Vasopressin increases Na-K-2Cl cotransporter expression in thick ascending limb of Henle's loop

Gheun-Ho Kim1, Carolyn A. Ecelbarger1, Carter Mitchell1, Randall K. Packer2, James B. Wade1,3, and Mark A. Knepper1

1 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892; 2 Department of Biological Sciences, George Washington University, Washington, District of Columbia 20052; and 3 Department of Physiology, University of Maryland College of Medicine, Baltimore, Maryland 21201

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

To investigate whether the enhancement of thick ascending limb (TAL) NaCl transport in response to long-term increases in circulating vasopressin concentration is associated with increased expression levels of the apical Na-K-2Cl cotransporter in the rat TAL, we have carried out immunoblotting and immunofluorescence studies using affinity-purified, peptide-directed antibodies. Semiquantitative immunoblotting studies demonstrated a marked increase (193% of controls) in Na-K-2Cl cotransporter band density in response to restriction of water intake to 15 ml/day for 7 days. In contrast, the expression levels of two other apical proteins of the TAL (the type 3 Na/H exchanger and Tamm-Horsfall protein) were unchanged in the outer medulla. A 7-day subcutaneous infusion of the V2 receptor-selective vasopressin analog, 1-desamino-[8-D-arginine]vasopressin (DDAVP), to Brattleboro rats also markedly increased Na-K-2Cl cotransporter expression in the outer medulla (183% of controls). Immunofluorescence localization in outer medullary tissue sections confirmed the increase in Na-K-2Cl cotransporter expression in response to DDAVP. We conclude that vasopressin strongly upregulates the expression of the Na-K-2Cl cotransporter of the TAL and that it is likely to play an important role in the long-term regulation of the countercurrent multiplication system.

antidiuretic hormone; countercurrent multiplication; sodium chloride transport

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE NEUROHYPOPHYSIAL hormone, vasopressin, has been demonstrated to increase the rate of active NaCl absorption in isolated perfused thick ascending limbs (TAL) from rodent kidneys (13, 15, 27), an effect seen within a few minutes of exposure of the TAL epithelium to the hormone. This effect is believed to be mediated by V2 receptors, which are coupled to adenylyl cyclase activation in the TAL (18). In addition, long-term infusions of vasopressin in vasopressin-deficient Brattleboro rats have been shown to be associated with stable increases in the rate of Cl absorption by the TAL (3). This long-term adaptation of TAL transport is likely to play a role in the long-term enhancement of urinary concentrating ability that occurs in response to a prolonged antidiuretic stimulus, a phenomenon originally demonstrated in the 1950s by Jones and DeWardener (20) and Epstein and colleagues (9). The molecular target for the long-term regulation of TAL NaCl absorption has not been identified. However, the short-term action of vasopressin to increase NaCl absorption by the TAL has been demonstrated to be in part due to direct stimulation of the apical Na-K-2Cl cotransporter (14, 24), the chief apical entry pathway for Na and Cl in the TAL. We conjecture that the long-term effect of vasopressin to increase NaCl absorption in the TAL may also be a result of regulation of the apical Na-K-2Cl cotransporter. Specifically, we hypothesize that long-term increases in circulating vasopressin levels may increase the expression level of the Na-K-2Cl cotransporter in the TAL, a process hypothetically analogous to the previously demonstrated long-term regulation of the collecting duct water channel, aquaporin-2, by vasopressin (6, 25, 31). To address this hypothesis, we have employed polyclonal antibodies to the apical Na-K-2Cl cotransporter (and to other TAL transporters) in semiquantitative immunoblotting and immunofluorescence localization studies in rats.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Polyclonal Antibodies

This study employs two different peptide-directed rabbit polyclonal antibodies that specifically recognize the TAL isoform of Na-K-2Cl cotransporter in rat: 1) L224 (8), which recognizes amino acids 109-129 of the rat Na-K-2Cl cotransporter sequence reported by Gamba et al. (11), and 2) L320, which is raised against a synthetic peptide corresponding to amino acids 33-55 of the rat cotransporter characterized here (see RESULTS). All Na-K-2Cl cotransporter immunoblots presented in this study were done initially with L320 and repeated with L224 to ensure that the findings are not dependent on the epitope recognized. In addition, the present studies utilized rabbit polyclonal antibodies against the type 3 Na/H exchanger (NHE3) (10), Tamm-Horsfall protein (19), aquaporin-2 (6), aquaporin-3 (7), and the alpha 1-subunit of rat Na-K-ATPase (Upstate Biotechnology, Lake Placid, NY), as well as a rabbit antiserum raised against the beta 1-subunit of rat Na-K-ATPase (Upstate Biotechnology). The antisera to the Na-K-2Cl cotransporter, NHE3, aquaporin-2, and aquaporin-3 were affinity-purified using columns on which 2 mg of the appropriate synthetic peptides were immobilized via covalent linkage to maleimide-activated agarose beads (immunobilization kit no. 2; Pierce, Rockford, IL).

