0013-7227/99/$03.00/0
4
Endocrinology
Copyright © 1999 by The Endocrine Society
Role of Thyroid Hormone in Regulation of Renal
Phosphate Transport in Young and Aged Rats*
ANA I. ALCALDE, MANUEL SARASA, DEMETRIO RALDÚA, JOSÉ ARAMAYONA,
ROSA MORALES, JÜRG BIBER, HEINI MURER, MOSHE LEVI, AND
Vı́CTOR SORRIBAS
Departments of Physiology (A.I.A.), Anatomy (M.S., D.D.), Toxicology (R.M., V.S.), and Pharmacology
(J.A.), University of Zaragoza, 50013 Zaragoza, Spain; Institute of Physiology (J.B., H.M.), University
of Zürich-Irchel, CH-8057 Zürich, Switzerland; and Department of Internal Medicine (M.L.),
University of Texas Southwestern Medical Center and Department of Veterans Affairs Medical Center,
Dallas, Texas 75216
ABSTRACT
In the present study, we have examined the cellular mechanisms
mediating the regulation of renal proximal tubular sodium-coupled
inorganic phosphate (Na/Pi) transport by thyroid hormone (T3) in
young and aged rats. Young hypothyroid rats showed a marked de-
crease in Na/Pi cotransport activity, which was associated with par-
allel decreases in type II Na/Pi cotransporter (NaPi-2) protein and
messenger RNA (mRNA) abundance. In contrast, administration of
long-term physiological and supraphysiological doses of T3 resulted in
significant increases in Na/Pi cotransport activity, protein, and
mRNA levels. Nuclear run-on experiments indicated that thyroid
hormone regulates NaPi-2 mRNA levels by a transcriptional mech-
H IGHER ORGANISMS use inorganic phosphorus (Pi)
for several vital functions, including bone matrix and
phospholipid synthesis, blood buffering, intracellular signal
transduction, synthesis of energetic bonds in nucleotides,
and others. These important and multiple functions imply
that organisms must have precise and efficient mechanisms
for controlling Pi homeostasis. Intestinal absorption and re-
nal excretion/reabsorption of Pi are the two main targets for
the mechanisms of control (e.g. Refs. 1 and 2). Whereas in-
testinal absorption has only a role on long-term regulation of
Pi homeostasis, the control of renal excretion/reabsorption is
important for both short-term and long-term regulation of Pi
homeostasis (3).
Short-term regulation of Pi reabsorption is mainly medi-
ated by PTH and alterations in dietary Pi intake, both mech-
anisms involving shuttling or recycling of Na/Pi cotrans-
porter-containing vesicles between the cytoplasm and the
brush border membrane (BBM). Long-term regulation in-
cludes many different hormonal and nonhormonal mecha-
nisms that alter the cell content of the specific messenger
RNA (mRNA) (3). To understand properly the complex net-
work controlling Pi renal excretion/reabsorption, it is im-
Received September 9, 1998.
Address all correspondence and requests for reprints to: Vı́ctor Sor-
ribas, Ph.D., Departamento de Toxicologı́a, Facultad de Veterinaria,
Universidad de Zaragoza, Calle Miguel Servet, 177, E-50013 Zaragoza,
Spain. E-mail: sorribas@posta.unizar.es.
* This work was supported by Grant PB93– 0585 from the Spanish
Minister of Education and Science (to V.S.).
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Vol. 140, No.
Printed in U.S.A.
anism. In aged rats, although there were no changes in T3 serum
levels (when compared with young animals), there were significant
decreases in serum Pi concentration, renal Na/Pi cotransport activity,
and NaPi-2 protein and mRNA abundance. These effects were me-
diated, at least in part, by a reduction in the transcriptional rate of
the NaPi-2 gene, probably caused by, among other factors, a smaller
response to the stimulatory action of T3. Compared with young rats,
the old rats exhibited less sensitivity of the Na/Pi cotransporter to
thyroid hormone, with decreased effects in both hypothyroid (inhib-
itory) and hyperthyroid (stimulatory) animals. (Endocrinology 140:
1544 –1551, 1999)
portant to determine precisely the physiological role of each
one of these regulatory mechanisms.
