Division of Endocrinology and Metabolism, 1 Department of Medicine, Georgetown University, Washington, District of Columbia 20007; and 2 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Renal sodium retention, as a result of increased
abundance of sodium transporters, may play a role in the development
and/or maintenance of the increased blood pressure in obesity. To
address this hypothesis, we evaluated the relative abundances of renal sodium transporters in lean and obese Zucker rats at 2 and 4 mo of age
by semiquantitative immunoblotting. Mean systolic blood pressure was
higher in obese rats relative to lean at 3 mo, P < 0.02. Furthermore, circulating insulin levels were 6- or 13-fold higher
in obese rats compared with lean at 2 or 4 mo of age, respectively. The
abundances of the 1-subunit of Na-K-ATPase, the
thiazide-sensitive Na-Cl cotransporter (NCC or TSC), and
the
-subunit of the epithelial sodium channel (ENaC) were
all significantly increased in the obese rats' kidneys. There were no
differences for the sodium hydrogen exchanger (NHE3), the
bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2 or BSC1), the type
II sodium-phosphate cotransporter (NaPi-2), or the
-subunit of
ENaC. These selective increases could possibly increase sodium
retention by the kidney and therefore could play a role in
obesity-related hypertension.
sodium-phosphate cotransporter type II; sodium-hydrogen exchanger type III; bumetanide-sensitive sodium-potassium-2 chloride cotransporter; insulin resistance; hypertension; sodium-chloride cotransporter; epithelial sodium channel; adenosine 5'-triphosphatase
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INTRODUCTION |
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OBESITY IS ASSOCIATED WITH hypertension, and several studies have shown clear effects of weight loss on reducing blood pressure (8, 28, 55). Multiple mechanisms have been proposed to explain this correlation, including (in the obese) increased sympathetic activity; increased activity of the renin-angiotensin-aldosterone system; increased cardiac output; and increased mechanical pressure from interstitial fat around organs, hyperinsulinemia, and/or insulin resistance (14, 26, 40, 56).
Sodium retention by the kidney could result from any of these mechanisms. Sodium balance in the body is maintained by regulation of renal sodium reabsorption, with fine tuning accomplished in the post-macula densa segments from the distal convoluted tubule through the collecting duct system. In these distal segments, aldosterone plays a central role in regulating sodium reabsorption.
Recently, cDNAs for most of the major renal sodium transporter and channel proteins expressed along the renal tubule have been cloned (6, 7, 23, 44, 47, 54). This knowledge allowed us to produce a variety of specific antibodies (18, 20, 35-38, 45) against these proteins for use in immunoblotting and immunohistochemical studies. These new tools now allow direct investigation of sodium transport regulation at the molecular level.
The basolateral Na-K-ATPase pump is expressed along the entire length
of the renal tubule and actively pumps sodium from the cell into the
interstitium to set up the electro-chemical gradient to allow sodium to
be reabsorbed (31, 59). In the kidney, the pump is
composed of two subunit proteins, 1 and
1
(42). In the proximal tubule, the primary route for apical
sodium transport is via the sodium-hydrogen exchanger subtype III
(NHE3) (1, 4). Also, present in the apical membrane of the
proximal tubule cells are various sodium cotransport proteins such as
the sodium-phosphate cotransporter subtype II (NaPi-2)
(3). In the thick ascending limb, apical sodium transport
is via both NHE3 and the bumetanide-sensitive Na-K-2Cl cotransporter
(NKCC2 or BSC1) (33). In the distal convoluted tubule,
sodium is primarily reabsorbed through the apical, thiazide-sensitive Na-Cl cotransporter (NCC or TSC) (33). In the connecting
tubule and the collecting duct, sodium reabsorption occurs through
the apical amiloride-sensitive Na channel (ENaC) (23a). The channel is
a heterooligomer composed of three distinct subunit proteins,
,
,
and
(23a).