Animals and Experimental Protocols

Pathogen-free male Sprague-Dawley rats (Taconic Farms, Germantown, NY) weighing 195-225 g were used in a chronic water restriction study, and male Brattleboro (di/di) rats (Harlan Sprague Dawley, Indianapolis, IN) weighing 210-260 g were used in a long-term infusion study of 1-desamino-[8-D-arginine]vasopressin (DDAVP). These rats were placed in metabolism cages for the physiological studies described here. All animal procedures have been approved by the National Heart, Lung, and Blood Institute Animal Care and Use Committee.

Water restriction study. Water restriction for 7 days was employed to produce a physiological increase in the circulating vasopressin level. Six control rats and six treated rats were chosen randomly, and the two groups were provided with the same amount of rat chow containing a different amount of water. For control rats, 15 g/200 g body wt per day of finely ground rat chow (NIH-07; Zeigler, Gardners, PA) was made into a paste by the addition of water (37 ml/200 g body wt per day). For water-restricted rats, the same rat chow (15 g/200 g body wt per day) was mixed well in a small amount of water (15 ml/200 g body wt per day). The amount of water given to water-restricted rats was determined empirically to be sufficient to replace nonrenal water losses (chiefly respiratory losses) plus a small amount of urine daily. Animals were fed once daily and ate all of the offered food-water slurry. Aside from the water added to the food, the rats did not have access to drinking water. All the rats showed a steady increase in body weight during the 7-day period of observation. On the final day, a 24-h urine collection was made for determination of the urine osmolality using a vapor pressure osmometer (Wescor, Logan, UT). After 7 days of treatment, the rats were killed for semiquantitative immunoblotting as described below.

DDAVP infusion study. Under methoxyflurane anesthesia (Metofane; Pitman-Moore, Mundelein, IL), osmotic minipumps (model 2002; Alzet, Palo Alto, CA) were implanted subcutaneously in six Brattleboro rats to deliver 20 ng/h of DDAVP (Rhone-Poulenc Rorer, Collegeville, PA), a V2 vasopressin receptor-selective agonist. Another six rats (control) were implanted with minipumps containing vehicle (saline) alone. After 7 days of DDAVP or vehicle infusion, during which time rats received water and pelleted chow ad libitum, all rats were killed by decapitation for semiquantitative immunoblotting as described below.

Semiquantitative Immunoblotting

Preparation of protein samples for immunoblotting. Semiquantitative immunoblotting was carried out to assess relative expression levels of transporter protein in homogenates prepared from the outer medulla of rat kidneys prepared as described below. The rats were killed by decapitation, and right kidneys were rapidly removed and placed in chilled isolation solution containing 250 mM sucrose, 10 mM triethanolamine (Calbiochem, La Jolla, CA), 1 µg/ml leupeptin (Bachem, Torrance, CA), and 0.1 mg/ml phenylmethylsulfonyl fluoride (US Biochemical, Toledo, OH) titrated to pH 7.6. Next, the kidneys were dissected to obtain the inner stripe of the outer medulla. Tissue samples were homogenized in ice-cold isolation solution using a tissue homogenizer (Omni 2000 fitted with a micro-sawtooth generator). After homogenization, protein concentration was measured using the Pierce bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL) and adjusted to 1.2 µg/µl with isolation solution. The samples were then solubilized by adding 1 vol 5× Laemmli sample buffer per 4 vol of sample and heating to 60°C for 15 min.

