Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724
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ABSTRACT |
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The effects of chronic administration of methylprednisolone (MP) were studied on the ontogeny of the renal type II Na-Pi transporter (NaPi-2). Immunoblot analysis showed that MP did not alter the expression of NaPi-2 protein levels in suckling and weanling rats; however, there was an ~50% decrease in adolescent and adult rats. There was no change in Na-dependent Pi uptake in brush-border membrane vesicles in suckling rats, but there was an almost twofold decrease in adolescent rats induced by MP treatment. MP administration did not alter mRNA levels in suckling or adolescent rats. Dual injections with the glucocorticoid receptor blocker RU-486 (mifepristone) and MP did not reverse the downregulation of NaPi-2 immunoreactive protein levels in adolescent rats. To control for RU-486 antagonism efficiency, Na/H exchanger isoform 3 (NHE3) protein levels were also assayed after injection with RU-486 and MP. As expected, NHE3 protein levels increased after MP injection; however, the increase was blocked in adolescent rats by RU-486. We conclude that there is an age-dependent responsiveness to glucocorticoids and that the marked decrease in NaPi-2 immunoreactive protein levels and activity in adolescent rats is due to posttranscriptional mechanisms.
sodium-hydrogen exchanger isoform 3; RU-486; methylprednisolone; rat development; kidney
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INTRODUCTION |
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THE TYPE II RENAL Na-Pi transporter is a protein that is specific for the brush-border membranes (BBMs) of the kidney, and it is responsible for the Na-dependent, unidirectional, transepithelial transport of Pi across the proximal tubules (9, 23, 35). It is regulated by a number of metabolic and hormonal stimuli, which include diet, growth factors, and a variety of peptide and steroid hormones. Some of the factors that upregulate transepithelial renal Pi transport are low-phosphate diet (8, 14, 28), thyroid hormone (32), and 1,25-dihydroxyvitamin D3 (34). Parathyroid hormone (29), epidermal growth factor (3), and glucocorticoids (2, 24, 25, 30, 31, 36, 37), in contrast, show inhibitory effects on the renal Na-Pi transporter expression and activity.
Glucocorticoids are an important class of steroid hormones, which modulate a large number of behavioral, immunologic, and inflammatory responses. Most of the known physiological effects of glucocorticoids involve regulation of gene transcription and are mediated by an intracellular glucocorticoid receptor (GR), which belongs to a superfamily of steroid/thyroid hormone receptors (4). Nongenomic actions of glucocorticoids have also been described. These may involve binding of the hormone to a membrane-associated steroid receptor, which initiates a second-messenger cascade (38), or modulating membrane fluidity, which results in a change in the activity of certain membrane proteins (24).
During chronic administration of synthetic glucocorticoids and in certain disease states such as Cushing's disease, the urinary excretion of Pi is increased and serum levels of Pi are significantly reduced (21, 22). The effects of glucocorticoids on the type II renal Na-Pi transporter have been investigated previously. Na-dependent Pi uptake studies performed on renal, proximal tubular BBM vesicles (BBMVs) isolated from glucocorticoid- and vehicle-injected rats exemplified a decreased maximal reaction velocity without any change in the Michaelis-Menten constant (24). Other studies showed that, under similar glucocorticoid administration parameters, uptake of Pi and immunoreactive protein levels were significantly decreased in adult rats (24, 36), neonatal rabbits (31), and opossum kidney (OK) cells (20, 37). This decrease in NaPi protein expression was abolished in adrenalectomized rats, such that NaPi-2 protein abundance was shown to increase on immunoblots compared with sham-operated animals (25).
Other studies have documented the effects of glucocorticoids on the renal Na-Pi transporter at the level of gene transcription. Northern blot analysis indicated that glucocorticoids do not change mRNA levels in neonatal rabbits (31), whereas a 2.5-fold decrease in mRNA abundance was shown in adult rats after injection with dexamethasone (24). Given the difference in species and the different ages of these animals, these results are difficult to correlate with one another.