In these studies, we examine the abundance of renal sodium transporters and channels in the young, prediabetic, obese Zucker rat. The obese Zucker rat (fa/fa) is a well-characterized strain of rat (21, 22, 24, 27, 29, 30, 34, 61) in which a mutation in the gene for the leptin receptor (30) results in hyperphagia and obesity. Rats homozygous for the mutation develop obesity. Rats either heterozygous or homozygous for the normal receptor gene remain relatively lean. Obese rats become hyperinsulinemic at an early age, and they manifest mild to moderate hypertension. With advanced age, they become severely obese and diabetic.
Therefore, to address the mechanism of hypertension in these rats, we hypothesized that the obese Zucker rat might have increased abundance of one or more of the critical sodium transporters/channels of the renal tubule. Increased abundances of any of these transporters would, theoretically, increase the sodium transport capacity of the renal tubule and predispose these animals to hypertension. Our approach was to use semiquantitative immunoblotting to examine the relative abundances of eight different sodium transport proteins expressed along the renal tubule in obese Zucker rats compared with their lean age mates at two different ages.
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METHODS |
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Animals and study design. Twelve "lean" and 12 "obese" male Zucker rats were obtained (Charles River Laboratories, Wilmington, MA) at 6 wk of age. Rats were grouped at Charles River Laboratories according to weight at 5 wk of age. Thus lean rats included both heterozygous and homozygous rats for the normal gene. Six rats of each type were euthanized at 2 or 4 mo of age. These ages were selected because the rats would be fully mature, but still quite young, adult animals. Thus the kidney would be mature, and transporter/channel expression would be stabilized [transporter expression occurs relatively late in kidney development (58)]. Furthermore, the animals would not be predicted to have diabetes or any signs of diabetic nephropathy, which would likely complicate the interpretation of the data. Basic tests of renal salt and water handling were performed on all rats 1-2 wk before they were to be euthanized, including 1) a basal urine excretion test, 2) a urinary concentrating test, and 3) a sodium chloride loading test. Systolic blood pressure was measured by tail cuff (Manual Tail Electro-sphygmomanometer, PE-300, Narco BioSystems, Houston, TX) at 3 mo of age in surviving rats. Rats were given ad libitum access to standard commercial rat chow and water during the entire study, except where indicated. All animals were maintained at all times under conditions and protocols approved by the Georgetown University Animal Care and Use Committee, an American Association for Accreditation of Laboratory Animal Care approved facility.
Renal salt- and water-handling tests. For the basal urine excretion test, urine was collected for a 48-h period while rats were housed in metabolic cages (Nalgene, Harvard Apparatus, Holliston, MA) to assess urine volume, osmolality (freezing-point depression, Advanced Osmometer, model 3D3, Advanced Instruments, Norwood, MA), and creatinine (Jaffe rate method, Creatinine Analyzer 2, Beckman Diagnostic Systems Group, Brea, CA) under untreated conditions. To assess the rats' ability to maximally concentrate their urine, a urinary concentrating test was administered, as described previously (19). Briefly, rats were given an intramuscular injection of 2 nmol of 1-desamino-8-D-arginine vasopressin, a V2-selective vasopressin receptor agonist. After a 1-h-waiting period, urine was collected and analyzed for osmolality (Advanced Osmometer). At both 2 and 4 mo of age, to assess the rats' ability to excrete a saline load, 1 wk before euthanasia surviving rats were fasted and deprived of water for 3 h and then gavaged with 6 (2 mo) or 8 (4 mo) ml of 0.9% saline while under methoxyflurane anesthesia (Metofane, Schering-Plough Animal Health, Union, NJ). Urine was collected for the next 4 h (in 1- or 2-h increments) in metabolic cages, and volume and sodium concentration were measured (ion-selective electrode system, EL-ISE Electrolyte System, Beckman Instruments, Brea, CA).
Plasma and kidney sample preparation. Rats were euthanized by decapitation, and heparinized trunk blood was obtained for measurement of creatinine (Creatinine Analyzer 2), glucose, (clinical chemistry analyzer, Spectrum, Abbott Laboratories, Dallas, TX), insulin (RIA kit, Linco Research, St. Charles, MO), glucagon (RIA kit, Linco Research), aldosterone (RIA kit, Diagnostic Products, Los Angeles, CA), and vasopressin (utilizing an RIA and our own anti-vasopressin antibody) (57). Both kidneys were rapidly removed, and the whole left kidney and outer medulla and cortex of the right kidney were each prepared for immunoblotting according to previously published protocols (16, 17).