Electrophoresis and immunoblotting of membranes. In all cases, to confirm equality of loading among lanes, electrophoresis was initially run for the entire set of samples in a given experiment on a single 12% polyacrylamide-SDS gel, which was then stained with Coomassie blue. Selected bands from these gels were analyzed by densitometry (Molecular Dynamics, San Jose, CA) to provide quantitative assessment of loading. These loading gels established that subsequent immunoblots (loaded proportionately) were uniformly loaded. For immunoblotting, outer medullary homogenate samples were solubilized at 60°C for 15 min in Laemmli sample buffer, and SDS-PAGE was performed. We used 7.5% polyacrylamide minigels to assess Na-K-2Cl cotransporter or Na-K-ATPase alpha 1-subunit protein expression, 10% polyacrylamide minigels for assessment of NHE3 or Tamm-Horsfall protein expression, and 12% polyacrylamide minigels to assess aquaporin-2, aquaporin-3, or Na-K-ATPase beta -subunit expression. The proteins were transferred from the gels electrophoretically to nitrocellulose membranes. After blocking with 5 g/dl nonfat dry milk, we probed proteins overnight at 4°C with the desired antibody at the following IgG concentrations (in µg/ml): 0.12 for Na-K-2Cl cotransporter antibodies, 0.12 for aquaporin-2, 0.10 for aquaporin-3, 0.40 for NHE3, and 0.20 for Na-K-ATPase alpha 1-subunit. Whole antiserum was used for Na-K-ATPase beta -subunit immunoblots at a dilution of 1:1,000. The antibodies were prepared in an antibody diluent containing 150 mM NaCl, 50 mM sodium phosphate, 10 mg/dl sodium azide, 50 mg/dl Tween 20, and 1 g/dl BSA, pH 7.5. The secondary antibody was donkey anti-rabbit IgG conjugated to horseradish peroxidase (Pierce 31458), used at a concentration of 0.16 µg/ml. Sites of antibody-antigen reaction were visualized using luminol-based enhanced chemiluminescence (LumiGLO; Kirkegaard and Perry Laboratories, Gaithersburg, MD) before exposure to X-ray film (Kodak 165-1579 scientific imaging film).

Statistical analysis. Relative quantitation of the band densities from immunoblots was carried out by densitometry using a laser densitometer (Molecular Dynamics) and ImageQuaNT software (Molecular Dynamics). To facilitate comparisons, we normalized the band density values by dividing by the mean value for the control group. Thus the mean for the control group is defined as 100%. Normalized band densities for treated rats were compared with controls using an unpaired t-test when SDs were the same or by Welch t-test when SDs were significantly different (INSTAT; Graphpad Software, San Diego, CA). P < 0.05 was considered statistically significant.

Immunofluorescence Immunocytochemistry

Tissue for immunocytochemistry was taken from eight 140-200 g male Brattleboro rats treated either with a chronic infusion of DDAVP (4 rats) or vehicle (4 rats) as described in Animals and Experimental Protocols. The kidneys were fixed with 2% paraformaldehyde by perfusion (2 min with PBS and 5 min with 2% paraformaldehyde in PBS) followed by perfusion for 2 min with a cryoprotectant consisting of 10% EDTA in 0.1 M Tris. Each fixed kidney was sliced into 3-4 pieces and further incubated in the EDTA cryoprotectant for 1 h. Kidney slices were then wrapped in aluminum foil, frozen on dry ice, and stored at -70°C in an airtight container. Cryostat sections 12-15 µm thick were made and picked up on coverslips treated with HistoGrip (Zymed, San Francisco, CA). Sections were treated with 6 M guanidine for 10 min to uncover antigenic sites and washed three times with high-salt solution (50 ml PBS, 0.5 g BSA, and 1.13 g NaCl). A blocking agent for nonspecific binding sites (50 ml PBS, 0.5 g BSA, and 0.188 g glycine, pH 7.2) was then added to the sample for 20 min followed by primary antibody (L320) diluted to 10 µg/ml with incubation medium (50 ml PBS, 0.05 g BSA, and 200 ml 5% NaN3). After overnight incubation at 4°C, these sections were rinsed three times for 5 min, once for 15 min, and once for 30 min with high-salt solution before incubation with the secondary antibody, donkey anti-rabbit IgG conjugated to FITC, diluted to 1:100 with incubation medium (Jackson Immunoresearch Labs, West Grove, PA), for 2 h at 4°C. The samples were again washed five times with high-salt solution over the course of 1 h and then with PBS to remove the excess salt before mounting. Labeled sections were examined with a Zeiss LSM410 confocal microscope.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Na-K-2Cl Cotransporter Antibody

In this study, we describe a new rabbit polyclonal anti-Na-K-2Cl cotransporter antibody, L320, raised to a synthetic peptide corresponding to a portion of the amino-terminal tail of the cotransporter. Figure 1 shows two immunoblots each loaded with renal cortical and outer medullary homogenates from a rat. The blots were probed with L320 (left) and with L320 preadsorbed with an excess of the immunizing peptide (right). The antibody recognizes a predominant band of ~160 kDa in outer medulla, as previously described with another anti-Na-K-2Cl cotransporter antibody, L224 (8). With longer exposures (not shown), this band can also be identified in cortex. In addition, a weak higher molecular weight band is seen in outer medulla. This has been identified as a dimer of the cotransporter (23). Both the 160-kDa band and the higher molecular weight band disappeared when the L320 antibody was preadsorbed with an excess of the immunizing peptide. Further characterization using immunoprecipitation (data not shown) and immunocytochemistry (see below) showed that labeling with L320 produces results virtually identical to those seen with L224.