Total corticosterone levels in rat serum are lowest during the first 6 days of life, then they surge and peak at around days 21-24 (19, 26). Paradoxically, this pattern is identical to the ontogenic expression of immunoreactive NaPi-2 protein, with lowest levels in suckling rats, highest levels in weanling rats, and decreasing levels with age (33). This, therefore, raises the possibility that the responsiveness of the NaPi-2 to glucocorticoid hormones is correlated during development, with NaPi-2 expression being least sensitive to glucocorticoids during the suckling and weaning periods.
The purpose of the present study was to compare and contrast the effects of chronic administration of glucocorticoids on renal NaPi-2 during ontogeny. Furthermore, we sought to contribute new insights into the mechanism by which glucocorticoids alter NaPi-2 expression.
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MATERIALS AND METHODS |
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Experimental animals.
Sprague-Dawley male rats (Harlan, Madison, WI) in the following age
groups were used: suckling (2 wk or 14 days old), weanling (3 wk or 21 days old), adolescent (6 wk or 42 days old), and adult (96-115
days old) (33). Animals were kept in overhanging cages and fed a normal
diet. Rats were treated with methylprednisolone sodium succinate (MP;
Upjohn, Kalamazoo, MI) at 30 µg/g body wt (5) or equal volumes of
vehicle (PBS). The rats were subcutaneously injected four times once
per 12 h. The final injection was performed 2 h before the animals were
killed. Rats were killed at the desired day (3rd day after the 1st
injection, when suckling rats were exactly 14 days old, weanling rats
were 21 days old, adolescent rats were 42 days old, and adult rats were
115 days old) by CO2 anesthesia
followed by cervical dislocation, and kidneys were harvested
immediately. Similarly, adolescent rats were injected subcutaneously
with mifepristone (RU-486; Sigma Chemical, St. Louis, MO) at 25 µg/g
body wt. This compound was suspended in PBS and sonicated thoroughly
before each injection (1, 18). The injections were performed four
times, with each one being given before the administration of MP.
Chemicals and reagents.
Poly(A)+ RNA was isolated using
the FastTrack kit (InVitrogen, La Jolla, CA). RNA mass standard was
obtained from GIBCO-BRL (Grand Island, NY). Isotope for Northern blot
analyses
([-32PO4]dCTP,
3,000 Ci/mmol; Redivue) was purchased from Amersham (Piscatawy, NJ).
Radioactive probes for Northern blot analyses were generated by random
prime labeling with use of the MegaPrime system (Amersham).
Nitrocellulose membranes (Nitroplus) were obtained from Micron
Separations (Westboro, MA), protein gel electrophoresis reagents from
Bio-Rad (Hercules, CA) and Novex (San Diego, CA), Rainbow Marker and
Kaleidoscope Prestained Standards from Amersham and Bio-Rad,
respectively, and X-ray film (X-Omat Blue XB-1, Kodak) and horseradish
peroxidase-linked secondary antibodies from Amersham. Mouse
anti-
-actin monoclonal antibody (clone AC-74) was obtained from
Sigma Chemical. The Renaissance chemiluminescent system (NEN-DuPont, Boston, MA) was used for immunoblot processing. Data from Northern and
Western blot analyses were quantitated by an imaging densitometer (model GS-700, Bio-Rad).
KH232PO4
(1 Ci/nmol) for uptake studies was purchased from New England Nuclear
(Boston, MA). All other chemicals and reagents were purchased from
Fisher Biotechnology (Pittsburgh, PA), Sigma Chemical, or Bio-Rad.
BBMV isolation and transport measurements. Three adolescent (6 wk) or 12 suckling (2 wk) rats were killed per group, and the kidneys were removed and decapsulated. Cortical tissue was excised and compiled from each group of rats. Rat renal BBMVs were prepared by a well-established Mg2+ precipitation method, as previously described (5, 8). Purified BBMVs were resuspended in 280 mM mannitol and 20 mM HEPES-Tris (pH 7.4). Protein concentration was assayed by a modified Bradford method.
