels to those of WT. The protein level
of calbindin-D9kin ArKO was not significantly different
than that of WT (p ? 0.1). Estradiol therapy raised the
mean level of calbindin-D9kin ArKO mice closer to the
WT mean. In agreement with the mRNA data, there was no
obvious difference in PTHR1 protein expression, by immu-
nofluorescent histochemistry, between the three groups
(Fig. 3). Moreover, we did not detect any difference in the
cellular distribution of the protein.
Given the resolution of hypercalciuria after estradiol
treatment of ArKO mice with a modest increase of
PMCA1b in mRNA and no normalization of NCX1, we
measured PMCA1 activity in all three experimental groups.
The interaction between groups was significant (ANOVA,
p ? 0.02). Untreated ArKO mice had lower mean levels of
PMCA activity than WT mice, but the difference was not
significant (p ? 0.08; Fig. 4). On the other hand, ArKO
mice treated with estradiol had significantly higher PMCA
activity levels compared with WT (p ? 0.04) and untreated
ArKO mice (p ? 0.01).
Increased level of klotho protein in ArKO mice
The klotho protein has recently been shown to be a regu-
lator of renal calcium handling through its regulation of
TRPV5. Therefore, we studied its expression in the experi-
mental groups. Immunohistochemistry was performed to
examine renal klotho expression. The staining was ob-
served in the distal convoluted tubule. Positive staining was
both more diffuse and consistently more intense in ArKO
kidneys (Fig. 5A). This increased expression was confirmed
by real-time PCR and Western blot analysis (Figs. 5B–5D).
The klotho standard used does not have the transmem-
brane domain and thus is smaller than the endogenous pro-
tein, accounting for the smaller apparent molecular weight
on the gels. Estradiol treatment of ArKO mice decreased
klotho expression in the kidney at both the mRNA and
protein levels (Figs. 5B–5D).
DISCUSSION
In the mammalian kidney, 90% of calcium reabsorption
occurs in the proximal tubule and cortical thick ascending
limb by paracellular diffusion. The remaining 10% takes
place in the distal convoluted tubule through active trans-
cellular transport and is believed to be responsive to cal-
cium-regulating hormones such as PTH and vitamin D. The
lack of significant change in vitamin D or PTH levels in the
ArKO model suggests that estrogen deficiency is the cause
of the hypercalciuria in the ArKO mice. The normal level
of urinary phosphorous suggests that the proximal nephron
function is intact in ArKO mice. The mechanism(s) by
which estrogens may regulate renal calcium reabsorption in
the DCT is not completely understood, but decreased ex-
pression of proteins involved in renal calcium reabsorption
may lead to increased urinary calcium. Indeed, the ArKO
mice showed hypercalciuria in both spontaneous voids or
FIG. 2.
estradiol supplementation on protein ex-
pression levels of calbindin-D28kand calbin-
din-D9k. Detergent lysates were prepared
from whole kidneys and equal amounts of
protein subjected to SDS-PAGE followed
by Western blotting. Membranes were
probed with antibody specific for calbindin-
D28k(A) or calbindin-D9k(C). Quantitative
analysis (B and D) of protein expression was
made by calculating the ratio of the band
intensity for protein of interest to that for
WT mean. WT, wildtype; ArKO, aromatase-
deficient mice, ArKO + E, ArKO mice
treated with estradiol, 20 ?g/mouse three
times per week for 3 wk.aArKO vs. WT and
bArKO + E vs. WT.
Effect of aromatase deficiency and
ÖZ ET AL. 1898

