Increased renal Na-K-ATPase, NCC, and beta -ENaC abundance in obese Zucker rats

Crystal A. Bickel1, Joseph G. Verbalis1, Mark A. Knepper2, and Carolyn A. Ecelbarger1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 1-subunit of Na-K-ATPase, the thiazide-sensitive Na-Cl cotransporter (NCC or TSC), and the beta -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 alpha -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, alpha 1 and beta 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, alpha , beta , and gamma  (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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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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) alpha  (45)-, 6) beta  (45)-, or 7) gamma -subunit (45); or 8) the alpha 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.


    RESULTS
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ABSTRACT
INTRODUCTION
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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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Fig. 1.   A: systolic blood pressures in lean and obese Zucker rats at 3 mo of age as assessed by tail cuff. Obese rats had significantly increased blood pressures relative to lean age mates (n = 6/rat type). B and C: urinary sodium excretion (expressed as percentage of sodium load) in the 4 h after an acute NaCl load by gavage in 2 (B)- and 4 (C)-mo-old lean and obese Zucker rats shown as hourly or bihourly increments and as total sum of sodium excreted in the 4-h period. At 2 mo, obese rats had a significantly greater ability to excrete a saline load. Differences were significant in the 1- to 2-collection, 2- to 4-h collection and overall. At 4 mo, obese rats had decreased excretion of sodium in the 4-h period. Differences were significant overall, and in the 1- to 2-h collection (n = 6 rats/rat type/age). *Significantly different compared with lean rats, P < 0.05.

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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Table 1.   Physiological data in lean and obese Zucker rats

Plasma composition is summarized in Table 2. Obese Zucker rats were markedly hyperinsulinemic at both 2 and 4 mo of age, with the degree of hyperinsulinemia increasing dramatically between 2 and 4 mo of age. The lean Zucker rats did not show a similar increase in insulin levels between 2 and 4 mo. Glucagon levels were similarly elevated in the obese Zucker rats, relative to their lean age mates, but only in the rats killed at 4 mo of age. Plasma aldosterone levels were not affected by rat type, but aldosterone levels were significantly higher and vasopressin levels were significantly lower in the rats killed at 4 mo of age compared with 2 mo. Furthermore, vasopressin levels were significantly lower in the obese rats at 4 mo of age compared with lean age mates. Corticosterone and plasma creatinine levels were not affected by rat type. No difference in plasma creatinine levels between lean and obese rats would suggest that the kidneys of the obese rats at 4 mo did not have deficient filtration capability. Glucose levels were significantly elevated in the 4-mo-old obese rats relative to their lean age mates, although the mean in the obese rats was not high enough to be considered clinically diabetic (fed level > 11 mmol/l). Creatinine clearance for rats at 4 mo of age was not different between lean and obese rats (1.80 ± 0.27 and 2.06 ± 0.13 l/day, respectively). Because creatinine clearance provides an estimate of glomerular filtration rate (GFR), this suggests that GFR was not different between lean and obese rats at 4 mo of age. (Note that insufficient plasma was collected from the 2-mo-old rats to measure creatinine, glucose, and corticosterone.)

                              
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Table 2.   Plasma parameters in lean and obese Zucker rats

Abundance of the alpha 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-alpha -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 alpha 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; alpha -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 alpha -Na-K-ATPase, 169 ± 22%, P = 0.027. 


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Fig. 2.   Immunoblots (A-C) and summary of immunoblot data (D) for proximal tubule sodium transporters. Representative blots are from 4-mo-old rats; 2-mo data are presented in D. For each blot, each lane was loaded with cortex homogenate from a different rat. Equal amounts of protein were loaded in all lanes. Immunoblots were probed with anti-sodium-phosphate cotransporter type II (NaPi-2; A) (37), anti-sodium-hydrogen exchanger type III (NHE3; B) (20, 35), or anti-alpha -Na-K-ATPase antibodies (C). The density for the specific bands for alpha -Na-K-ATPase was increased in obese rats at both 2 and 4 mo of age. Relative abundance of NaPi-2 and NHE3 was not affected by rat type. *Significantly different compared with lean rats, P < 0.05.

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 alpha -Na-K-ATPase, respectively. The summary is shown in 3D. There were no significant changes in the abundances for NKCC2, alpha -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; alpha -Na-K-ATPase, 85 ± 7%, P = 0.21; 4-mo rats, NKCC2, 116 ± 27%, P = 0.65; NHE3, 79 ± 8%, P = 0.39; and alpha -Na-K-ATPase, 129 ± 8%, P = 0.12. 


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Fig. 3.   Immunoblots (A-C) and summary of immunoblot data (D) for outer medullary thick ascending limb sodium transporters. Representative blots are from 4-mo-old rats, 2-mo data (D). For each blot, each lane was loaded with outer medullary homogenate from a different rat. Equal amounts of protein were loaded in all lanes. Immunoblots were probed with anti-bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2; A) (18, 36), anti-NHE3 (B) (20, 35), or anti-alpha -Na-K-ATPase antibodies (C). There were no differences in relative abundances for any of these proteins.