Semiquantitative immunoblotting and primary antibodies.
Semiquantitative immunoblotting techniques were the same as previously
described (16, 17). Immunoblots prepared from either whole
kidney, outer medulla, or cortex homogenates were probed with
antibodies against one of the following proteins: 1) NaPi-2 (37); 2) NHE3 (20, 35);
3) NCC (38); 4) NKCC2 (18,
36); ENaC 5) (45)-, 6)
(45)-, or 7)
-subunit (45);
or 8) the
1-subunit of Na-K-ATPase (Upstate
Biotechnology, cat. no. 074-1806, Lake Placid, NY).
Statistics. Relative intensities of the resulting immunoblot band densities were determined by laser scanning (Scanjet IIC scanner, Deskscan software, Hewlett Packard, Palo Alto, CA) followed by densitometry (NIH Image, Bethesda, MD). Densitometry data were normalized to percentage of lean mean. To assess effect of rat type within an age, the data were analyzed by unpaired t-test when they were normally distributed, otherwise by a Mann-Whitney Rank Sum Test (Sigma Stat, Chicago, IL). The effect of age or rat type (considering both ages) on plasma and urine parameters was determined by a two-way ANOVA (age × rat type) followed by a multiple comparisons test (Tukey's). P < 0.05 was considered statistically significant for all tests.
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RESULTS |
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Obese Zucker rats had increased blood pressure and responded
abnormally to a saline challenge.
At 3 mo of age, systolic blood pressure was significantly elevated in
the obese Zucker rats relative to their lean age mates (118 ± 4 vs. 104 ± 3 mmHg, P = 0.019, Fig.
1A). At 2 mo of age, obese
rats, in response to an acute saline load, had an increased ability to
excrete NaCl (Fig. 1B). In the 4-h period after the saline
load, obese rats excreted 131 ± 13% of the sodium load in their
urine, whereas the lean rats excreted only 58 ± 9%. (Data shown
are from rats killed at 2 mo of age; n = 6/rat type.)
In contrast, at 4 mo of age, the obese rats had a decreased ability to
excrete an acute saline load (Fig. 1C). The obese rats
excreted only 20 ± 10% of the entire sodium load in the entire
4-h period, and they produced virtually no urine between hours
1 and 2. In contrast, the lean rats excreted 45 ± 4% of their sodium load during the same 4-h period (P < 0.05).
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Obese rats had normal basic renal function but were markedly
hyperinsulinemic.
Table 1 shows a summary of body and
kidney weights, as well as various urinary parameters. As expected,
obese rats were significantly heavier than their lean age mates at both
2 and 4 mo of age. Also, as expected, basal urine volume was
significantly higher in the obese rats relative to their lean age mates
at both 2 and 4 mo. Urinary osmolality was reduced in the obese rats at
4 mo of age relative to their lean age mates. At 2 mo of age, obese
rats excreted significantly more osmoles in their urine than did their
lean age mates. Thus there was no difference in urine osmolality
between the two groups despite a nearly twofold greater urine volume in the obese rats. Urine creatinine excretion was greater in obese rats
relative to lean rats, suggesting greater muscle mass. There were no
differences in maximal urinary concentrating capacity between lean and
obese rats or between 2- and 4-mo-old rats.
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Abundance of the 1-subunit of Na-K-ATPase is
increased in the kidney cortex of obese Zucker rats whereas NHE3 and
NaPi-2 are unchanged.
Immunoblotting of kidney cortex homogenates was used to assess the
relative abundance of sodium transporters in the proximal tubule.
Before running immunoblots, Coomassie-stained "loading gels" were
performed on all sample sets, as previously described (14,
15) to ensure that equal amounts of total protein were loaded in
all lanes. Figure 2,
A-C, displays representative immunoblots of
cortex homogenate samples of rats euthanized at 4 mo of age. Each lane
was loaded with a sample from a different rat (6 lean and 6 obese), and
the immunoblots were probed with anti-NaPi-2, (Fig. 2A),
anti-NHE3 (Fig. 2B), and anti-
-Na-K-ATPase (Fig.