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Fig. 1.   Immunoblots probed with anti-Na-K-2Cl cotransporter antibody L320 (left) and antibody L320 preadsorbed with immunizing peptide (right). Immunoblots were loaded with homogenates from cortex (2 µg) and outer medulla (OM; 2 µg) of Sprague-Dawley rat. IgG concentration of L320 was 0.12 µg/ml for both blots. For preadsorption, 1 mg of peptide was incubated with antibody.

Chronic Water Restriction in Sprague-Dawley Rats

Restriction of water intake normally triggers a rise in the circulating vasopressin level, which results in a homeostatic decrease in renal water excretion. To assess the effect of chronic water restriction on medullary TAL Na-K-2Cl cotransporter expression in Sprague-Dawley rats, we ran semiquantitative immunoblots using outer medullary homogenates from six control rats receiving 37 ml/200 g body wt of water per day and from six water-restricted rats receiving 15 ml/200 g body wt of water per day for 7 days.1 Urine osmolality was markedly higher in water-restricted rats compared with controls, as expected (2,913 ± 175 vs. 549 ± 22 mosmol/kgH2O, respectively, P < 0.0001).

To ensure that this protocol reproduced the previously observed long-term effects of fluid restriction on aquaporin-2 (25, 31) and aquaporin-3 (7, 31) abundance, we carried out semiquantitative immunoblotting for both water channels (Fig. 2). As shown in Fig. 2A, water restriction for 7 days indeed resulted in a large increase in aquaporin-2 expression in the outer medulla. Normalized band densities for water-restricted and control rats were 575 ± 146 and 100 ± 21%, respectively (P < 0.05). Furthermore, as shown in Fig. 2B, the 7-day water restriction protocol also resulted in a large increase in aquaporin-3 expression in the outer medulla (455 ± 125% for water-restricted rats vs. 100 ± 32% for control rats, P < 0.05). The changes in aquaporin-2 and -3 expression were qualitatively similar to those previously obtained with a 24-h thirsting protocol (31). Consequently, we conclude that the moderate fluid restriction protocol introduced in this study is efficacious in bringing about adaptive changes in aquaporin expression similar to those that have previously been attributed to long-term elevation in circulating vasopressin levels due to thirsting.


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Fig. 2.   Effect of restriction of water intake on expression of aquaporin-2 (A) and aquaporin-3 (B) in rat renal outer medulla. Immunoblots of outer medullary homogenates from Sprague-Dawley rats. Control rats received 37 ml/200 g body wt per day of water, and water-restricted rats received 15 ml/200 g body wt per day of water for 7 days. Each lane was loaded with protein sample from a different rat. Parallel 12% Coomassie blue-stained SDS-polyacrylamide gels confirmed equal loading among lanes. A: blot was loaded with 5 µg total protein/lane and was probed with rabbit polyclonal anti-aquaporin-2 antibody L127. Band density for aquaporin-2 protein was significantly increased by chronic restriction of water intake (see RESULTS). B: blot was loaded with 10 µg total protein/lane and was probed with the rabbit polyclonal anti-aquaporin-3 antibody L178. Band density for aquaporin-3 protein was significantly increased by chronic restriction of water intake (see RESULTS).

The same outer medullary samples were subjected to semiquantitative immunoblotting for the apical Na-K-2Cl cotransporter using anti-Na-K-2Cl cotransporter antibody L320 (Fig. 3). Consistent with the major hypothesis of this study, the normalized Na-K-2Cl cotransporter band density in water-restricted rats was significantly increased to 193 ± 32% (P < 0.05) compared with controls (100 ± 12%), which is indicative of a large increase in cotransporter protein abundance. A similar increase was also seen when the blot was repeated with a different anti-Na-K-2Cl cotransporter antibody (L224, data not shown). A parallel Coomassie-stained SDS-PAGE gel demonstrated uniform loading among all samples (data not shown), ruling out the possibility that the increase in Na-K-2Cl cotransporter band density in either outer medulla or cortex could be due to differences in loading.


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Fig. 3.   Effect of restriction of water intake on expression of thick ascending limb (TAL) form of Na-K-2Cl cotransporter in rat renal outer medulla. Immunoblot of same outer medullary homogenates from Sprague-Dawley rats as used for Fig. 2. Blot was loaded with 2 µg total protein/lane and was probed with rabbit polyclonal anti-Na-K-2Cl cotransporter antibody L320. Band density for Na-K-2Cl cotransporter protein was significantly increased by chronic restriction of water intake (see RESULTS).