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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Fig. 4.   Immunoblots [2 (A)- and 4-mo (B)] and band density summary (C) for the thiazide-sensitive Na-Cl cotransporter (NCC). For each blot, each lane was loaded with whole kidney homogenate from a different rat. Equal amounts of protein were loaded in all lanes. Blots were probed with anti-NCC antibody (38). NCC abundance was significantly increased in obese rats at both ages. *Significantly different compared with lean rats, P < 0.05.

beta -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-alpha (Fig. 3A)-, anti-beta (Fig. 5B)-, or anti-gamma (Fig. 5C)-ENaC antibodies. alpha - and gamma -ENaC abundances were not statistically different in the obese rats at 2 or 4 mo of age (Fig. 5, A, C, D). However, beta -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, alpha -ENaC, 129 ± 16%, P = 0.16; beta -ENaC, 161 ± 11%, P = 0.008; gamma -ENaC, 105 ± 4%, P = 0.47; 4-mo rats, alpha -ENaC, 101 ± 5%, P = 0.91; beta -ENaC, 162 ± 21%, P = 0.023; and gamma -ENaC, 120 ± 18%, P = 0.41. 


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Fig. 5.   Immunoblots (A-C) and band density summary (D) for the 3 subunits of the epithelial sodium channel (ENaC). Representative blots are from 4-mo-old rats; 2-mo data are presented in D. For each blot, each lane was loaded with whole kidney homogenate from a different rat. Equal amounts of protein were loaded in all lanes. Blots were probed with anti-alpha -ENaC antibody (A) (45), anti-beta -ENaC antibody (B) (45), or anti-gamma -ENaC antibody (C) (45). The mean density for the band corresponding to beta -ENaC (90 kDa) was significantly increased in the obese rats at both 2 and 4 mo of age. There were no significant differences in band densities for alpha - or gamma -ENaC. *Significantly different compared with lean rats, P < 0.05.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -subunit of Na-K-ATPase, 2) NCC, and 3) the beta -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 alpha -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 alpha 1-subunit of Na-K-ATPase was significantly increased in rat kidney cortex at both 2 and 4 mo of age. The alpha -subunit is the catalytic and transporting subunit of the alpha -beta complex (42). The increase in alpha -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 alpha 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 alpha -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.

In addition, when predicting how changes in Na-K-ATPase abundance might affect sodium reabsorption, one must consider another factor. The Zucker rat is considered a low-renin model of hypertension (27), and in other such models, e.g., the "one-kidney, one-wrap" model, there is an increase in the circulating levels of ouabain- or digitalis-like substances that inhibit Na-K-ATPase activity (9, 25, 48, 67). These substances are proposed to contribute to the hypertension by causing blood vessel constriction and increased cardiac contractility (67). However, whether renal Na-K-ATPase activity is inhibited in these models is controversial. Pamnani et al. (48) reported that in one-kidney, one-wrap dogs, although circulating levels of an ouabain-like substance were increased, renal Na-K-ATPase activity was unchanged. However, de Mendonca et al. (9) have reported decreased renal Na-K-ATPase activity in rats that developed hypertension after renal ablation and high-salt intake. Therefore, the role of endogenous inhibitors of Na-K-ATPase in determining renal Na-K-ATPase activity and thus sodium reabsorption in the obese Zucker rat is not clear. Overall, further studies are needed to address potential mechanisms of the observed increase in cortical Na-K-ATPase alpha 1-subunit abundance and how this may relate to Na-K-ATPase activity, sodium reabsorption, and blood pressure in these animals.

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 beta -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: alpha , beta , and gamma . 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 beta -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 beta -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, alpha  and gamma . The alpha -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, gamma -ENaC abundance, like beta , 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 beta -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 alpha -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 beta -ENaC. Further studies will be needed to address this question.

Physiological impact of increased sodium transporter abundances. Overall, we found increases in cortical alpha 1-Na-K-ATPase abundance and increases in the abundances of two post-macula densa sodium transport proteins, the NCC and the beta -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 alpha -subunit of ENaC (45) in these regions.

With regard to our findings in obese Zucker rats, we would, therefore, predict that a greater percentage of the filtered sodium load would be reabsorbed throughout the renal tubule. In agreement with this prediction, at 4 mo of age the obese rats had a markedly blunted ability to excrete sodium during the saline challenge relative to the lean rats, despite what appears to be normal GFR. This finding is also in agreement with previous investigators (22) who have demonstrated that pressure natriuresis is shifted and/or blunted in obese Zucker rats. Furthermore, West et al. (66) and DiBona et al. (10) have demonstrated decreased excretion of an acute saline load in obese dogs and rats with mild congestive heart failure, respectively.

Nevertheless, at 2 mo of age our obese Zucker rats had an apparent increased capacity to excrete an acute saline load (Fig. 1B). This phenomenon of "exaggerated natriuresis" has been well described in the spontaneously hypertensive rat (11, 50) and in hypertensive humans (43). It has been proposed to be due to decreased reabsorption in the thick ascending limb (11, 43), likely the result of enhanced inhibition of renal sympathetic nerve activity (50). It is not clear what is different in our Zucker rats at 4 vs. 2 mo of age that might cause this loss of ability to rapidly excrete an acute saline load. DiBona and colleagues (10) have suggested that angiotensin II may play a role in this same defect in rats with mild congestive heart failure. Overall, these findings suggest that there are clear differences in relative abundance of renal sodium transporters and channel subunits in obese vs. lean Zucker rats, which are evident in young adulthood and persist. These changes may play a role in the development and maintenance of elevated blood pressure in the obese Zucker rat.


    ACKNOWLEDGEMENTS

We thank Dr. Harold Preuss and his laboratory for use of tail-cuff blood pressure equipment.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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