2C) antibodies, respectively. The relative densities of the
resulting specific bands were analyzed by laser densitometry, and the
summary of both the 2- and 4-mo data is shown in the bar graph (Fig.
2D). The relative abundances of the apical
sodium-transporter proteins NaPi-2 and NHE3 were not different between
the lean and obese rats at either age. However, the abundance of the
1-subunit of Na-K-ATPase was significantly increased in
the obese rats at both 2 and 4 mo of age. Densitometry values for the
obese rats as a percentage of the mean for the lean rats were as
follows: 2-mo rats, NaPi-2, 80 ± 7%, P = 0.090; NHE3, 95 ± 5%, P = 0.66;
-Na-K-ATPase, 196 ± 14%, P = 0.0019; 4-mo
rats, NaPi-2, 122 ± 16%, P = 0.30; NHE3,
119 ± 38%, P = 0.39; and
-Na-K-ATPase,
169 ± 22%, P = 0.027.
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No differences were found in abundances of sodium transporters of
the thick ascending limb.
The outer medullary homogenates were used to assess relative
differences in sodium transporter abundance(s) in the medullary thick
ascending limb (Fig. 3). Sample
immunoblots prepared from the rats euthanized at 4 mo of age are shown
in Fig. 3, A-C, probed with antibodies against
NKCC2, NHE3, and -Na-K-ATPase, respectively. The summary is shown in
3D. There were no significant changes in the abundances for
NKCC2,
-Na-K-ATPase, and NHE3 at either age. Densitometry values for
the obese rats as a percentage of the mean for the lean rats were as
follows: 2-mo rats, NKCC2, 145 ± 10%, P = 0.090; NHE3, 104 ± 10%, P = 0.76;
-Na-K-ATPase, 85 ± 7%, P = 0.21; 4-mo rats,
NKCC2, 116 ± 27%, P = 0.65; NHE3, 79 ± 8%, P = 0.39; and
-Na-K-ATPase, 129 ± 8%, P = 0.12.
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Abundance of the NCC is increased in obese Zucker rats.
Figure 4, A-B,
shows representative immunoblots loaded with cortex homogenate samples
probed with anti-NCC antibody. In Fig. 4, A and
B, respectively, are blots from 2- and 4-mo-old rats. At
both ages, the abundance of NCC was significantly higher in obese rats.
A summary of the densitometry obtained from immunoblotting is shown in
Fig. 4C. Densitometry values for obese rats as a percentage of the mean for the lean rats were as follows: 2-mo rats, 233 ± 38%, P = 0.010; and 4-mo rats, 164 ± 19%,
P = 0.020.
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-ENaC abundance is increased in obese Zucker rats.
Kidney abundances of ENaC subunits were evaluated in these rats
utilizing whole left kidney homogenates (Fig.
5). Figure 5, A-C,
shows immunoblots from 4-mo-old rats probed with anti-
(Fig. 3A)-, anti-
(Fig. 5B)-, or anti-
(Fig.
5C)-ENaC antibodies.
- and
-ENaC abundances were not
statistically different in the obese rats at 2 or 4 mo of age (Fig. 5,
A, C, D). However,
-ENaC abundance
was significantly increased in obese rats at both ages (Fig. 5,
B and D). Densitometry values for obese rats as a
percentage of the mean for lean rats were as follows: 2-mo rats,
-ENaC, 129 ± 16%, P = 0.16;
-ENaC, 161 ± 11%, P = 0.008;
-ENaC,
105 ± 4%, P = 0.47; 4-mo rats,
-ENaC,
101 ± 5%, P = 0.91;
-ENaC, 162 ± 21%,
P = 0.023; and
-ENaC, 120 ± 18%,
P = 0.41.