Page 7

with fasting. Although we believe that the observed hyper-
calciuria in the ArKO female is caused principally by a
renal calcium leak in the distal nephron, the current experi-
mental design does not rule out the possibility that an in-
creased renal filtered load of calcium could contribute to
the observed hypercalciuria. On the other hand, increased
intestinal calcium absorption is not expected to be contrib-
uting to an increased filtered load, because serum 1,25-
dihydroxyvitamin D concentration was observed to be nor-
mal in these mice and fasting hypercalciuria was evident.
The decreased expression of TPRV5, calbindin-D28k,
PMCA1b, and NCX1, proteins involved in transcellular re-
absorption pathway of calcium, during estrogen deficiency
as shown here is consistent with a coordinated regulation.
Our findings are reminiscent of the coordinated regulation
of the transcellular calcium reabsorption pathway after
parathyroidectomy in rats. Similar to these findings during
estrogen deficiency, van Abel et al.(18)showed that para-
thyroidectomized rats have decreased mRNA and protein
levels of TRPV5 and calbindin-D28k, and decreased mRNA
levels of NCX1. Although urine calcium was not reported
in the study of van Abel et al., serum calcium levels were
low, and presumably the animals were hypercalciuric. One
difference between the parathyroidectomized rats and the
ArKO mice is that PMCA1b mRNA levels were lower in
ArKO mice but unchanged in the rats. The cause for this
difference between animal models is not known, but
clearly, marked deficiency in either PTH or estrogens is
sufficient to cause simultaneous decrease of some proteins
involved in transcellular calcium transport.
TRPV5 is thought of as a gatekeeper of epithelial cal-
cium uptake(19,20)and was upregulated at the mRNA level
by estradiol treatment of the ArKO mouse. This finding is
similar to those of van Abel et al., who showed that estra-
diol treatment of ovariectomized rats increased renal
TRPV5 mRNA and protein expression.(14)In addition,
17?-estradiol was shown to increase renal mRNA levels of
calbindin-D28Kand the basolateral membrane calcium
pump PMCA1b. However, when similar studies were per-
formed in male 25-hydroxyvitamin D3-1?-hydroxylase-
knockout mice, only TRPV5 expression remained respon-
sive to estrogen. Combined, the two studies suggest a
coordinated regulation pattern by estradiol that is indepen-
dent of vitamin D. In our study, estradiol therapy of ArKO
mice also restored TRPV5, calbindin-D28k, and PMCA1b
mRNA levels. Because ArKO mice have normal serum
1,25-dihydroxyvitamin D concentrations, this study sup-
ports the conclusion that estrogen regulation of the gate-
keeper TRPV5, calbindin-D28k, and PMCA1b in the kidney
occurs in a coordinated fashion and can occur indepen-
dently of vitamin D.
The TRPV6 calcium channel is also expressed in the kid-
FIG. 3.
estradiol supplementation on PTHR1 recep-
tor expression. Kidneys were fixed and bi-
sected. Immunofluorescent histochemistry
was performed using negative control (no
primary antibody, panel A) or rabbit anti-
PTHR1 primary (B–D) followed by detec-
tion with biotinylated goat anti-rabbit IgG
and streptavidin-FITC. WT, wildtype;
ArKO, aromatase-deficient mice; ArKO +
E, ArKO mice treated with estradiol, 20 ?g/
mouse three times per week for 3 wk.
Effect of aromatase deficiency and
ESTROGEN AND RENAL CALCIUM REABSORPTION1899