To determine whether the demonstrated increase in Na-K-2Cl cotransporter expression in the rat renal outer medulla was selective for the Na-K-2Cl cotransporter or was part of a generalized increase in expression of all apical plasma membrane proteins of the TAL, we carried out semiquantitative immunoblotting for two other apically located proteins expressed in the TAL, NHE3 (Fig. 4A) and Tamm-Horsfall protein (Fig. 4B). There was little or no change in the expression level of either of these proteins. NHE3 band densities for water-restricted rats vs. control rats were 128 ± 21 vs. 100 ± 6%, respectively. Tamm-Horsfall band densities for water-restricted rats vs. control rats were 98 ± 15 vs. 100 ± 33%, respectively. Therefore, among apical membrane proteins assessed in this study, a large increase in abundance was seen only for the Na-K-2Cl cotransporter, indicating that water restriction had a relatively selective effect on Na-K-2Cl cotransporter expression.


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Fig. 4.   Effect of restriction of water intake on expression of type 3 Na/H exchanger (NHE3, A) and Tamm-Horsfall protein (B) in rat renal outer medulla. Immunoblots of same outer medullary homogenates from Sprague-Dawley rats as used for Figs. 2 and 3. A: blot was loaded with 10 µg total protein/lane and was probed with rabbit polyclonal anti-NHE3 antibody L546. Band density for NHE3 protein was not significantly altered by chronic restriction of water intake (see RESULTS). B: blot was loaded with 5 µg total protein/lane and was probed with rabbit polyclonal Tamm-Horsfall protein antibody. Band density for Tamm-Horsfall protein was not significantly changed by chronic restriction of water intake (see RESULTS).

The primary active transporter that drives net NaCl absorption across the TAL epithelium is the Na-K-ATPase. Among nephron segments in the inner stripe of the outer medulla, the TAL has the greatest Na-K-ATPase abundance and TAL segments are thought to account for at least 90% of the total Na-K-ATPase in the outer medulla.2 Therefore, measurements of Na-K-ATPase subunit abundance in the outer medulla are tantamount to measurement of the abundance in the TAL. Figure 5A shows a semiquantitative immunoblot for the alpha 1-subunit of Na-K-ATPase in the same outer medullary samples as used for Figs. 2-4. There was a moderate increase in band density in water-restricted rats vs. controls. Normalized band densities for water-restricted rats vs. controls were 157 ± 14 vs. 100 ± 11%, respectively (P < 0.05). Figure 5B shows a semiquantitative immunoblot for the beta 1-subunit of Na-K-ATPase in the same samples. The expression level of the beta 1-subunit did not change significantly. Normalized band densities for water-restricted rats vs. controls were 201 ± 75 vs. 100 ± 13%, respectively (P > 0.05).


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Fig. 5.   Effect of restriction of water intake on expression of alpha 1-subunit (A) and beta -subunit (B) of Na-K-ATPase in rat renal outer medulla. Immunoblots of same outer medullary homogenates from Sprague-Dawley rats as used for Figs. 2-4. A: blot was loaded with 20 µg total protein/lane and was probed with rabbit polyclonal anti-alpha 1-subunit antibody. Band density for anti-alpha 1-subunit of Na-K-ATPase was significantly increased by chronic restriction of water intake (see RESULTS). B: blot was loaded with 15 µg total protein/lane and was probed with rabbit polyclonal antiserum to beta -subunit of Na-K-ATPase. Band density was not significantly changed by chronic restriction of water intake (see RESULTS).

Long-Term Administration of DDAVP in Brattleboro Rat

The demonstrated changes in Na-K-2Cl cotransporter expression in response to restriction of water intake for 7 days could be due to the associated increase in circulating vasopressin or to other concomitant factors. To provide a test of the role of vasopressin, the V2 receptor-selective vasopressin analog, DDAVP, was subcutaneously infused to Brattleboro rats for 7 days. Infusion of DDAVP markedly decreased urine volume from 230 ± 21 to 11 ± 2 ml/day (P < 0.0005). Urinary osmolality (mosmol/kgH2O) was markedly elevated in DDAVP-infused Brattleboro rats vs. controls (1,168 ± 164 vs. 111 ± 7, respectively, P < 0.005), demonstrating the efficacy of the DDAVP infusion in creating an antidiuretic state. Immunoblots to assess the expression levels of collecting duct aquaporins showed that aquaporin-2 band density was dramatically increased to 1,074 ± 62% of vehicle-infused controls (100 ± 7%), whereas aquaporin-3 band density was increased to 2,156 ± 560% of the vehicle-infused controls (100 ± 8%) (data not shown). These findings are wholly consistent with expectations from prior studies (6, 31) and reinforce the view that the DDAVP infusions resulted in a chronic increase in V2 receptor occupation.