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DISCUSSION |
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In this report we evaluate the protein abundance of sodium
transporters and channels along the nephron and collecting duct by
semiquantitative immunoblotting in the obese Zucker rat at two
different ages. We found selective increases in the abundances of three
different sodium transporter proteins in the obese rats: 1)
the -subunit of Na-K-ATPase, 2) NCC, and 3)
the
-subunit of the ENaC. The relative increases in abundance of
these sodium transporters, without decreases in the other sodium
transporters, would be predicted to result in enhanced tubular sodium
reabsorption. As a result, these alterations in renal sodium
transporter abundance might play a role in the development and/or
maintenance of elevated blood pressures in these animals.
Increased cortical -Na-K-ATPase abundance.
Normally, ~65% of the filtered sodium load is reabsorbed in the
proximal tubule (39, 62). Active sodium extrusion from the
epithelial cell into the interstitium is carried out by the Na-K-ATPase
pump, which is essential for transepithelial sodium reabsorption. The
proximal tubule has been proposed to be a key site of sodium balance
dysregulation in both hypertensive humans and animals
(13). In our obese Zucker rats, the abundance of the
1-subunit of Na-K-ATPase was significantly increased in
rat kidney cortex at both 2 and 4 mo of age. The
-subunit is the catalytic and transporting subunit of the
-
complex
(42). The increase in
-subunit abundance in our
cortical samples is likely due to an increase in the proximal tubule,
because of the predominance of proximal tubules in the cortex. However,
increases in other segments cannot be ruled out by these studies.
Na-K-ATPase activity and/or abundance has been shown to be regulated by
several factors, including corticosteroids, catecholamines, insulin,
and vasopressin (59). Acute regulation is thought to be
primarily accomplished by trafficking and membrane insertion. Long-term or chronic regulation of Na-K-ATPase in the form of increased pump
number on the surface of the cells has been demonstrated in response to
insulin and steroids, such as aldosterone (59). Several
groups (21, 24, 29, 51) have studied regulation of
Na-K-ATPase in the obese Zucker rat. However, no one, to our knowledge,
has reported an increase in the abundance of kidney Na-K-ATPase. Using
immunoblotting techniques, Ferrer-Martinez et al. (21)
have shown increased abundance of the
1-subunit of
Na-K-ATPase in the intestinal mucosa and liver but not the kidney of
~2-mo-old obese Zucker rats. Hussain et al. (29) have shown a blunted Na-K-ATPase inhibition in response to dopamine, which
results in natriuresis in normal rats. However, they attributed this
difference to a decrease in the number of D1-like dopamine receptors and decreased activation of G proteins by dopamine and dopamine agonists, because they did not see an increase in the abundance of Na-K-ATPase with immunoblotting. Both insulin and aldosterone could be candidates for mediating the increased abundance of Na-K-ATPase in the present study. However, aldosterone levels were
not significantly different between lean and obese rats at either age
(Table 2). Insulin, on the other hand, was increased at both 2- and
4-mo of age in obese rats. However, our studies, in which we infused
insulin (4.4 U · kg
1 · day
1)
by osmotic minipump to normal Sprague-Dawley rats for 3 days, did not
result in an increase in the abundance of the
-subunit of
Na-K-ATPase (Ecelbarger CA, unpublished observations).
Furthermore, it is unclear how insulin resistance, which occurs in
these obese rats, affects the overall response to this high circulating insulin.
Increased NCC abundance. Fine tuning of sodium reabsorption occurs in the post-macula densa segments of the renal tubule, including the distal convoluted tubule and the collecting duct. The abundance of NCC of the distal convoluted tubule was significantly increased in the obese rats at both 2 and 4 mo of age. The abundance of this protein appears to be quite highly regulated. NCC abundance has already been shown to be increased in rat kidney by aldosterone infusion or use of a low-salt diet (38), nitric oxide synthase inhibition (60), estrogen infusion (63), insulin infusion (15), during escape from the antidiuretic action of vasopressin (17), and after streptozotocin treatment (65). As mentioned above, in our studies, plasma aldosterone levels were not measurably different at either age in the obese rats relative to their lean age mates. However, the obese rats were markedly hyperinsulinemic, due to compensation for insulin resistance. Increased sodium delivery to the distal tubule may also increase the abundance of this transporter. Bachmann et al. (2) have shown that furosemide treatment of mice increased NCC mRNA expression and protein abundance. Regardless of the mechanism, increased expression of this distal sodium transporter might predispose the obese rats to inappropriate sodium retention or blunted pressure natriuresis.