Because Pi is intensively used in general metabolism, Pi
homeostasis should be regulated by factors controlling the
rate of metabolism itself. One such factor is thyroid hormone,
and its role in Pi reabsorption regulation has been extensively
analyzed (4 – 6). Pharmacological doses of T3 have been
shown to increase Na/Pi cotransport in BBM vesicles from
rat proximal tubules (4, 5). In addition, T3 concentrations
approximating the association constant (Km) of the thyroid
hormone nuclear receptor also elicited a similar increase in
Pi transport in opossum kidney (OK) cells (6). In both cases,
the increase in transport rate was caused by an increase in the
capacity of the transport system, whereas the affinity was not
modified. Recently, Euzet et al. (8, 9) have shown an impor-
tant role for T3 in the maturation of the renal Na/Pi cotrans-
porter, which was associated with changes in both Km and
Vmax, as well as in the recently cloned type II Na/Pi cotrans-
porter (NaPi-2) (7) protein and mRNA abundance.
In the present study, we have determined the role of phys-
iological concentration of thyroid hormone in renal phos-
phate transport in vivo; more exactly, the molecular mecha-
nisms of enhancement of phosphate reabsorption by thyroid
hormone. In addition, we have tried to determine the po-
tential role of thyroid hormone in impairment of phosphate
reabsorption that accompanies the aging kidney. Our results
show that chronically treated hypothyroid rats, using a phys-
iological dose of T3, exhibit increases in phosphatemia,
NaPi-2 mRNA and protein content, and Na/Pi cotransport in
 THYROID HORMONE MODULATES PHOSPHATE TRANSPORT
superficial and juxtamedullary renal cortex, all these effects
by means of enhanced transcription of the corresponding
NaPi-2 gene. The stimulatory effect of the hormone is less
evident in the aging kidney, which shows a lower level of
basal phosphate reabsorption. In our experimental design,
only pharmacological hyperthyroidism is able to restore par-
tially the level of phosphatemia observed in young animals.
Materials and Methods
Animals and experimental designs
All studies were conducted in accordance with the European Union
legislation and the NIH Guide for the Care and Use of Laboratory
Animals. The experiments were performed with 3-month-old (young) or
24-month-old (old) male Wistar rats. Animals were thyroparathyroid-
ectomized (TPTX) in our laboratory and were left for 1 month in their
cages to reach an approximately zero concentration of T3 in blood serum
and kidney. These animals were drinking water containing calcium
gluconate (350 mg Ca21/liter). After this time, rats were divided into
four groups: group A, nonoperated control rats; group B, TPTX-hypo-
thyroid rats, treated during 20 days with sc 21-day release placebo
pellets of T3, obtained from Innovative Research of America(IRA, Sara-
sota, FL); group C, TPTX-euthyroid rats, treated during 20 days with a
physiological dose of T3, 0.5 mg/100 mg BWzday by using sc pellets of
21-day release (IRA); group D, TPTX-hyperthyroid rats, treated for 20
days with 5 times the physiological dose, 2.5 mg T3/100 mg BWzday, with
sc pellets (IRA). In some experiments, a treatment with 10 times the
physiological dose of T3 was also performed (group E, 5 mg T3/100 mg
BWzday). In the experiments with old animals, group C was excluded,
because we did not find differences with group A old animals; instead,
group E was always used in experiments with old rats. Normal levels
of T3 in kidneys of hypothyroid rats are reached after 12 days of treat-
ment with 0.5 mg T3/100 mg BWzday (personal communication,
G. Morreale de Escobar, Institute for Biomedical Research-Consejo Su-
perior de Investigaciones Cientifica, Madrid, Spain). On the day of the
experiment, 24-h urine was collected, the animals were euthanatized by
CO2 narcosis, and blood was drawn from the aorta. TPTX condition was
systematically checked by postmortem inspection of the thyroid gland,
weight changes of the animals, and RIA of T3 and T4 serum levels. Urine
and serum Pi, calcium, and creatinine were measured as previously
described (10). Fractional excretion of Pi (FEPi) was calculated as (UPi/
SPi)/(UCr/SCr), where U is urine, S is serum, and Cr is creatinine.
Thyroid hormone assays
T3 was measured in whole plasma by specific and highly sensitive
RIA, as described (11). The standard curve is prepared using plasma
from severely hypothyroid thyroidectomized rats, the limit of detection
being 15 ng T3/dl.
BBM preparations
On the day of the experiment and after CO2 narcosis, blood was
drawn from the aorta, and the kidneys were rapidly removed. Thin slices
were cut, at 4 C, from the superficial and juxtamedullary cortex and were
homogenized with a Disperser DIAX 600 (Heidolph, Kelheim, Ger-
many) in a buffer consisting of the following (in mm): 300 DL-mannitol,
5 EGTA, 0.5 phenylmethylsulfonyl fluoride, and 16 HEPES (pH 7.5 with
Tris). BBM were purified from this homogenate by Mg21 precipitation
and differential centrifugation, as described (12). The final pellet was
resuspended in a buffer of 300 mm mannitol and 16 mm HEPES-Tris (pH
7.5). Purity of BBM preparations was enzymatically assayed as described
(12).