Page 8

ney but at much lower levels compared with TRPV5. We-
ber et al.(21)studied the response of renal TRPV6 in ovari-
ectomized mice with and without estradiol treatment at
doses similar to ours. Expression was higher in the ovari-
ectomized mice. After estradiol administration, the expres-
sion level decreased to a point between that of the sham
and ovariectomized groups. Unfortunately, the authors did
not perform statistical analysis to determine the significance
of the increase but concluded that expression of TRPV6 in
the kidney is independent of estradiol levels. In contrast, we
observed significantly decreased TRPV6 expression in
ArKO mice that was unchanged with estradiol therapy.
Taken together, the results suggest that ovarian factors
other than estradiol maybe involved in regulation of renal
TRPV6 and that TRPV6 is not important for normalizing
transepithelial calcium transport after an estradiol chal-
lenge.
Although TRPV5 and TRPV6 are involved in calcium
uptake at the apical surface, PMCA1b and NCX1 are re-
sponsible for transport of calcium into the blood at the
basolateral surface. In the ArKO mice, estradiol therapy
increased the mRNA levels of PMCA1b to a point not
statistically different from WT levels, although mean levels
were still lower. Moreover, this treatment increased
PMCA1b activity significantly above that in WT and un-
treated ArKO mice. This observation is reminiscent of in
vitro studies showing estradiol can enhance the activity of
PMCA1b in a renal tubule cell line without changes in ex-
pression levels.(22)On the other hand, the same dose failed
to increase NCX1 expression. These observations, together
with normalization of urine calcium level in treated ArKO
mice, suggested that, whereas both NCX1 and PMCA1b
are important in calcium extrusion under basal conditions,
it is PMCA1b that is responsible for extruding calcium in
response to an estrogen challenge. However, we can not
exclude that NCX1 is an estrogen regulated protein be-
cause increased expression of NCX1 mRNA has been re-
ported by van Abel et al.(14)after estradiol therapy at doses
of 250 ?g/d and higher, a dose that exceeded ours by 4-fold.
Nevertheless, the observed increased enzymatic activity
and modest changes in PMCA mRNA level after estradiol
treatment seem sufficient for normal calcium efflux.
Recent reports of the glucuronidase klotho regulating
TRPV5 activity(23)prompted us to compare the levels of
klotho expression in WT and ArKO kidneys. The ArKO
mice had increased levels of the klotho protein. Clues to the
function of the klotho protein were originally discovered as
a result of random insertional mutagenesis in mice resulting
in many phenotypes seen in aging.(24)To our knowledge,
this is the first report that estrogens regulate renal klotho
expression. The increased expression of klotho in aro-
matase deficiency may be a compensatory physiological re-
sponse to increased urinary calcium to compensate for de-
creased expression of the TRPV5 gatekeeper by prolonging
its presence and activity on the surface membrane.(23)The
fact that mRNA levels decreased after estradiol therapy
suggests that estradiol regulates klotho expression at the
transcriptional level. In addition to the results shown in this
report, estradiol regulates klotho expression in the MDBK
and MDCK kidney cell lines (A Hajibeigi and OK Öz,
unpublished results, 2007). In contrast to our findings of
suppression of klotho expression by estradiol, others have
shown upregulation of klotho expression after a single in-
jection of vitamin D.(25)Although klotho-deficient mice
have been shown to have defective PTH-induced luminal
calcium reabsorption,(26)to our knowledge, the regulation
of klotho protein expression in the kidney by PTH in the
intact mouse has not been reported. Thus, klotho regula-
tion by calcitropic hormones needs further study but is
likely to be different for each hormone.
Estrogens act through two receptors, namely ER? and
ER?. Estrogen receptors have been found in the mamma-
lian kidney.(27)A prior study in ?ERKOAF-1−/−mice re-
ported mRNA levels of renal calcium transport proteins
were also decreased.(28)The ER? splice variant expressed
by these mice lacks the AF-1 domain but retains the ability
to signal through classical genomic mechanisms. To our
knowledge, there are no published studies that show
changes in expression of calcium transport proteins in the
kidneys of ER? knockout mice. Therefore, it seems that
ER? is the mediator of estrogen action at the kidney, and
regulation of the calcium reabsorption pathway by estro-
gens may not involve classical genomic signaling. One ad-
vantage to the ArKO model is the ability to study effects on
molecular expression after estradiol treatment, which al-
lowed us to determine the estrogen responsiveness of uri-
nary calcium excretion and protein expression abnormali-
ties. The facts that both ArKO and ?ERKOAF-1−/−mice
show decreased levels of the same calcium transport pro-
teins and ?ERKOAF-1−/−female mice have elevated estra-
FIG. 4.
tation on PMCA activity. Membrane vesicles (50 ?g of membrane
protein) prepared from whole kidneys were added to a buffer
containing CaCl2and inhibitors of Na-K ATPase, SERCA pumps,
and mitochondrial calcium transport.45CaCl2was added at an
activity of 50 ?Ci/mol of total Ca. The PMCA reaction was initi-
ated by the addition of 6 mM ATP, allowed to proceed for 10 min,
and terminated by filtration through 0.45-?m filters. WT, wild-
type; ArKO, aromatase-deficient mice; ArKO + E, ArKO mice
treated with estradiol, 20 ?g/mouse three times per week for 3 wk.
aArKO + E vs. WT andbArKO + E vs. ArKO.
Effect of aromatase deficiency and estradiol supplemen-
ÖZ ET AL.1900

