Regulation of stanniocalcin in MDCK cells by hypertonicity and
extracellular calcium
David
Sheikh-Hamad,
Diane
Rouse, and
Yu
Yang
Renal Section, Department of Medicine, Baylor College of Medicine,
Houston, Texas 77030
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ABSTRACT |
Differential display
RT-PCR cloning method was applied to poly(A)+ RNA isolated
from Madin-Darby canine kidney (MDCK) cells in isotonic or hypertonic
medium. A differentially expressed 360-bp PCR fragment was isolated,
subcloned, sequenced, and used to screen an MDCK cDNA library
constructed in
ZapII. A composite sequence of two overlapping cDNA
clones provided 1,053 bp of sequence that was 93% identical to human
stanniocalcin and corresponded to the 3'-end of the mRNA.
Although the fish homolog of this hormone inhibits calcium uptake by
the gill and intestine, the function of mammalian stanniocalcin remains
unknown. Stanniocalcin cDNA probe hybridizes to a 4.4-kb mRNA that is
induced eightfold by hypertonicity, in a manner that is dependent on
medium organic osmolytes. The mRNA induction correlates with increased
total cellular content of the protein and its concomitant release to
the medium, consistent with secretion for autocrine or paracrine
activity. Furthermore, induction of the mRNA by hypertonicity is
dependent on extracellular calcium and displays a threshold phenomenon.
The data suggest that kidney stanniocalcin may have a role in the
adaptation of kidney cells to osmotic stress, in a manner that is
extracellular calcium dependent.
kidney; osmotic stress; calcium; Madin-Darby canine kidney cells; thick ascending limb
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INTRODUCTION |
IN OUR PURSUIT OF IDENTIFYING mRNAs that are induced by
hyperosmotic stress in Madin-Darby canine kidney (MDCK) cells, we used
differential display RT-PCR cloning method to display and clone partial
cDNA products corresponding to down- or upregulated mRNA (19, 20). Here
we report cloning of a partial stanniocalcin cDNA from MDCK cells.
Stanniocalcin mRNAs are induced by hypertonicity in a manner that is
dependent on extracellular calcium. In addition, hypertonicity
increases the total cellular content of stanniocalcin protein and leads
to its release to the medium, consistent with secretion for autocrine
or paracrine activity.
Stanniocalcin was originally described as a calcium-regulating hormone
in bony fish (11). It is produced and stored in the corpuscles of
Stannius (29), organs associated with the kidneys in fish (37).
Although the hormone is detected in freshwater fish species, it is
found in higher concentrations in saltwater species that are exposed to
high external calcium concentrations (10 mM). In vivo,
elevation of plasma ionized calcium induces synthesis and secretion of
stanniocalcin (16, 36). On the other hand, studies in cultured cells
isolated from corpuscles of Stannius have been inconsistent. Wagner and
co-workers (34, 35) reported increased stannioclacin release and higher
mRNA levels in response to changes in ionized calcium concentration within the physiological range (0.32-1.78 mM). However, Hanssen et
al. (16) reported such an increase only when calcium concentrations exceeded the physiological range (2.5-3.75 mM). Because of its rapid action to lower total body and plasma calcium in fish,
stanniocalcin is considered a major regulator of body calcium. It is
secreted into the blood in response to an increase in ambient water
calcium concentration, and rapidly acts to inhibit the uptake of
calcium by the gills (21, 28) and the gut (30). In the gill,
stanniocalcin inhibits calcium influx but does not affect its efflux
(18). These results suggest that the hormone interacts with a receptor on the serosal membrane of the gill cells and, through an unknown second messenger system, blocks calcium entry through the apical membrane (surface exposed to ambient water) (37).
In mammals, stanniocalcin is expressed in multiple organs including
heart, kidney, prostate, placenta, lung, skeletal muscle, pancreas,
thymus, testes, small intestine, colon, thyroid, spleen, and ovary (3,
4). Immunohistochemical studies in rat, mouse, and human kidney
localize stanniocalcin to distal nephron segments including thick
ascending limb, distal convoluted tubules, and collecting duct cells
(principal and type A intercalated) (9, 15, 23, 38, 39). To date,
despite evidence to suggest the presence of stanniocalcin in mammalian
blood and its expression in multiple organs (23), the function of this
hormone remains unknown. Although the expression of stanniocalcin in
distal nephron segments, where calcium reabsorption occurs, suggests a
regulatory role in renal calcium handling, the following data suggest
that stanniocalcin may play a role in the adaptation of kidney cells to
osmotic stress.