Transport assays
Sodium gradient-dependent phosphate transport (Na/Pi cotrans-
port) measurements were performed, in freshly isolated BBM vesicles,
by uptake of 0.1 mm PO4 (a mix of K2HPO4 plus KH2PO4, pH 7.4), plus
K2H32PO4 (DuPont NEN Research Products, Boston, MA) as radio tracer
1545
(4 mCi/ml uptake medium, 3,000 Ci/mmol) and an inwardly directed
sodium gradient (120 mm NaCl), followed by rapid filtration. Uptake
was measured at 10 sec, which still represents the initial linear phase of
phosphate transport at 25 C.
SDS-PAGE and immunoblots
Aliquots of superficial and juxtamedullary BBM vesicles were dena-
tured for 2 min at 95 C in 2% SDS, 10% glycerol, 0.5 mm EDTA, and 95
mm Tris-HCl (pH 6.8) (final concentrations), and 10 mg BBM protein per
lane were separated on 10% polyacrylamide gels according to the
method of Laemmli (13) and electrotransferred on nitrocellulose paper
(14). Immunodetection with antiserum against NaPi-2 (15) and sodium-
sulfate cotransporter NaSi-1 (16) was performed by chemiluminescence
using the BM Western Blotting Kit from Boehringer Mannheim (Mann-
heim, Germany) and visualized with x-ray films (Hyperfilm MP) from
Amersham International (Buckinghamshire, UK). Image analysis and
quantification were done with a Gel Doc 1000 Video Gel Documentation
System (Bio-Rad Laboratories, Inc., Hercules, CA).
Immunohistochemistry
These experiments were performed as described (10). Briefly, anes-
thetized rats were perfused retrogradely with a fixative of paraformal-
dehyde and picrinic acid, and 5-mm-thick sections were cut at 220 C
with a cryostat. Anti-NaPi-2 antibody was used at 1:500 dilution in
PBS/milk powder buffer and detected with goat antirabbit IgG antibody
linked to Cy2 (Amersham International) at 1:200 dilution. In some ex-
periments, b-actin was visualized with rhodamin-conjugated phalloidin
(Sigma Chemical Co., St. Louis, MO) at 1:500 dilution in PBS/milk
powder. Sections were coverslipped using mounting media plus 2.5%
1,4-diazabi-cyclo{2.2.2}octane (DABCO; Sigma Chemical Co.) as a fading
retardant and studied with epifluorescence microscopy using a narrow-
band filter system for fluorescein isothiocyanate (BX60, Olympus Op-
tical Co., Tokyo, Japan) or a confocal Zeiss LSM 410 (Carl Zeiss, Jana,
Germany).
RNA isolation and Northern blotting
Superficial and juxtamedullary cortex were cut out of the kidney at
4 C and homogenized with a Disperser DIAX 600 in a denaturation
solution containing 4 m guanidium thiocyanate, 25 mm sodium citrate
(pH 7.0), 0.5% sarcosyl, and 0.1 m 2-mercaptoethanol. RNA was ex-
tracted by the guanidium thiocyanate-phenol acid-chloroform method,
as described (10, 17). Twenty micrograms of total RNA were denatured,
electrophoresed in a formaldehyde agarose gel, transferred onto Hy-
bond N1 nylon membranes (Amersham International) and UV cross-
linked (UVC 500, Hoefer Pharmacia Biotech Inc., San Francisco, CA).
Full-length complementary DNA (cDNA) probes of NaPi-2, NaSi-1,
b-actin, and 18S ribosomic RNA were labeled with [a-32P]-deoxycyti-
dine triphosphate (3,000 Ci/mmol) by random priming (RadPrime DNA
Labeling System; Gibco BRL-Life Technologies, Grand Island, NY), and
hybridization was carried out at high stringency, as described (18).
Signals were quantified by image analysis with Gel Doc 1000 Video Gel
Documentation System (Bio-Rad Laboratories, Inc.) after exposure to
x-ray films (Hyperfilm MP).
Nuclear run-on
Cell nuclei were isolated from superficial and juxtamedullary cortex,
as described (19). Briefly, 0.5 g tissue was mixed with 5 ml lysis buffer
(0.32 m sucrose, 5 mm CaCl2, 3 mm magnesium acetate, 0.1 mm EDTA,
0.1% Triton X-100, 1 mm dithiothreitol (DTT), 1 mm Tris-Cl, pH 8.0) and
homogenized in a loose-fitting pestle. After filtration through several
layers of cheesecloth, the filtrate was rehomogenized with 10 strokes in
a tightly fitting pestle. The homogenate was mixed with 1 vol 2 m sucrose
solution (2 m sucrose, 3 mm magnesium acetate, 0.1 mm EDTA, 1 mm
DTT, 10 mm Tris-Cl, pH 8.0) and centrifuged over a cushion of the same
solution at 30,000 3 g for 45 min at 4 C. Nuclei were resuspended in
glycerol storage buffer (40% glycerol, 5 mm magnesium acetate, 0.1 mm
EDTA, 5 mm DTT, 50 mm Tris-Cl, pH 8.0) at 100 3 106 nuclei/ml.