Figure 6A shows the effect of DDAVP infusion on the expression level of the apical Na-K-2Cl cotransporter in the outer medulla. As seen previously with chronic restriction of water intake, DDAVP infusion resulted in a large increase in Na-K-2Cl cotransporter expression. Normalized band densities for DDAVP infusion vs. controls were 183 ± 16 vs. 100 ± 9%, respectively (P < 0.005). An immunoblot for the TAL Na-K-2Cl cotransporter in the cortex of the same animals also revealed a large increase in expression (Fig. 6B). Normalized band densities for DDAVP infusion vs. controls were 247 ± 30 vs. 100 ± 13% (P < 0.005). Parallel Coomassie-stained SDS-PAGE gels demonstrated uniform loading among outer medullary samples and among cortical samples (data not shown), ruling out the possibility that the increase in Na-K-2Cl transporter band density in either outer medulla or cortex could be due to differences in loading.


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Fig. 6.   Effect of 1-desamino-[8-D-arginine]vasopressin (DDAVP) infusion on expression of TAL form of Na-K-2Cl cotransporter in renal outer medulla (A) and renal cortex (B) of Brattleboro rats. Immunoblots of outer medullary homogenates (A) and cortical homogenates (B) from Brattleboro rats receiving a subcutaneous infusion of DDAVP (20 ng/h) or vehicle by osmotic minipump. Each lane was loaded with protein sample from a different rat. Parallel 12% Coomassie blue-stained SDS-polyacrylamide gels confirmed equal loading among lanes. Blots were probed with rabbit polyclonal anti-Na-K-2Cl cotransporter antibody L320. A: blot of outer medulla was loaded with 2 µg total protein/lane. Band density for Na-K-2Cl cotransporter was significantly increased by DDAVP infusion (see RESULTS). B: blot of cortex was loaded with 7 µg total protein/lane. Band density for Na-K-2Cl cotransporter protein was significantly increased by DDAVP infusion (see RESULTS).

Figure 7 shows immunofluorescence labeling for the Na-K-2Cl cotransporter (L320 antibody) of outer medullary (inner stripe) tissue sections from vehicle-infused (Fig. 7A) and DDAVP-infused (Fig. 7B) Brattleboro rats. Labeling conditions and settings for the confocal microscope were identical for Fig. 7, A and B. The intensity of Na-K-2Cl cotransporter labeling was substantially greater in the TAL of DDAVP-treated than vehicle-infused Brattleboro rats, consistent with the findings obtained by semiquantiative immunoblotting. Note also that the TAL tubule diameters tended to be greater in the DDAVP-treated Brattleboro rats, confirming previous observations that chronic vasopressin treatment causes hypertrophy of this segment (1, 4, 22). In addition, examination of the data from the immunoblotting studies revealed that the total amount of outer medullary protein measured in homogenates of the inner stripes of outer medullas dissected from kidneys of DDAVP-treated rats (n = 6) was substantially greater than corresponding values for vehicle-treated rats (5.31 ± 0.30 vs. 3.85 ± 0.18 mg, respectively, P < 0.005), consistent with the previously demonstrated DDAVP-induced outer medullary hypertrophy (1, 4, 22).


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Fig. 7.   Immunofluorescence localization of Na-K-2Cl cotransporter in inner stripe of outer medulla from Brattleboro rats infused with vehicle (A) or DDAVP (B), showing an increase in Na-K-2Cl cotransporter labeling of medullary TAL from DDAVP-treated rats. Labeling conditions and confocal microscope settings were identical for A and B. Scale bar, 25 µm.

As in the water-restriction experiment, outer medullary samples from DDAVP-infused and vehicle-infused rats were subjected to semiquantiative immunoblotting for two other apically located proteins expressed in the TAL, NHE3 (Fig. 8A) and Tamm-Horsfall protein (Fig. 8B). In contrast to the increase seen for the Na-K-2Cl cotransporter, the expression levels of these two proteins were not increased by the DDAVP infusion. In fact, the expression level of the Tamm-Horsfall protein was markedly decreased in response to DDAVP infusion. Normalized band densities for DDAVP infusion vs. controls were 32 ± 6 vs. 100 ± 13%, respectively (P < 0.05), consistent with prior observations with AVP infusions (8). Therefore, among apical membrane proteins of the TAL examined, a large increase in abundance was seen only for the Na-K-2Cl cotransporter, indicating that DDAVP infusion had a relatively selective effect and that the increase in Na-K-2Cl cotransporter expression was not a consequence of generalized amplification of the apical plasma membrane.


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Fig. 8.   Effect of DDAVP infusion on expression of type 3 Na/H exchanger (A) and Tamm-Horsfall protein (B) in renal outer medulla of Brattleboro rats. Immunoblots of same outer medullary homogenates from Sprague-Dawley rats as used for Fig. 6A. A: blot was loaded with 10 µg total protein/lane and was probed with rabbit polyclonal anti-NHE3 antibody L546. Band density for NHE3 protein was not significantly altered by DDAVP infusion (see RESULTS). B: blot was loaded with 5 µg total protein/lane and was probed with rabbit polyclonal antibody to Tamm-Horsfall protein. Band density was significantly decreased by DDAVP infusion (see RESULTS).