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METHODS |
Cell culture.
MDCK cells (American Type Culture Collection, Rockville, MD) were used
in passages 62-68. Cells were grown in a serum-free, defined
medium, containing 1.55 mM Ca2+ (315 mosmol/kgH2O) (31). This medium contains 120 µM
myo-inositol (inositol) and no betaine or choline. In some
experiments, the media used were as above but with one of the following
modifications: 1) no inositol, to deplete medium osmolytes;
2) addition of 5 mM inositol and 5 mM betaine, to supplement
medium osmolytes; and 3) addition of 200 mosmol/kgH2O of one of the following, for osmotic stress:
NaCl, raffinose, or urea. These osmotically active constituents were
added to the basic medium, making it hyperosmotic to varying extents
(515-525 mosmol/kgH2O). For another portion of the
experiments, EGTA (0.0-1.5 mM) was added to the experimental medium for chelation of calcium. Alternatively, measured amounts of
calcium were added to calcium-deficient medium to control for EGTA
effects. All cultures were maintained in 5% CO2-95% air
at 37°C.
Differential mRNA display.
Confluent MDCK cells were exposed to isotonic medium (315 mosmol/kgH2O) lacking betaine and inositol, or the same
medium made hypertonic (515 mosmol/kgH2O) by addition of
NaCl. Total RNA was purified using RNAzol (Tel-Test, Friendswood, TX)
(7). Poly(A)+ RNA was isolated as previously described
(14). Differential mRNA display (19, 20) was performed using RNAmap Kit
A (GeneHunter, Brookline, MA) according to manufacturer's
instructions. Briefly, poly(A)+ RNA was subjected to DNase
I treatment, reverse-transcribed, and amplified by PCR using a modified
14-mer oligo(dT) and a 10-mer arbitrary primer, in the presence of
[
-35S]dATP (Amersham, Arlington Heights,
IL). Radiolabeled PCR products were resolved on denaturing 6%
polyacrylamide DNA sequencing gels and analyzed for differentially
expressed products (19, 20). Each gel piece, corresponding on the
autoradiographs to hypertonically induced cDNA, was cut, and the DNA
was extracted. The cDNA was then reamplified by PCR using the same set
of primers, cloned in pCR-II vector (TA Cloning Kit, Invitrogen, San
Diego, CA), and sequenced.
Northern blot analyses.
Total and poly(A)+ RNA were isolated as described above.
Electrophoresis was performed, loading equal amounts of
poly(A)+ RNA per lane in a 1% agarose/2.2 M formaldehyde
gel, followed by transfer to a GeneScreen membrane (New England
Nuclear) (14). Human (2 kb) full-length
-actin cDNA (Clontech) and
mRNA-display PCR products were labeled with
[
-32P]dCTP (Random Primed DNA Labeling Kit,
Boehringer-Mannheim, Indianapolis, IN) for use as probes. The probes
were hybridized to the blots overnight at 42°C in a solution
containing 40% formamide, 5× SSC (750 mM NaCl, 75 mM trisodium
citrate), 5× Denhardt's solution [0.5% (wt/vol)
polyvinylpyrrolidone, 0.5% (wt/vol) BSA, and 0.5% (wt/vol) Ficoll
400], 0.5% SDS, 250 µg/ml salmon sperm DNA, 10 mM Tris (pH
7.5), and 10% dextran sulfate. The blots were then washed at 65°C
as follows: for 30 min twice in 2× SSC, 0.5% SDS; 1 h in
0.5× SSC, 0.5% SDS; 30 min twice in 0.1× SSC, 0.5% SDS. Autoradiographs were prepared using X-Omat AR film (Kodak; Rochester, NY) with an intensifying screen. Bands on Northern blots were scanned
using UMAX Astra 1200S scanner (Fremont, CA) and quantitated using
Adobe Photoshop 4 and UTHSCSA Image Tool software. Band intensities
were determined relative to the corresponding
-actin bands.