Usually, about 30 million nuclei were obtained per 0.5 g renal tissue.
Labeling of nascent RNA transcripts was performed exactly as described
1546 THYROID HORMONE MODULATES PHOSPHATE TRANSPORT Endo • 1999
Vol 140 • No 4

(19). A final heterogeneous RNA probe of 8 –10 3 106 cpm/2 ml hy-
bridization solution [10 mm N-Tris[hydroxymethyl]methyl-2-aminoeth-
anesulfonic (TES; pH 7.4), 10 mm EDTA, 0.2% SDS, 300 mm NaCl] was
incubated at 65 C for 36 h with single nylon strips containing 5 mg of each
denatured cDNA (NaPi-2, NaSi-1, b-actin, and linearized pBluescript as
negative control) and 10 mg 18S, that were fixed by slot blotting and UV
cross-linking. After posthybridization washes and ribonuclease A treat-
ment, nylon strips were exposed to BioMax MS films and their corre-
sponding intensifying screens. Specific signals were quantified using the
Gel Doc 1000 Video Gel Documentation System (Bio-Rad Laboratories,
Inc.).
Statistical analysis
All the data are expressed as mean 6 se. A one-way ANOVA, with
Fisher’s protected least-significant difference as a multiple-comparison
method, was used to compare data among groups and was considered
statistically significant at P , 0.05.
Results
Thyroid hormone increases renal reabsorption of
inorganic phosphate
Preliminary experiments were performed to establish the
experimental conditions for further assays. In these experi-
ments, chronic treatment of rats with thyroid hormone was
assayed up to a concentration 10 times the physiological dose
of the hormone, as explained in Materials and Methods. The
plasma concentrations of T3 are shown in Table 1, together
with phosphate and calcium serum levels and the fractional
excretion of phosphate. Plasma concentration of phosphate
was progressively increased by T3 and reduced in hypothy-
roid animals that also showed the highest fractional excretion
of phosphorus.
Renal phosphate reabsorption was also measured in these
animals by using BBM vesicles from both superficial and
juxtamedullary cortex (Fig. 1). Inward sodium-coupled
phosphate transport was increased by the hormone in a
dose-dependent manner in both superficial and juxtamed-
ullary vesicles, although no significant difference was ob-
served between both hyperthyroid animals [those receiving
2.5 (group D) and 5 (group E) mg T3/100 g bwzday]. The
stimulation of phosphate transport in juxtamedullary BBM
vesicles tended to be higher than the stimulation measured
in BBM vesicles from the superficial cortex; however, such
differences were not statistically significant.
Interestingly, hypothyroid young rats showed a reduction
of approximately 50% in phosphate transport, with respect
to euthyroid rats. Hyperthyroidism elicited a 40% increase in
Pi transport over euthyroid animals and 175% increase over
hypothyroid animals.
Thyroid hormone specifically increases NaPi-2 protein
To understand the effect of thyroid hormone on phosphate
reabsorption, we have analyzed the content of Na/phos-
phate cotransporter in each of the treatments, by Western
blotting assay, using the same BBM vesicles as in the trans-
port experiments. As expected, we have found a correspon-
dence between the changes in transport activity elicited by
the hormone and the changes in NaPi-2 protein. Fig. 2 shows
the three conditions analyzed: euthyroid, hypothyroid, and
hyperthyroid animals. These results indicate that chronically
administered triiodothyronine increased NaPi-2 transporter
in a way similar to that of phosphate transport, with more
intensity in juxtamedullary than in superficial renal cortex,
although this difference was, again, statistically not distin-
guishable. Whereas hypothyroidism reduced NaPi-2 protein
to about 40% of euthyroid animals (60% reduction), hyper-
thyroidism increased this protein 350%, compared with
hypothyroidism; and 65%, compared with euthyroidism.
FIG. 1. Effect of T3 on phosphate transport in young and old rats.