Uptake of phosphate and glucose was measured by a rapid filtration technique. Pi transport was initiated by incubating 20 µl of the vesicular suspension with 80 µl of 100 mM NaCl or KCl, 100 mM mannitol, 10 mM HEPES-Tris, pH 7.4, 0.1 mM KH2PO4, and tracer amounts of KH232PO4. The reaction was terminated at 10 s, which is within the linear rate phase (27, 28), by addition of 2 ml of ice-cold stop solution (100 mM NaCl, 10 mM mannitol, 10 mM HEPES-Tris, pH 7.4, and 10 mM KH2PO4). Na-dependent glucose uptake measurements were performed to determine whether the changes in Pi transport were specific to Na-Pi cotransport. For these experiments, 20 µl of the vesicular suspension were mixed with 80 µl of 100 mM NaCl, 100 mM mannitol, 10 mM HEPES-Tris (pH 7.4), 0.1 mMProtein gel electrophoresis and Western blot analysis.
BBMVs were purified as described above (see
BBMV isolation and transport
measurements) from suckling, weanling, adolescent, and adult rats. The BBMVs isolated from the renal cortex were diluted
in at least an equal volume of Laemmli solubilization buffer (2% SDS,
10% glycerol, 1 mM EDTA, and 2 mM -mercaptoethanol, pH 6.8) and
placed on ice for 30 min. After the protein standards and samples were
fractionated on a 4-12% gradient Tris-glycine gel, they were
electroblotted onto nitrocellulose membranes at 4°C. The
nitrocellulose membranes were blocked overnight in PBS with 0.05%
Tween 20 (PBST) and 5% nonfat dry milk. On the next day the membranes
were rinsed with PBST-0.1% milk and incubated for 40 min at room
temperature with the primary antibody [rabbit polyclonal antibody
raised against mouse Na-Pi
transporter-specific COOH-terminal peptide (8, 11)] at 1:4,000
dilution. This antibody has been shown previously to cross-react with
high specificity with the rat NaPi-2 (33). The membranes were washed
with PBST-0.1% milk four times for 10 min each and then incubated with
the secondary antibody (anti-rabbit IgG) at 1:2,000 dilution for 40 min. Finally, the membranes were washed with PBST-0.1% milk four times
for 10 min each, reacted with the chemiluminescence reagent for 60 s, and then exposed to film. The membranes were incubated with
-actin antibody in the same manner at 1:5,000 dilution of the primary antibody
and 1:2,000 of the secondary antibody (anti-mouse IgG).
-Actin
signal was used as an internal standard to depict the changes in
immunoreactive NaPi-2 levels. Furthermore, Na/H exchanger isoform 3 (NHE3)-specific antibody (12, 13) labeling was utilized to control for
efficiency of RU-486 antagonism at a dilution of 1:3,000. Anti-rabbit
IgG was utilized as a secondary antibody at a dilution of 1:2,000 for
these experiments.
mRNA isolation and Northern blot analysis. Six suckling and three adolescent rats were killed from each group. Kidney cortexes were removed and snap-frozen in liquid nitrogen. Poly(A)+ RNA was isolated by using a commercially available kit according to the manufacturer's protocol. With use of 4 µg/lane, the poly(A)+ RNA was fractionated by denaturing agarose gel electrophoresis. After the samples were blotted onto nitrocellulose membranes, the blots were hybridized with rat NaPi-2-specific probe (33). High-stringency washes were performed as previously described (33). The NaPi-2-specific hybridization signal was visualized on X-ray films. The 18S ribosomal RNA, as it appeared under ultraviolet light on the membranes, was used to control for loading and transfer efficiency.
Statistical analysis of results. The data were analyzed for statistical significance by Student's t-tests or ANOVA followed by Fisher's protected least significant difference post hoc test by using the Statview software package (version 4.53, Abacus Concepts, Berkeley, CA).
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RESULTS |
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Western blot analysis of BBM proteins with use of NaPi-2- and
-actin-specific antisera.
A predominant band at 73 kDa for NaPi-2 was seen in all age groups, as
previously described (8, 11, 33). This predominant band was normalized
with respect to
-actin at 47 kDa (Fig.