Immunoprecipitation and SDS-PAGE.
Cells were lysed for 30 min in buffer containing [20 mM Tris (pH
7.4), 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM
phenylmethylsulfonyl fluoride (PMSF), and 1 µg/ml leupeptin]. Lysates were cleared from insoluble material by 10-min centrifugation at 10,000 g and 4°C, and protein was quantitated. After
immobilization of anti-human stanniocalcin antibodies (Human Genome
Sciences, Rockville, MD) (23) to protein A&G agarose, an aliquot of
cell lysate (150 µg protein) or culture medium (1 ml) was added, and stanniocalcin was immunoprecipitated. After repeated washes in lysis
buffer, immunoprecipitate was suspended in Laemmli sample buffer and
run on 12% SDS-PAGE under reducing conditions. Immunoblots were
reacted with 1:250 dilution of antistanniocalcin antibodies in
Tris-buffered saline (20 mM Tris base, pH 7.6, 137 mM NaCl) containing
1% BSA and 0.05% Tween-20, followed by incubation with horseradish
peroxidase-conjugated anti-rabbit antibodies. Stanniocalcin protein was
quantitated using ECL-Plus (Amersham International, Little Chalfont
Buckinghamshire, UK). For control, MDCK cells lysates, prepared as
above, were immunoprecipiated with anti-
1-integrin (K20,
Immunotech, Westbrook, ME) monoclonal antibodies. Precipitates were
resolved on 12% SDS-PAGE under reducing conditions and immunoblots reacted with anti-stanniocalcin.
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RESULTS |
Clone HT9 corresponds to dog kidney stanniocalcin.
HT9, a 360-bp PCR fragment amplified with primers AP-3 and T12MG
(RNAmap Kit A) using MDCK cells poly(A)+ RNA, appeared to
be differentially induced by extracellular hyperosmolality. When used
as a probe on Northern blots, the cDNA hybridized to a 4.4-kb mRNA that
is induced sixfold (after 24 h) by the addition of NaCl to the medium
(Fig. 1). The sequence of HT9 was
determined after subcloning into pCRII (Invitrogen). A search with the
BLAST network service of the National Center for Biotechnology
Information (NCBI) found 94 and 93% identity (Fig.
2A) to
3'-untranslated region of mouse (accession no. AF099098) and
human (accession no. U25997) stanniocalcin-1 (STC1) mRNAs, respectively
(5, 10). The cDNA has no sequence homology with the product of STC2 gene (6). HT9 was then used to screen an MDCK cDNA library constructed
in
Zap-II. This screening yielded few small cDNA clones, not
exceeding 600 bases in length each. A composite sequence of 1,053 bp
(Fig. 2B) was obtained from two overlapping clones, using
ASSEMGEL program of PC/GENE sequence analysis software
(Intelligenetics, Mountain View, CA). Reprobing Northern blots using
these partial cDNAs as probes yielded identical results to those
obtained with the 360-bp PCR product. We conclude that HT9 is induced
by hypertonicity in MDCK cells and corresponds to the 3'-end of
STC1 mRNA.

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Fig. 1.
HT9 (stanniocalcin) mRNA is induced by hypertonicity. Madin-Darby
canine kidney (MDCK) cells were grown to confluence in isotonic medium
(315 mosmol/kgH2O). Cells were placed for 24 h in medium,
which was made hyperosmotic (515 mosmol/kgH2O) by the
addition of NaCl (HT). Control cells were maintained in isotonic medium
(ISO). Cells were then analyzed for HT9 mRNA abundance.
Poly(A)+ RNA (3 µg) was loaded per lane on RNA gels, and
Northern blots were probed with HT9 display product. Data are based on
at least 3 independent determinations.
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Fig. 2.
A: sequence comparison between dog stanniocalcin (STC) cDNA
(HT9) and human stanniocalcin-1 mRNA (STC), using Blastn
program of the National Center for Biotechnology Information.
B: composite sequence of partial dog kidney stanniocalcin
cDNA.
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Stanniocalcin mRNA is induced by hypertonicity.