Phosphate transport was measured in BBM vesicles from different
animal groups, as explained in Materials and Methods. Group A,
Nonoperated, control rats; group B, TPTX-hypothyroid rats receiving
placebo pellets; group C, TPTX-euthyroid rats receiving a physiolog-
ical dose of T3 (0.5 mg/100 mg BWzday); group D, TPTX-hyperthyroid
rats receiving 5 times the physiological dose of T3; group E, TPTX
hyperthyroid rats receiving 10 times the physiological dose of T3;
black bars, BBM from superficial cortex; lined bars, BBM from jux-
tamedular cortex; Y, young rats; O, old rats; *, significant difference
from the respective animal group A.

TABLE 1. Effect of T3 on serum phosphate concentration and fractional excretion in young and aged rats

Young rats Aged rats

Animals T3 (ng/dl) Pi (mg/dl) Ca (mg/dl) FEPi (%) T3 (ng/dl) Pi (mg/dl) Ca (mg/dl) FEPi (%)
A 79.2 6 8.6 10.5 6 0.8 a
9.8 6 0.6 7.2 6 1.1 a
81.3 6 6.7 6.6 6 0.3 9.3 6 0.5 13.6 6 0.8
B ,15.0b 4.8 6 0.2a,b 8.4 6 0.4 15.2 6 0.9b ,15.0b 4.2 6 1.2b 8.9 6 0.8 16.5 6 2.0b
C 45.0 6 8.3 11.1 6 2.6 9.0 6 0.5 6.8 6 0.4
D 481.6 6 10.0c 12.8 6 2.1 8.9 6 0.8 4.8 6 0.6c,a 260.0 6 18.3c 7.2 6 1.7 8.7 6 0.9 9.4 6 1.2c
E .1000c 13.1 6 0.3c 9.3 6 0.6 3.8 6 0.4a,c .1000c 8.0 6 0.8c 8.5 6 1.0 8.7 6 0.9c
Group A, Nonoperated, control rats; group B, TPTX-hypothyroid rats receiving placebo pellets; group C, TPTX-euthyroid rats receiving
physiological dose of T3 (0.5 mg/100 g BW per day); group D, TPTX-hyperthyroid rats receiving 5 times the physiological dose of T3; group E,
TPTX hyperthyroid rats receiving 10 times the physiological dose of T3. The treatment was maintained for 20 days. Relevant statistic differences
(95%). FEPi, Fractional excretion of inorganic phosphate.
a
Significant between the corresponding values of young and aged rats (same lane).
b
Significant with all other data from the same column.
c
Significant with value from line A of the same column.
 THYROID HORMONE MODULATES PHOSPHATE TRANSPORT 1547

FIG. 2. Effect of thyroid hormone on NaPi-2 protein content in BBM vesicles from proximal tubules in young and old rats. NaSi protein content
is also shown as a nonmodified control. SC, Superficial cortex; JM, juxtamedullary cortex. Samples, run in duplicate for each condition, are the
same as in Fig. 1. Ratios between optical densities of NaPi-2 and NaSi are shown in bars, and P , 0.001 in all groups. All differences with group
B in the histograms are significant (95%); group D is significant with A and C in young animals, and group E with A in old rats.

FIG. 3. Confocal immunohistochemical

analysis of T3 effect on NaPi-2 abun-
dance in proximal tubules of superficial
cortex from young rat kidneys. Hyper-
thyroidism elicits an increase in NaPi-2
protein content in both apical mem-
brane and intracellular stores (dotted
pattern) of proximal tubular epithelial
cells, in comparison with the hypothy-
roid status. b-Actin was stained with
phalloidin as an unmodified membrane
protein. Bar, 10 mm.

Signals corresponding to NaSi are shown as unmodified
controls.
These results were confirmed by immunohistochemistry.
As Fig. 3 shows, hyperthyroidism elicited a strong increase
of NaPi-2 protein in the luminal membrane of proximal tu-
bules. This increase was also extended to the intracellular
stores of the transporter, shown in the figure as fluorescent-
dotted cytoplasm of the epithelial cells (2). Fig. 3 also shows
b-actin filament labeling of tubular plasma membrane,
which is not changed by thyroid hormone.
Thyroid hormone increases NaPi-2 mRNA by
transcriptional stimulation
Most cellular effects of thyroid hormone are mediated by
increases in mRNA transcription and consequent protein
synthesis. Therefore, several Northern blotting assays were
performed to check for an increase in NaPi-2 mRNA steady-
state in rat kidney cortex. The results are summarized in Fig.