1A). Groups of six suckling rats, four weanling rats, and two adolescent and
adult rats were used for each repetition. After administration of MP,
there was no change in NaPi-2 immunoreactive protein levels in suckling
and weanling rats, whereas there was an almost twofold decrease in
adolescent and adult rats (Fig. 1B).
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Effect of MP on Na-dependent Pi and
D-glucose uptake by rat renal BBMVs.
BBMVs were prepared from suckling and adolescent animals by the
Mg2+ precipitation method, as
described in MATERIALS AND METHODS. Alkaline phosphatase
activity was 10- to 12-fold enriched in all groups compared with the
crude homogenate (data not shown). There was no change in
Pi uptake by BBMVs isolated from
suckling rats (0.186 ± 0.022 and 0.246 ± 0.061 nmol
Pi · mg
protein1 · 10 s
1 for control and MP,
respectively, n = 3); however, there
was a significant decrease after MP injection in adolescent rats (0.619 ± 0.012 and 0.323 ± 0.034 nmol
Pi · mg
protein
1 · 10 s
1 for control and MP,
respectively, n = 5, P < 0.0001; Fig.
2). There was no difference between control
and injected groups in terms of glucose uptake in suckling (0.284 ± 0.038 and 0.439 ± 0.089 nmol
glucose · mg
protein
1 · 10 s
1 for control and MP,
respectively, n = 3, P = 0.1842) or adolescent rats (0.215 ± 0.041 and 0.191 ± 0.052 nmol
glucose · mg
protein
1 · 10 s
1 for control and MP,
respectively, n = 4; Fig. 2,
inset). This finding is in agreement
with previously published data (15) and confirms the specificity of MP
action on NaPi-2 activity.
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Northern blot analysis.
To determine the age-specific response to glucocorticoids on the level
of NaPi-2 mRNA abundance, suckling and adolescent rats were studied.
After hybridization with NaPi-2-specific cDNA probe, a 2.6-kb band (8,
14) was quantitated by densitometry and normalized with respect to 18S
ribosomal RNA (Fig.
3A). The
hybridization signal obtained for NaPi-2 demonstrates that
glucocorticoids do not alter mRNA expression in suckling or adolescent
rats. These data are depicted graphically in Fig.
3B.
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Effect of RU-486 on NaPi-2 and NHE3 immunoreactive protein levels.
Adolescent rats were injected with RU-486 and MP to determine whether
the GR antagonist would block the glucocorticoid-induced repression of
NaPi-2 protein abundance. Immunoreactive NaPi-2 protein levels
decreased, as expected, with MP injection (Fig. 4). This effect of MP was not neutralized
after MP and the GR antagonist RU-486 were injected in close succession
(Fig. 4). The antagonist was not able to reverse the downregulation of
NaPi-2 protein levels (P = 0.0012 for control vs. MP, P = 0.0002 for control vs. RU-486 + MP, n = 8;
Fig. 5). NHE3 is known to be upregulated by
glucocorticoids. To control for RU-486 antagonism efficiency, NHE3
protein levels were also assayed by Western blot analysis after dual
injection with MP and RU-486. The induction in NHE3 expression after MP
administration was abolished by RU-486
(P = 0.032 for control vs. MP,
P = 0.011 for MP vs. RU-486 + MP; Fig.
5, inset).
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DISCUSSION |
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Proper maintenance of Pi homeostasis is crucial for development and bone formation. Conditions such as hypophosphatemic rickets (9, 10), which develops early in life, are associated with growth retardation and lower skeletal abnormalities. Chronic administration of synthetic glucocorticoids and disease states such as Cushing's disease (adrenal hyperplasia) increase the risk of development of osteoporosis in part because of a continuous renal leak of Pi. The present investigation sought to determine the effects of chronic MP injection on the ontogeny of the renal NaPi-2 and to determine the mechanism by which glucocorticoids affect renal epithelial Pi transport.