Extracellular hyperosmolality exerted by a nonpermeable solute (e.g.,
NaCl or raffinose, but not urea) has a hypertonic effect; it shrinks
cells and increases intracellular potassium and sodium concentrations
(ionic strength). There is evidence to suggest that increased
intracellular ionic strength is among the initial signals for induction
of genes responsible for organic osmolyte accumulation (27, 32).
However, the mechanism through which increased intracellular ionic
strength ultimately induces transcription of specific genes remains to
be determined. MDCK cells were exposed to medium that was made
hyperosmotic (final osmolality of 515 mosmol/kgH2O) by the
addition of NaCl, raffinose, or urea, and the abundance of
stanniocalcin mRNA was determined using Northern hybridization.
Addition to the medium of equiosmolal amounts (200 mosmol/kgH2O) of either NaCl or raffinose, but not the
membrane permeable solute urea, induces stanniocalcin mRNA sixfold
at 24 h (Fig. 3, A and
B). We conclude that stanniocalcin is induced by hypertonic
stress imposed by impermeable solutes and may have a role in the
adaptation of kidney cells to osmotic stress.


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Fig. 3.
Hypertonicity induces stanniocalcin mRNA in MDCK cells. This effect is
attenuated by cell accumulation of betaine and inositol. MDCK cells
were grown to confluence in isotonic medium (315 mosmol/kgH2O). A: at time 0, cells were
placed in medium containing either no betaine or inositol (B,I) or 5 mM
of each, in the presence or absence of NaCl or raffinose (final
osmolality of 515 mosmol/kgH2O). Control cells were
maintained in isotonic medium without betaine and inositol. B:
at time 0, cells were switched to medium containing no betaine
and inositol, in the presence or absence of urea (final osmolality of
515 mosmol/kgH2O). Control cells were maintained in
isotonic medium without betaine and inositol (ISO). Cells were then
analyzed for stanniocalcin mRNA abundance. Poly(A)+ RNA (3 µg) was loaded per lane on RNA gels, and Northern blots were probed
with dog stanniocalcin cDNA. Representative Northern blots are shown.
Data are based on at least 3 independent determinations.
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Induction of stanniocalcin mRNA correlates with increased abundance
of the protein and its release to the medium.
To determine the kinetics of stanniocalcin mRNA induction by
hypertonicity, we used Northern hybridization to measure stanniocalcin mRNA abundance in MDCK cells at various time points after the addition
of NaCl (200 mosmol/kgH2O) to the medium, or at a fixed time point (16 h) after the addition of various amounts of NaCl to the
medium. Induction of the mRNA occurs as early as 6 h after exposure of
the cells to hypertonicity, peaks (8-fold) after 16 h (Fig.
4A), and shows maximal induction
with 100 mosmol/kgH2O increment in medium osmolality (Fig.
4B). This pattern of induction is similar to that of other
hypertonicity-induced mRNAs in its timing, but not in its dose
response. For example, peak induction of mRNAs corresponding to genes
involved in the accumulation of organic osmolytes occurs after
16-24 h of exposure to hypertonicity (8, 33). In addition, they
demonstrate further increase in abundance as medium tonicity increases
beyond 100 mosmol/kgH2O (8, 33). We then used
immunoprecipitation and Western blotting to determine the effect of
hypertonicity on the total cellular content of stanniocalcin protein
and examined for its presence in the medium. As shown in Fig.
5, stanniocalcin protein (broad band
spanning 29-34 kDa) is detected simultaneously in cells and medium
after 16 h of exposure to hypertonicity. Stanniocalcin was not detected
in immunoprecipitates of an irrelevant antibody (anti-
1-integrin, K20). Of note, although the mRNA is
detectable in unstressed cells and at all time points after exposure to
hypertonicity, the protein is measurable only after prolonged exposure
to hypertonicity (16 h). The significance of this discrepancy remains
to be determined. Collectively, these data are consistent with
induction and release of stanniocalcin protein to the medium upon
exposure of the cells to hypertonicity and suggest that stanniocalcin
acts as a paracrine or juxtacrine hormone in mammalian kidney in a
hypertonicity-dependent manner.


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Fig. 4.