Figure 9 shows semiquantitative immunoblots for the two Na-K-ATPase subunits in the outer medulla. There was no significant change in the band densities of either the alpha 1-subunit (Fig. 9A; 108 ± 4% for DDAVP vs. 100 ± 11% for controls, NS) or the beta 1-subunit (Fig. 9B; 117 ± 6% for DDAVP vs. 100 ± 7% for controls, NS).


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Fig. 9.   Effect of DDAVP infusion on expression of alpha 1-subunit (A) and beta -subunit (B) of Na-K-ATPase in renal outer medulla of Brattleboro rats. Immunoblots of same outer medullary homogenates from Sprague-Dawley rats as used for Figs. 6A and 8. A: blot was loaded with 20 µg total protein/lane and was probed with rabbit polyclonal anti-alpha 1-subunit antibody. Band density for anti-alpha 1-subunit of Na-K-ATPase was not significantly changed by DDAVP infusion (see RESULTS). B: blot was loaded with 15 µg total protein/lane and was probed with rabbit polyclonal antiserum to beta 1-subunit of Na-K-ATPase. Band density was not significantly changed by DDAVP infusion (see RESULTS).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The bumetanide-sensitive Na-K-2Cl cotransporter is the chief apical Na and Cl entry pathway in TAL cells and has been identified as a target for short-term regulation of NaCl absorption by vasopressin (14, 24). We have demonstrated that this cotransporter is also a target for long-term regulation by vasopressin. Specifically, we have demonstrated a large increase in the abundance of Na-K-2Cl cotransporter protein in the TAL either in response to increases in endogenous vasopressin levels resulting from restriction of fluid intake or to infusions of the V2 receptor-selective agonist DDAVP. Because the expression levels of two other apically targeted proteins in the TAL (NHE3 and Tamm-Horsfall protein) were not similarly increased, the vasopressin-mediated regulation appears to be selective for the Na-K-2Cl cotransporter. Because the increase in Na-K-2Cl cotransporter abundance was seen in response to DDAVP (a V2-selective agonist) and the V2 receptor is coupled to activation of adenylyl cyclase, we infer that the upregulation of cotransporter abundance is likely to be a result of elevated levels of intracellular cAMP. Consistent with this possibility are the findings of Igarashi et al. (17), who have recently cloned and sequenced a large portion of the 5'-flanking region of the mouse NKCC2 gene, which codes for the apical Na-K-2Cl cotransporter of the TAL. Analysis of the sequence revealed the presence of a cAMP-regulatory element (as well as several other potential regulatory elements) that could potentially be a mediator of cAMP-mediated transcriptional regulation. It is also possible that the observed increase in Na-K-2Cl cotransporter expression could be due to indirect actions of vasopressin, possibly related to changes in interstitial osmolality or vasopressin-induced production of autacoids. Further studies are needed to determine the molecular mechanisms involved in vasopressin-mediated regulation of Na-K-2Cl cotransporter abundance.

Active NaCl absorption across the TAL epithelium provides the driving force for generation of a renal corticomedullary NaCl gradient via the countercurrent multiplier mechanism (21). This gradient provides the osmotic driving force for osmotic water absorption from the medullary collecting duct. Hence NaCl absorption by the TAL is crucial to the urinary concentrating process and to renal water conservation. Accordingly, acceleration of active transport of NaCl across the TAL epithelium can be expected to enhance urinary concentrating ability. Previous studies by Bessighir and colleagues (3) have clearly demonstrated that long-term DDAVP infusion in Brattleboro rats enhances the rate of Cl absorption by the TAL. In the present study, DDAVP infusion resulted in increased expression of the transporter responsible for most of the apical entry of sodium and chloride in the TAL, namely the Na-K-2Cl cotransporter. There was no effect of DDAVP infusion on the expression of the apically expressed NHE3, an alternative pathway for sodium entry into the TAL cells. We suggest, on the basis of these observations, that the long-term enhancement of active NaCl absorption from the TAL in response to increases in circulating vasopressin is largely due to increased sodium entry resulting from an increased number of Na-K-2Cl cotransporters in the TAL cells.

Although the present results demonstrate a role for vasopressin in the long-term regulation of Na-K-2Cl cotransporter expression in the TAL, they do not rule out long-term regulation of other TAL ion transporters involved in net NaCl absorption by the TAL. Previous studies have already demonstrated short-term actions of vasopressin or cAMP in the short-term regulation of the apical K conductance (14, 26, 33) and the basolateral chloride conductance (28) of TAL cells. These conductances have been proposed to be mediated by the ROMK channel (16) and ClC-K chloride channel (32), respectively. Conceivably, the abundances of these two transporter proteins are regulated in parallel with the Na-K-2Cl cotransporter to account for the overall long-term response of the TAL to vasopressin.