4: In parallel with the results of protein content, thyroid
hormone induced similar changes in specific NaPi-2 mRNA
1548 THYROID HORMONE MODULATES PHOSPHATE TRANSPORT
FIG. 4. Effect of T3 on NaPi-2 mRNA
content in superficial kidney cortex of
young and old rats. Samples are run in
duplicate, and the filters were probed
with the cDNAs indicated at the left of
the figure. The histograms show ratios
of NaPi-2/NaSi optical densities. Treat-
ments are the same as in previous fig-
ures. ANOVA, P , 0.001. All differences
with group B in the histograms are sig-
nificant (95%); group D is significant
with A and C in young animals, and E
with A and D in old rats.
abundance in all conditions assayed. In this case, the stim-
ulation by T3 on juxtamedullary NaPi-2 mRNA was also
higher than in superficial cortex mRNA (data not shown). As
unmodified controls, RNA levels of sodium-sulfate cotrans-
porter, b-actin, and ribosomic 18S are also shown.
In a previous paper (6), we have shown that the effect of
thyroid hormone on phosphate transport in OK cells is not
mediated by changes in the stability of the specific NaPi-4
mRNA. Now, we have checked for changes in transcription
rate of the NPT-2 gene (21). Nuclear run-on experiments were
performed from several conditions using nuclei isolated
from full kidney cortex. Results are shown in Fig. 5A, with
comparison between hypothyroid and hyperthyroid condi-
tions: an approximately 2-fold stimulatory effect of T3 in
NPT-2 gene transcription was found between these two con-
ditions. However, an additional change in the NaPi-2 mRNA
stability in the rat cannot be excluded.
Thyroid hormone stimulation of phosphate transport is
impaired in the aged
As expected from our previous results (10), 24-month-old
rats exhibited lower plasma phosphate concentration and
increased fractional excretion of phosphate than young rats
(Table 1). This decrease was paralleled by a proportional
reduction in phosphate transport capacity by BBM vesicles
from superficial and juxtamedullary proximal tubular cells
(Fig. 1). The effect of thyroid hormone to modulate Na/Pi
cotransport activity was less dramatic than in young animals.
Preliminary experiments with old rats, including group C,
showed (as expected) no differences with group A of the
same age animals. Therefore, this group was omitted when
using old rats. Instead, we always used group E to find a dose
of T3 that resulted in effects similar to those seen in the young
animals. Chronic hypothyroidism elicited a 30% reduction in
phosphate reabsorption and plasma concentration, that was
Endo • 1999
Vol 140 • No 4
restored with thyroid hormone treatment, whereas chronic
hyperthyroidism increased phosphate transport by only 20%
over control levels and 50% over the hypothyroid status,
even when plasma concentration of the hormone was more
than 10 times the physiological levels. Interestingly, these
thyrotoxic doses of T3 in aged rats were still not able to restore
the basal level of phosphate transport present in control
young animals (Figs. 1 and 6), although additional factors
(different from a reduced sensitivity to T3) may be respon-
sible for the blunted stimulatory effect of this hormone.
Western blot analysis of NaPi-2 protein in old rats showed
a parallelism with transport (Fig. 2). Densitometric analysis
revealed a 40% reduction of NaPi-2 protein in hypothyroid,
compared with euthyroid animals, whereas hyperthyroid-
ism increased NaPi-2 protein 110%, with respect to hypo-
thyroidism, and 40% over euthyroid animals. Similar results
were obtained for the specific NaPi-2 mRNA content, by
Northern blotting, in both superficial and juxtamedullary
kidney cortex (Fig. 4), that was also caused by an increase in
transcription of the NPT-2 gene, although this increase was
less effective than in young animals (Fig. 5).
Figure 6 summarizes the influence of aging on the T3
stimulatory effect on phosphate transport rate, NaPi-2 pro-
tein, and mRNA contents, and it compares young-control
nonoperated animals with old-control and old-hyperthroid
TPTX-animals. On the one hand, transport rate and NaPi-2
protein and RNA contents were less than half in control
(nonoperated) old animals, compared with control young
rats. On the other hand, hyperthyroidism in old rats in-
creased NaPi-2 RNA content above the level in young ani-
mals. However, protein content was similar in hyperthyroid
old animals and euthyroid young animals, and in terms of
phosphate transport rate, these old animals exhibited even
less transport rate per protein unit than the young rats.
Therefore, these results seem to indicate that the aging pro-
 THYROID HORMONE MODULATES PHOSPHATE TRANSPORT
FIG. 5. Effect of T3 on gene NPT-2 transcription rate, measured in
nuclear run-on from renal cortex of young and old rats. Total labeled
nascent RNA was labeled and probed against denaturized-linear plas-
mids containing the cDNAs indicated at the left of the figure. HypoT3,
Hypothyroid animals, group B; Hyper T3, hyperthyroid animals,
group D; pBlu, pBluescript.
cess affects all the steps of the stimulatory effect of thyroid
hormone on the cell physiology of phosphate transport, from
gene transcription to protein function.