Page 9

diol levels, whereas ArKO mice have undetectable estra-
diol levels, establish that ligand-dependent pathways acting
through ER? are important regulators of renal calcium
transport in vivo.
In summary, estrogen deficiency, produced by aromatase
inactivation, is sufficient to cause renal calcium loss. The
calcium loss is independent of changes in vitamin D and
PTH. The decreased calcium reabsorption is associated
with coordinated decreased expression of the genes for
some renal calcium transport proteins including TRPV5
and 6, calbindin-D28k, NCX1, and PMCA1b; however, nor-
mocalciuria after estradiol therapy is associated with a co-
ordinated increase in expression only for TRPV5, calbin-
din-D28k, and PMCA1b. This implies that, whereas TPRV6
and NCX1 maybe involved in basal calcium transport in the
kidney, they are not responsible for estradiol-mediated in-
creases in renal calcium reabsorption. Taken together with
previous work by other groups, we suggest that estrogen
regulates renal calcium reabsorption at the transcriptional
level through ER? acting to regulate these genes in a co-
ordinated ligand dependent fashion but the regulation may
not be solely through classical estrogen response elements.
In addition, estradiol may regulate the process through
nontranscriptional based regulatory mechanisms such as
klotho regulating activity of TRPV5 and estradiol regula-
tion of PMCA enzymatic activity. The study findings may
provide an explanation for the increased risk of nephroli-
thiasis and the observed increased incidence in females at
the menopause. Patients with congenital aromatase defi-
ciency or those being treated with aromatase inhibitors for
breast cancer may also be at increased risk for hypercalci-
uria and nephrolithiasis. The findings also suggest attention
to urinary calcium levels in patients being treated with aro-
matase inhibitors for delaying growth plate closure. Finally,
deficiency of calcitropic hormones seems to have common
and unique effects on the expression of renal calcium trans-
port proteins.(29)Understanding the molecular basis for dif-
ferences in expression of calcium transport proteins after
parathyroidectomy, aromatase inactivation, or in vitamin D
insufficiency may be helpful in understanding the complex
physiological regulation of renal calcium reabsorption in
the distal convoluted tubule leading to improved health
care for patients at risk for nephrolithiasis as well as bone
loss.
ACKNOWLEDGMENTS
The authors thank Alexia Thomas for colony manage-
ment and genotyping animals, Jan Koska and Kathy Rodg-
ers for analytical studies on urine, Alan Stewart for serum
analysis, Patrick Keller for performing quantitative real-
time RT-PCR of the ERs, Angie Bookout for designing the
klotho primers, Jocelyn Chafouleas for manuscript prepa-
ration, and Virginia Barnett, Glenn Katz, and Dorothy
Smith for graphics support. This work was supported in part
by a grant from the seed account of the Center for Mineral
Metabolism and Clinical Research at UT Southwestern
Medical Center, in part by NIH HD01463, P01DK20543,
and unrestricted funds from the endowment of the Effie
and Wofford Cain Distinguished Chair in Diagnostic Imag-
ing.
FIG. 5.
estradiol supplementation on klotho expres-
sion. (A) Immunohistochemical analysis.
Kidneys were fixed and bisected. Immuno-
histochemistry (a–f) was performed using a
rat monoclonal anti-klotho (a and d); no pri-
mary antibody (b and e); and H&E stain of
renal tissue (c and f). The magnification bar
represents 50 ?m. (B) Anti-klotho Western
blot. Detergent lysates were prepared from
whole kidneys and subjected to SDS-PAGE
followed by Western blotting. The blot was
probed with rat anti-klotho antibody fol-
lowed by quantitative analysis of the West-
ern blot. (C) Real-time PCR analysis of
klotho expression. WT, wildtype; ArKO,
aromatase-deficient mice; ArKO + E, ArKO
mice treated with estradiol, 20 ?g/mouse
three times per week for 3 wk.aArKO vs.
WT andbArKO + E vs. WT.
Effect of aromatase deficiency and
ESTROGEN AND RENAL CALCIUM REABSORPTION 1901

Page 10

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Address reprint requests to:
Orhan K Öz, MD, PhD
Department of Radiology
UT Southwestern Medical Center at Dallas
5323 Harry Hines Boulevard
Dallas, TX 75390-9153, USA
E-mail: Orhan.Oz@UTSouthwestern.edu
Received in original form October 26, 2006; revised form August 2,
2007; accepted August 10, 2007.
ÖZ ET AL. 1902