Increased -ENaC abundance.
The ENaC is vital for the reabsorption of sodium in the connecting
tubule and the collecting duct, especially when serum aldosterone levels are high. ENaC is found on the apical membrane of the principal cells and is composed of three distinct subunits:
,
, and
. Hormones that have been shown to increase the physiological activity of
ENaC include aldosterone (5, 41, 64), vasopressin,
(49), and insulin (5, 49, 64). In these
studies, we found a significant increase in the abundance of the
-subunit for ENaC in the obese Zucker rats. Previously this subunit
has been shown to be upregulated in rats by vasopressin or water
restriction (16), as well as during sodium-bicarbonate
loading in rats (37). The increase in
-ENaC in the
present studies, however, most likely was not vasopressin mediated,
because at 2 mo of age there was no difference in circulating
vasopressin levels and, in fact, at 4 mo of age, vasopressin levels
were significantly lower in the obese rats relative to their lean age
mates. However, the acid-base status of these rats was not evaluated.
Interestingly, we found no significant increase in the abundances for
the other two subunits of ENaC,
and
. The
-subunit of ENaC
has been shown to increase in abundance in response to a low-salt diet
or aldosterone infusion (45) during vasopressin escape
(17), as well as in response to insulin infusion
(15). Finally,
-ENaC abundance, like
, has been
shown to be increased by vasopressin (16) and
sodium-bicarbonate loading (37). Thus these results
provide another example of independent regulation of the ENaC subunit
proteins. The overall effect on collecting duct sodium transport of
increasing
-ENaC abundance without concomitant changes in the other
subunits is difficult to interpret. Studies by May et al.
(46) have suggested that the abundance of
-ENaC is
generally rate limiting for the assembly of the tetramer. Nevertheless,
in these complex in vivo models it would be difficult to ascertain the
relative abundance of these subunit proteins in relation to each other
and thus the physiological impact of increased
-ENaC. Further
studies will be needed to address this question.
Physiological impact of increased sodium transporter abundances.
Overall, we found increases in cortical 1-Na-K-ATPase
abundance and increases in the abundances of two post-macula densa sodium transport proteins, the NCC and the
-subunit of the
ENaC in the obese rats. Transport in the kidney can clearly be
regulated in ways other than by changes in the protein abundance of the transporters, such as via trafficking, phosphorylation, or proteolytic cleavage of the transporters. However, several examples exist in which
transport positively correlates with relative protein abundance, as
determined by immunoblotting. For example, increased aquaporin-2
expression in the principal cells of the collecting duct has clearly
been associated with increased water reabsorption from this segment
(12). Similarly, aldosterone increases the reabsorption of
sodium in the distal convoluted tubule and the collecting duct
(528), and aldosterone has recently been shown to increase
the abundance of NCC (38) and the
-subunit of ENaC (45) in these regions.
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ACKNOWLEDGEMENTS |
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We thank Dr. Harold Preuss and his laboratory for use of tail-cuff blood pressure equipment.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-38094 (J. G. Verbalis and C. A. Ecelbarger) and K01 DK-02672 (C. A. Ecelbarger), a George E. Schriener, MD Young Investigator Grant from the National Kidney Foundation (C. A. Ecelbarger), and a Research Award from the American Diabetes Association (C. A. Ecelbarger) at Georgetown University, as well as the intramural budget of the National Heart, Lung, and Blood Institute (M. A. Knepper).
Address for reprint requests and other correspondence: C. A. Ecelbarger, Bldg D, Rm. 232, Georgetown University, 4000 Reservoir Rd., NW, Washington, DC 20007 (E-mail: ecelbarc{at}georgetown.edu).
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. Section 1734 solely to indicate this fact.
Received 22 March 2001; accepted in final form 6 June 2001.
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