Discussion
Effect of T3 on phosphate reabsorption
The present study demonstrates a physiological role for
thyroid hormone in the control of phosphate homeostasis.
We have found that a physiological dose of T3 stimulates Pi
renal reabsorption (Fig. 1) to a level that is able to increase
serum Pi concentration (Table 1). Higher (pharmacological)
doses of T3 further increase Pi renal reabsorption and serum
phosphate level. This effect is mediated by parallel increases
in the amount of Na/Pi cotransporter (NaPi-2) in the brush
border of proximal tubular epithelial cells (Figs. 2 and 3). The
specific increase in protein content is, in turn, caused by an
increase in the intracellular content of the specific NaPi-2
mRNA (Fig. 4), which was produced by stimulation of the
transcription rate of the corresponding gene, NPT-2 (Fig. 5).
These results point to the classic mechanism of thyroid hor-
mone, acting through intracellular receptors and binding to
thyroid hormone response elements (TREs) in the corre-
sponding gene promoters (20). However, in spite of the re-
cent identification and sequence of several type II (NaPi-2,
NPT-2) gene promoters (21), no consensus sequence of classic
TREs has been found, but only a putative vitamin D-response
element (22). Therefore, the stimulatory effect on NaPi-2-
mediated transport could be explained by the existence of a
novel TRE in the NPT-2 gene or by an indirect T3 stimulation;
i.e. T3 could stimulate the synthesis of a protein responsible
for a direct effect on the NPT-2 gene. Future experiments,
using these gene promoters, should clarify this question.
To our knowledge, our study is the first that clearly shows
a physiological role for T3 on renal tubular phosphate reab-
1549
FIG. 6. Comparison of T3 effects on superficial kidney cortex from
young and old rats: Na/phosphate cotransport, NaPi-2 protein, and
NaPi-2 mRNA. A-Y, Control nonoperated young rats (group A, young);
A-O, control nonoperated old rats (group A, old); D-O, TPTX-hyper-
thyroid old rats (group D, old); *, significant difference with group AY;
#, significant difference between AO and DO. Values of RNA and
protein densitometries in histograms are arbitrary densitometric
units. Dens., Densitometric.
sorption: chronic hypothyroidism induces a substantial de-
crease in serum phosphate, as well as an inhibition of phos-
phate transport, that is reversed by the exogenous
physiological treatment with T3. It is interesting that the
reduction in phosphatemia occurs in spite of the existence of
alternative mechanisms for increasing phosphate reabsorp-
tion in the rat (e.g. acute and chronic adaptation to dietary
phosphate deprivation, and others), because none of them is
able to restore the euthyroid level of serum phosphate in
hypothyroid animals. This could be explained as either the
existence of additive effects of all these regulatory mecha-
nisms, T3 being a major regulator, and/or as the need of
thyroid hormone presence for a correct functioning of all
other additional mechanisms. The second possibility is more
likely to occur because, as it has been shown, euthyroid rats,
chronically fed with low (0.1% Pi) phosphate diet vs. control
(0.6% Pi) or high (1.2% Pi) phosphate diets, are able to main-
tain a normal phosphatemia, thanks to an avid renal adaptive
mechanism that induces an increase of almost 100% reab-
sorption of phosphate ultrafiltrate (18, 23). In this case, the
adaptation consists in both increases in specific NaPi-2
mRNA and protein content in tubular cells, although this
effect is not mediated by NPT-2 gene transcription stimula-
tion but by an increase of NaPi-2 mRNA stability (24, 25). In
1550 THYROID HORMONE MODULATES PHOSPHATE TRANSPORT
addition, though the expected transient hypophosphatemia
caused by dietary phosphate deprivation induces the men-
tioned increase in NaPi-2 mRNA and protein, in the case of
hypothyroidism, the effect is just the contrary (that is, re-
duction in NaPi-2 mRNA and protein content).
From these results, we can initially conclude that thyroid
hormone is a major controller of phosphate homeostasis in
long-term regulation, which should be distinguished from
acute regulation, a less frequent physiological condition. This
conclusion is also supported by a characteristic of our ex-
perimental design: to avoid interferences with PTH, the an-
imals were made TPTX, instead of just thyroidectomized.
Therefore, our animals possessed hypothyroid and hypo-
parathyroid status. As it has been recently shown, hypopar-
athyroidism induces an increase in phosphate reabsorption
through lack of the phosphaturic hormone (26). Therefore,
when the animals have normal levels of PTH in serum, the
actual effect of chronic hypothyroidism is, most likely, more
dramatic than we have shown in the present paper.