In our previous studies of Na-K-2Cl cotransporter expression (8), a complete elimination of water intake in rats for 48 h resulted in little or no change in Na-K-2Cl expression, in contrast to what we have observed here with more moderate water restriction for a longer period (7 days). The difference may be related to the duration of the stimulus. In addition, however, complete elimination of water intake results in substantial contraction of extracellular fluid volume and may, therefore, activate other regulatory processes that could counteract the direct action of vasopressin to upregulate Na-K-2Cl cotransporter expression, such as activation of the renin-angiotensin system and enhancement of renal sympathetic nerve activity. Furthermore, very large increases in circulating vasopressin levels may enhance activation of the V1a subtype of vasopressin receptors. Previous studies have reported that the V1a receptor is resident in the TAL (2, 30). This receptor, which is coupled to the phosphoinositide signaling pathway through activation of phospholipase C, may result in effects that oppose the effect of cAMP to upregulate Na-K-2Cl cotransporter expression in the TAL. Further investigation will be required to test these hypotheses.

The abundance of the alpha 1-subunit of Na-K-ATPase was significantly increased by water restriction but not altered in response to DDAVP infusion. This suggests that the increase seen in response to restriction of water intake is not mediated by the V2 vasopressin receptor but instead may be a response to some other regulatory factor triggered by water restriction. Furthermore, DDAVP infusion did not increase the expression level of the beta -subunit of the Na-K-ATPase. Hence, this study does not provide any support for the view that Na-K-ATPase expression is altered by vasopressin acting through the V2 receptor. In contrast, short-term effects of vasopressin to increase Na-K-ATPase activity in the TAL have been reported (5).

This study also confirms the observation originally made by Kriz and Bankir (22) that long-term infusion of vasopressin into Brattleboro rats causes significant hypertrophy of TAL cells in the outer medulla. We found a marked increase in luminal Na-K-2Cl cotransporter labeling of histological sections from DDAVP-infused rats relative to sections from vehicle-infused controls. Furthermore, measurements of the total amount of protein in the inner stripes of outer medullas revealed a marked increase in total protein in DDAVP-infused rats. Because samples for immunoblotting in this study were loaded to equalize the amount of total protein loaded per lane, the change in Na-K-2Cl cotransporter in the entire inner stripe is necessarily underestimated by the immunoblots relative to the actual change in the total amount of protein in the inner stripe. Thus it is likely that the Na-K-2Cl cotransporter protein per tubule in the inner stripe of DDAVP-infused Brattleboro rats was increased to considerably more than double the values in control rats.

In summary, the chief conclusion from the present study is that vasopressin strongly upregulates the expression of the bumetanide-sensitive Na-K-2Cl cotransporter of the TAL of Henle's loop, an effect that can explain the demonstrated action of long-term vasopressin infusions to increase the capacity of the TAL to reabsorb NaCl. This long-term action may contribute to the enhancement of urinary concentrating ability that is associated with sustained antidiuresis.

    ACKNOWLEDGEMENTS

The authors thank Dr. Lise Bankir of Paris for advice concerning experimental protocols, Daniel Wu for expert assistance with perfusion of kidneys with fixative, and Jie Liu for excellent technical assistance in carrying out immunolocalizations.

    FOOTNOTES

Funding for this study was derived from the intramural budget of the National Heart, Lung, and Blood Institute to M. A. Knepper (Project No. Z01-HL-01282-KE) and from National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-32839 to J. B. Wade. The Confocal Microscope Facility used for the immunolocalizations was funded by National Science Foundation Grant BIR9318061.

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.

1 Preliminary studies established that 200-g rats lose ~12 ml of water per day via nonrenal mechanisms. Most of this is presumably lost via respiration. Thus, by taking in 15 ml/day of water, the rats are able to maintain water balance by excreting a relatively small amount of urine.

2 The inner stripe of the outer medulla contains three tubule segments: thin descending limbs (tDL), thick ascending limbs (TAL), and outer medullary collecting duct (OMCD). The ratio of numbers of these three tubule types is 6:6:1 (29), whereas the ratio of Na-K-ATPase activities per individual tubule is approximately 1:31:5 (12). Multiplying these ratios, one obtains 6:186:5 as the ratio of total Na-K-ATPase in the outer medulla accounted for by each of the three structures.

Address for reprint requests: M. A. Knepper, National Institutes of Health, 10 Center Drive MSC 1603, Bldg. 10, Rm. 6N260, Bethesda, MD 20892-1603.

Received 29 July 1998; accepted in final form 1 October 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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