Time course (A) and dose response (B) of stanniocalcin
mRNA to hypertonicity. MDCK cells were grown to confluence in isotonic
medium (315 mosmol/kgH2O). A: at time 0,
cells were placed in medium containing no betaine or inositol, in the
presence of NaCl (final osmolality of 515 mosmol/kgH2O).
Control cells were maintained in isotonic medium without betaine and
inositol. At various time points, cells were harvested and analyzed for
stanniocalcin mRNA abundance. B: at time 0, cells were
placed in medium containing no betaine or inositol, in the presence of
varying NaCl concentrations. Control cells were maintained in isotonic
medium without betaine and inositol. After 16 h, cells were harvested
and analyzed for stanniocalcin mRNA abundance. Poly(A)+ RNA
(3 µg) was loaded per lane on RNA gels, and Northern blots were
probed with stanniocalcin cDNA. Representative Northern blots are shown
(B, in duplicate). Data are based on at least 3 independent
determinations.
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Fig. 5.
Hypertonicity induces stanniocalcin protein and leads to its release to
the medium. MDCK cells were grown to confluence in isotonic medium (315 mosmol/kgH2O). At time 0, cells were placed in
medium containing no betaine or inositol, in the presence of NaCl
(final osmolality of 515 mosmol/kgH2O). Control cells were
maintained in isotonic medium without betaine and inositol. At various
time points, cells were harvested and analyzed for stanniocalcin
protein abundance. Stanniocalcin was immunoprecipitated from 150 µg
of cell lysate (top panel) or 1 ml of medium (bottom
panel). Precipitates were run on 12% SDS-PAGE, and immunoblots
reacted with anti-human stanniocalcin. For control, 150 µg of protein
lysate or 1 ml of medium was immunoprecipitated with
anti- 1-integrin antibody (K20). Precipitates were run on
SDS-PAGE page, and immunoblots reacted with antistanniocalcin antibody.
Data are based on at least 3 independent determinations. Representative
blot is shown. Lanes 1-6, immunoprecipitation with
antistanniocalcin, reaction with antistanniocalcin; lane 7,
immunoprecipitation with K20, reaction with antistanniocalcin.
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Accumulation of intracellular organic osmolytes attenuates the
induction of stanniocalcin mRNA by hypertonicity.
As mentioned above, hypertonicity induces genes responsible for the
accumulation of organic osmolytes by the cell. Accumulation of organic
osmolytes in turn attenuates the induction of these genes, presumably
by decreasing intracellular ionic strength (25, 27). To test whether
stanniocalcin mRNA induction by hypertonicity is modulated by medium
organic osmolytes, MDCK cells were exposed to hypertonic medium under
conditions that would exaggerate or minimize organic osmolyes
accumulation (24). Under hypertonic conditions, MDCK cells accumulate
four major organic solutes: betaine, inositol, taurine, and
glycerophosphocholine (GPC) (13). Betaine, taurine, and inositol are
accumulated only when they are present in the medium, and the magnitude
of their transport is dependent on their concentration in the medium
and the extent of extracellular hyperosmolality (13, 22). Although GPC
can be synthesized from intracellular stores of choline, it does not accumulate in the cell in vitro to appreciable amounts in the absence
of medium choline (12). Hence, stanniocalcin mRNA abundance was
measured in cells exposed to medium containing high NaCl (final osmolality of 515 mosmol/kgH2O) and either no betaine and
inositol in the medium (to prevent their accumulation) or with 5 mM of each added (to enhance their accumulation) (25). Under hypertonic conditions, and in the absence of medium betaine and inositol, stanniocalcin mRNA is induced sixfold at 24 h. However, under hypertonic conditions, and in the presence of medium betaine and inositol, stanniocalcin mRNA induction is attenuated to a level equal
to that seen in cells under isotonic conditions (Fig. 3A). The
modulation of hypertonic induction of gene expression by cellular accumulation of organic osmolytes was originally considered unique to
genes that are directly involved in the accumulation of organic solutes, such as transporters for betaine, taurine, and inositol (25).