With respect to the stimulatory effect of T3, treatment with
T3 (5 and 10 times the physiological dose) further increased
Pi reabsorption through the same mechanisms as the phys-
iological dose (RNA and protein synthesis). The stimulatory
effect of chronic hyperthyroidism was already saturated at 5
times the physiological dose in young animals (see Fig. 1) and
was similar to the increase obtained with acute thyrotoxic
doses, 400 times the physiological dose (200 mg T3/100 mg
BWz12 h, for 3 days) shown in previous papers and in our
own lab (Refs. 4 and 23; data not shown). The equivalence of
our chronic hyperthyroid doses to acute thyrotoxic doses
(because of saturability) shown in the present study, with
respect to the stimulation of phosphate reabsorption, could
make feasible the use of low-dose thyroid hormone as a
long-term therapy in several disorders involving phosphate
homeostasis unbalance or bone diseases.
Previous works (4) reported that the main effect of T3 takes
place on the renal juxtamedullary cortex (mostly straight
tubules), in spite of the higher transport rate and NaPi-2
mRNA and protein content of the superficial cortex (mostly
convoluted tubules; Ref. 15). In the present paper, we have
not found such difference in T3 effect; that again, could be
caused by the differences in the experimental design: long-
term physiological T3 doses vs. acute thyrotoxic doses in the
previous work.
Finally, two comments must be made with respect to the
two controls used, the NaSi and b-actin. Two groups have
found discrepancies, in relation to the the effect of T3 on
sulfate transport in BBM vesicles: no effect in rat (4, 5); and
stimulation in mouse (27). Our results have shown a lack of
T3 effect on transcription and RNA and protein content of the
NaSi transporter. With respect to b-actin, it has been shown
recently that thyroid hormone induces a 2-times inhibition in
its mRNA levels in skeletal and white muscle tissue (28). In
the present work, we have also not found any effect on
mRNA or protein b-actin levels. This difference could be
explained by tissue-specific effects, but also by the differ-
ences in both experimental design and the dose: 0.5 mg T3/
100 mg BW vs. 100 mg/100 mg BW in the previous paper.
Endo • 1999
Vol 140 • No 4
Effect of aging on thyroid hormone action
There are two main results of the present study in relation
to the effect of aging: first, NPT-2 gene transcriptional activity
is decreased with the age of the animals; and second, the
stimulatory effect of thyroid hormone is also reduced in the
aging. In a previous paper (10), we have published that old
rats contain less NaPi-2 mRNA and protein per tissue unit
than young rats. Now, we have shown that such decrease
may be caused by a reduced transcription rate of the corre-
sponding gene, which might explain the lower serum phos-
phate concentration in old animals. Such a decrease is a
general characteristic of the aging process in mammals (29).
Old rats are also less sensitive to T3 than the young ones, and
this can be observed in both hypothyroid and hyperthyroid
animals. On the one hand, old hypothyroid animals show a
smaller reduction in phosphatemia than young hypothyroid
animals (in comparison with the respective euthyroid rats),
as well as proportional (smaller) alterations in transport rate,
protein, and mRNA levels of the renal phosphate transporter
NaPi-2. On the other hand, the stimulatory effect of T3 to
induce hyperthyroidism is also less effective in old than in
young animals, for all parameters analyzed.
In addition, the reduction in the basal transcriptional rate
of NaPi-2 mRNA in aged rats is not caused by an age-
dependent reduction in serum levels of T3. It should be noted
that some authors have reported a decrease in serum T3
levels with age in humans (30), caused by both decreases in
pituitary TSH release and peripheral degradation of T4 that,
in return, result in a decline in serum and tissue concentra-
tion of T3 (31). Our results are, however, in agreement with
other studies (e.g. Ref. 32), which have not found differences
in serum T3 concentrations between young and old rats, and
they suggest that the relative hypothyroid status of the aging
rat is not produced by a decreased concentration of T3 but is
caused by a reduced relative response to the hormone. In fact,
the effect of the aging can be detected in all the cellular and
molecular processes of phosphate transport regulation: tran-
scription, translation, and function of the NaPi-2 protein
(Fig. 6).
In summary, we have shown a physiological role of T3 in
the regulation of, mainly, basal level of renal phosphate
reabsorption, which is impaired with aging. Furthermore,
our study is the first that shows genomic regulation of the
corresponding gene, NPT-2, by thyroid hormone and the
aging process.
Acknowledgment
We thank Prof. Gabriela Morreale, from the Institute for Biomedical
Research-Consejo Superior de Investigaciones Cientifica, for T3 RIA and
comments on the manuscript.
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