The levels of immunoreactive NaPi-2 protein levels in the control groups showed the anticipated maturational decrease that has been documented by our group (i.e., lowest in suckling rats, highest in weanling rats, and declining with age) (33). As shown in Figs. 1 and 2, there was a 60% reduction in protein abundance after MP administration in adult and adolescent rats. This marked decrease closely resembles those that have been documented by other groups (24, 25). However, no change in NaPi-2 protein levels after glucocorticoid treatment of suckling and weanling animals is a novel observation (Fig. 1). Because suckling and weanling rats and adolescent and adult rats showed similar results in regard to MP responsiveness, we decided to pursue the remainder of the investigation with only the suckling (2 wk) and adolescent (6 wk) age groups. NaPi-2 activity, as estimated by initial rate 32P uptake studies with renal BBMVs, paralleled the changes observed in immunoreactive protein. There was a 48% decrease in Pi transport induced by MP in adolescent animals. These data match earlier studies performed in adult rats, where Pi transport was shown to decrease significantly after glucocorticoid treatment (24). Consistent with NaPi-2 protein levels, Pi uptake was unaffected by MP treatment in suckling rats. To control for the specificity of MP effect on Pi transport in adolescent rats, we performed Na-dependent glucose uptake experiments. As predicted and in agreement with previously published experiments (18), there was no effect of MP on glucose uptake in BBMVs isolated from adolescent rat kidneys. In addition, when the control groups are compared, there is about a threefold increase in Pi uptake in adolescent animals as opposed to sucklings, which is in agreement with our previously published results (33).
As determined by Northern blotting, the NaPi-2 mRNA levels were not
altered in response to MP in suckling and adolescent rats (Fig. 3). In
suckling rats, this is in agreement with the levels of immunoreactive
protein and Na-dependent Pi
uptake, neither of which was affected by MP. There is, however, an
inconsistency between increased NaPi-2 protein and its activity and
unaltered mRNA levels observed in adolescent rats. Because the data
conflict with previously published results (24), which showed an
~2.5-fold decrease in relative levels of NaPi-2 mRNA in response to
dexamethasone, we increased the number of repetitions of Northern
analyses to eight. It is difficult to explain the discrepancy between
the previously published results and the presented data. In both
experimental designs, rats of approximately the same age were utilized.
One could speculate that the potency of dexamethasone used by Levi et
al. (24) is much greater (5-fold higher) than that of MP used in our
studies. However, in our experimental design, we used MP at 30 µg/g
body wt, which should correct for and exceed the respective dose and
potency of dexamethasone. On the basis of the time effectiveness of
dexamethasone (long acting) and MP (intermediate acting), the
administration schedule [once a day for dexamethasone (24) and
every 12 h for MP (our studies)] is also unlikely to be the cause
of the described differences. In the studies by Levi et al., the
authors used deionized water for control injections, whereas
dexamethasone as a water-insoluble compound must have been injected as
ethanol solution. On the basis of published information, it is
difficult to assess the amount and concentration of ethanol administered in this study. In our present studies we used MP sodium
succinate, which is very soluble in aqueous solutions and was dissolved
in PBS, which was also used for control injections. It is, however,
difficult to speculate whether this discrepancy could account for the
observed differences in NaPi-2 mRNA levels in adolescent rats. In
support of the hypothesis that the rat NaPi-2 may be mainly regulated
at the posttranscriptional level are ontogenic changes in NaPi-2
expression. It has been documented that the developmental increase in
protein expression and Pi
transport does not involve changes in NaPi mRNA (33). An example of
another transport protein that is affected by glucocorticoids in a
similar fashion is the pancreatic cell glucose transporter GLUT-2.
Dexamethasone had no effect on the mRNA levels but decreased protein
abundance by 65% (17). In addition, when dexamethasone was
administered to neonatal (3- to 5-day-old) rabbits, the activity of the
Na-Pi transporter and protein
decreased, without any change in mRNA levels (31), which further
indicates a transcription-independent pathway for downregulation of
Na-Pi transporter expression.