However, recent data from this laboratory suggest that a number of
genes that are induced by hypertonicity and are not directly involved
in organic solute transport display such behavior. Among these genes
are the 70-kDa heat shock protein and the adhesion molecules CD9 and
1-integrin (24-26). This suggests a common
regulatory pathway for the plethora of genes that are induced by
hypertonicity, irrespective of their direct involvement in organic
osmolytes transport.
Induction of stanniocalcin by hypertonicity is extracellular calcium
dependent.
Stanniocalcin is considered a major regulator of body calcium in fish,
and the release of stanniocalcin from corpuscles of Stannius in vivo
and in tissue culture is regulated by variations in extracellular
calcium. For this reason, we asked whether stanniocalcin expression in
MDCK cells is regulated by extracellular calcium. Confluent MDCK cells
were exposed to hypertonic medium for 24 h in the presence of
increasing medium EGTA concentrations (0-1.55 mM), and
stanniocalcin mRNA abundance was determined using Northern hybridization. As shown in Fig. 6,
stanniocalcin mRNA induction by hypertonicity is blunted only when EGTA
concentration reaches 1.5 mM, suggesting a threshold response. With the
use of equations derived from Bulos and Sactor (2), this EGTA
concentration corresponds to calculated medium calcium of 0.05 mM.

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Fig. 6.
Hypertonic induction of stanniocalcin is EGTA dependent. MDCK cells
were grown to confluence in isotonic medium (315 mosmol/kgH2O) containing 1.55 mM calcium. Cells were then
placed in same medium containing no betaine and inositol, to which NaCl
(HT) was added (final osmolality of 515 mosmol/kgH2O), in
the presence or absence of increasing EGTA concentrations. Control
cells were maintained in isotonic medium without betaine and inositol,
which contained 1.55 mM calcium. After 24 h, cells were analyzed for
stanniocalcin mRNA abundance. Poly(A)+ RNA (3 µg) was
loaded per lane on RNA gels, and Northern blots were probed with
stanniocalcin cDNA. Representative Northern blot is shown (duplicate
experiments). Data are based on at least 3 independent
determinations.
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The above observations are based on the assumption that the effects
attributed to EGTA are related solely to decreasing calcium concentration in the medium. To control for nonspecific EGTA effects, MDCK cells were placed in calcium-deficient hypertonic medium, to which
varying concentrations of calcium were added. Stanniocalcin mRNA
induction by hypertonicity is blunted only when medium calcium concentration is lowered to the range of 0.1 mM (Fig.
7A). This concentration is nearly
identical to the calculated calcium threshold obtained from the EGTA
experiments (0.05 mM). To determine whether stanniocalcin expression
demonstrates similar calcium threshold phenomenon under isotonic
conditions, MDCK cells were exposed to isotonic medium containing
various calcium concentrations for 24 h, and the abundance of
stanniocalcin mRNA was measured. As shown in Fig. 7B, under
isotonic conditions, stanniocalcin mRNA expression is not affected by
medium calcium concentrations shown earlier to blunt its induction by
hypertonicity. From these data, it can be concluded that the induction
of stanniocalcin by hypertonicity requires an extracellular calcium
concentration greater than 0.1 mM. Whether this finding is pertinent to
the regulation of the adaptive response to high osmolality or to other
physiological functions of kidney stanniocalcin remains to be
determined.


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Fig. 7.
Hypertonic induction of stanniocalcin is calcium dependent. MDCK cells
were grown to confluence in isotonic medium (315 mosmol/kgH2O) containing 1.55 mM calcium. A: at
time 0, cells were placed in calcium-deficient medium,
containing no betaine and inositol, to which NaCl [(HT), final
osmolality of 515 mosmol/kgH2O] and increasing
amounts of calcium were added. B: control cells were maintained
in isotonic medium, containing no betaine and inositol, to which
increasing amounts of calcium were added. Cells were then analyzed for
stanniocalcin mRNA abundance, using stanniocalcin cDNA as a probe.
Poly(A)+ RNA (3 µg) was loaded per lane. Representative
Northern blots are shown (duplicate experiments). Data are based on at
least 3 independent determinations.
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Stanniocalcin mRNA distribution in rat tissue.