The insensitivity of NaPi-2 to glucocorticoids in suckling and weanling rats could represent a physiological adaptation to keep serum Pi levels at a maximum during early development, since it is crucial for proper formation and mineralization of bone. In light of the suppressive effects of glucocorticoids in adult rats, lack of responsiveness to these hormones around weaning may provide the necessary conditions for the developmental increase in NaPi-2 protein levels and Pi transport activity observed in rats (33; present data). We, therefore, hypothesize that it is not glucocorticoids per se that are responsible for developmental changes in NaPi-2 expression but, rather, an unidentified mechanism responsible for sensitization/desensitization of NaPi-2 to glucocorticoids. In an earlier study the response of neonatal rabbits to dexamethasone was investigated, and it was concluded that glucocorticoids did play a role in the maturational decrease in proximal tubule Pi transport (2). Given the difference in species, however, it is difficult to correlate these findings with those of the present investigation.
No change in NaPi-2 mRNA levels in response to MP in adolescent rats suggests that decreases observed in immunoreactive protein and Pi uptake represent nongenomic actions of glucocorticoids. Because transcriptional activation or repression by glucocorticoid hormones is mediated through the GR, we studied the influence of a specific inhibitor of GR, RU-486. As expected, the effect of MP was not inhibited by the RU-486 (Figs. 4 and 5), proving our hypothesis that rat renal Na-Pi transporter is not regulated at the level of gene transcription by glucocorticoids. The effectiveness of RU-486 inhibition of MP-induced transcription was confirmed by analyzing NHE3 protein, a transporter known to be regulated at a transcriptional level by glucocorticoids (6, 7, 12, 25). It was observed that RU-486 did indeed inhibit the induction of NHE3 (Figs. 4 and 5). These results further implicate a nongenomic action of MP on NaPi-2 and support the observed lack of change in NaPi-2 mRNA in response to MP.
In vitro studies on the effects of dexamethasone on Na-Pi cotransport in OK cells (37) showed that Pi uptake was significantly higher in cells treated with RU-486 and dexamethasone than in cells treated with dexamethasone only. This suggests that GR is the key element for glucocorticoid action in OK cells. This study was solely trying to assess the short-term (acute) regulation by glucocorticoids on the OK cell Na-Pi transporter, whereas the present investigation was based on long-term in vivo effects of MP administration.
One can only speculate on the exact mechanism of the nongenomic downregulation of NaPi-2. Glucocorticoids are known to alter cell membrane fluidity (24), which can affect the function of certain membrane transporters. However, evidence exists that changes of membrane fluidity do not specifically affect Pi transport (2). More attention is given to membrane-initiated actions of glucocorticoids mediated by a specific membrane-associated GR (38). In this model, glucocorticoids exert their effect via a second-messenger pathway. Recently, it was documented that dexamethasone-treated adult rats exhibit an increase in intracellular immunohistochemical staining of NaPi-2 (25). It is possible that glucocorticoids are altering the amount of apical membrane-associated NaPi-2 via a hormone-initiated cell-signaling cascade, leading to the internalization of the transporter. Internalization of the transporter may be followed by its degradation. Dexamethasone-induced degradation of GLUT-2 was demonstrated by pulse-chase experiments (17). This is particularly of interest, because dexamethasone decreases GLUT-2 protein levels without affecting mRNA levels (17). Furthermore, parathyroid hormone-induced degradation of NaPi-2 has been demonstrated as well (29), which shows that there is already a cascade of hormone-mediated events that can lead to the degradation of the type II renal Na-Pi transporter.
In view of our present data, we conclude that the responsiveness of rat NaPi-2 to glucocorticoids is age dependent, with suckling and weanling rats being least sensitive to MP-induced downregulation. We hypothesize that lack of responsiveness of this transporter to glucocorticoids around weaning plays a permissive role in the maturational increase in NaPi protein abundance and activity. The obtained results suggest also that the observed changes in NaPi-2 expression and activity in adolescent rats represent nongenomic actions of glucocorticoids and do not involve changes in NaPi-2 steady-state mRNA levels.
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ACKNOWLEDGEMENTS |
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This investigation was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-33209.
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FOOTNOTES |
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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: F. K. Ghishan, Dept. of Pediatrics, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, 1501 N. Campbell Ave., Tucson, AZ 85724 (E-mail: fghishan{at}peds.arizona.edu).
Received 7 May 1999; accepted in final form 12 July 1999.
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