To better understand the role of stanniocalcin in mammals, we examined
the expression and distribution of its mRNA in rat tissue. As shown in
Fig. 8, stanniocalcin mRNA (3.8 kb) is
detected in kidney cortex and medulla, adrenals, colon, and lungs,
whereas it is weakly expressed in heart, parathyroid, and cerebrum.
This pattern of expression is notably different from previous reports (3, 4) in its expression in brain, adrenals, and parathyroid glands and
in its absence in spleen, testes, and skeletal muscle. The expression
in parathyroid, colon, and kidney suggests involvement in calcium
homeostasis, whereas the significance of its expression in other organs
remains to be determined.

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Fig. 8.
Stanniocalcin mRNA expression in rat tissues. Twenty micrograms of
total RNA were loaded per lane, and Northern blot was probed with
stanniocalcin cDNA.
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DISCUSSION |
Our data suggest the involvement of stanniocalcin in the adaptation of
kidney cells to osmotic stress. In normal calcium media (1.55 mM),
stanniocalcin mRNA expression is increased by hypertonicity. However,
when medium calcium is decreased to 0.1 mM range, the induction by
hypertonicity is blunted. The data suggest dual regulation of
stanniocalcin mRNA in these cells by hypertonicity and extracellular calcium. MDCK cells are considered to be of distal nephron origin and
display characteristics of thick ascending limb cells (17). Thus the
expression of stanniocalcin mRNA in these cells is consistent with
literature reports of stanniocalcin expression in distal nephron. In
addition, it may be deduced from our data that MDCK cells are capable
of sensing extracellular calcium and modulate gene expression in
response to it. These cells have recently been reported to express
calcium receptors (1). However, it remains to be determined whether
calcium receptors are involved in the response of stanniocalcin to
hypertonicity or extracellular calcium.
The extracellular calcium threshold to which stanniocalcin responds in
MDCK cells is likely between 0.1 and 0.5 mM (the narrowest calcium
concentration range tested, through which induction of stanniocalcin by
hypertonicity is altered). This is still significantly lower than that
reported in fish, being 1-4 mM in the cells of Stannius corpuscles
(16, 34). It is suggested that, although the function of stanniocalcin
across the evolutionary tree from fish to mammals was maintained,
possibly to inhibit calcium entry into cells, the calcium levels to
which it responds vary. Furthermore, although stanniocalcin functions
as a true hormone in fish, it may be operating in the kidney as an
autocrine or paracrine substance.
Finally, we can only speculate about the physiological role of
stanniocalcin in the kidney. The calcium concentrations in the lumen of
the thick ascending limbs and in nephron segments beyond remain largely
unknown. Thus extrapolation about the physiological relevance of the
calcium concentration threshold at which stanniocalcin is regulated
remains speculative. However, assuming that the function of
stanniocalcin is preserved across the evolutionary tree, that is, to
inhibit calcium entry or uptake, we speculate that stanniocalcin may
function to inhibit calcium entry into cells under hypertonic conditions, for cytoprotection. Alternatively, it may function to
regulate calcium uptake in the distal nephron. In the latter case, we
speculate that stanniocalcin either directly inhibits calcium channels
or that it binds to a putative receptor and sets off a signaling
cascade that eventually leads to inhibition of calcium channels.
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ACKNOWLEDGEMENTS |
HT9 was a generous gift from Drs. Maurice Burg and Arlyn
Garcia-Perez. It was cloned by D. Sheikh-Hamad during fellowship in the
Lab of Kidney and Electrolytes Metabolism at the National Institutes of
Health. At that time, the homology between HT9 and stanniocalcin was
not known. D. Sheikh-Hamad acknowledges and thanks Drs. Maurice Burg,
Arlyn Garcia-Perez, and Joan Ferraris for their guidance and contribution.
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FOOTNOTES |
This work was supported in part by National Institute of Diabetes and
Digestive and Kidney Diseases Grant AGO 1-R01-DK-55137-01 and
junior faculty seed funds provided by Baylor College of Medicine (to D. Sheikh-Hamad).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. Sheikh-Hamad,
Renal Section, Dept. of Medicine, Baylor College of Medicine, 6535 Fannin St., MS F505, Houston TX 77030 (E-mail:
sheikh{at}bcm.tmc.edu).
Received 23 February 1999; accepted in final form 31 August 1999.
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