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Increased Abundance of Distal Sodium Transporters in Rat Kidney during Vasopressin Escape

CAROLYN A. ECELBARGER^*, MARK A. KNEPPER and JOSEPH G. VERBALIS^*

^* Department of Medicine, Division of Endocrinology and Metabolism, Georgetown University, Washington, D. C.
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland.

Correspondence to Dr. Carolyn A. Ecelbarger, Bulding D, Room 232, Georgetown University, 4000 Reservoir Road NW, Washington, DC. Phone: 202-687-0453; Fax: 202-687-2040; E-mail: ecelbarc{at}gunet.georgetown.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract. Hyponatremia is associated with inappropriately elevated vasopressin levels. A brisk natriuresis precedes the escape from this antidiuresis. Thus, the hypothesis was that the abundance of one or more of the sodium transporters of the distal tubule (a site for fine tuning of sodium balance) would be altered during vasopressin escape. Semiquantitative immunoblotting was used to examine the regulation of abundance of several sodium transporters/channels of the thick ascending limb through the collecting duct in the rat model. Osmotic minipumps to infuse dDAVP, the V2-selective vasopressin agonist (5 ng/h) for the entire experiment, were implanted in Male Sprague-Dawley rats. After 4 d, rats were divided into a control (dry AIN-76 diet/ad libitum water) or a water-loaded (gelled-agar-AIN-76 diet/ad libitum water) group. Rats were killed after 1, 2, 3, or 7 additional days. The water-loaded rats were hyponatremic (plasma Na⁺, 98 to 122 mmol/L) and manifested the expected early natriuresis and diuresis of vasopressin escape. Water loading (with dDAVP infusion) resulted in increased whole-kidney abundances of the thiazide-sensitive Na-Cl co-transporter, the -subunit of the epithelial sodium channel (ENaC), and the 70-kD dimer of the -subunit of ENaC. No changes were observed for the ß-subunit of ENaC. Similar protein changes have recently been associated with elevated aldosterone levels in rats. However, plasma aldosterone levels were significantly suppressed in this model. These data suggest that several distal sodium reabsorptive mechanisms are upregulated during vasopressin escape; this may help to attenuate the developing hyponatremia resulting from water loading when vasopressin levels are inappropriately elevated.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vasopressin is the major hormonal regulator of body water homeostasis. In certain clinical circumstances, e.g., the syndrome of inappropriate antidiuretic hormone secretion (SI-ADH), vasopressin levels are inappropriately high, resulting in free-water retention and hyponatremia. The degree of hyponatremia is limited, however, by a process that counters the water-retaining action of vasopressin. This process is the so-called "vasopressin escape" phenomenon.

In animal models of vasopressin escape (1,2,3), continued administration of vasopressin and water typically results in transient free-water retention followed by natriuresis, which is thought to contribute to the hyponatremia. However, after 1 to 3 d, urinary sodium excretion returns to baseline while urinary volume increases dramatically. Previously (4), we showed that this diuretic phase correlates very well with the downregulation of the apical water channel aquaporin-2 in the kidney collecting duct (CD) principal cells. Furthermore, the decreased abundance of aquaporin-2 protein was associated with an approximate twofold reduction in osmotic water permeability of perfused inner medullary CD (5). Thus, reduction in CD water reabsorption is most likely the predominant mechanism by which the diuresis is achieved. This reduction in water permeability of the CD would be expected not only to enhance diuresis but also to attenuate the developing hyponatremia and its associated symptoms.

Fine tuning of sodium balance in the body is achieved primarily by regulated sodium reabsorption in the distal convoluted tubule (DCT) through the CD. The thiazide-sensitive NaCl co-transporter (NCC) of the DCT and the epithelial sodium channel (ENaC) of the cortical and outer medullary CD principal cells are important apical sodium transport pathways in these segments. In the thick ascending limb (TAL), transport of sodium chloride across the apical plasma membrane occurs via secondary active transport through the bumetanide-sensitive Na-K-2Cl co-transporter (NKCC2). This protein is thought to be more important in water than in sodium balance. In tandem with decreased water permeability, one would predict that increased sodium chloride reabsorption in the distal segments would also assist in reducing the developing hyponatremia during vasopressin-induced water retention.

Little is understood about the regulation of sodium reabsorptive pathways during vasopressin escape at the molecular level. Aldosterone, a major hormonal regulator of sodium reabsorption in the distal nephron, increases the abundance of NCC (6) as well as the -subunit of the ENaC (7). However, the manner in which the renin-angiotensin-aldosterone axis responds during SIADH or in animal models of vasopressin escape is not entirely clear (see the Discussion section). Overall, an increase in aldosterone levels or a relative increase in mineralocorticoid receptor signaling would be predicted to increase sodium transport in these renal segments.

In addition, increased sodium delivery to the distal tubule during vasopressin escape could potentially upregulate NaCl transport proteins. It is clear from physiologic studies that a rapid natriuresis occurs in the early phases of vasopressin escape (1,2,3,8), although increased sodium excretion does not persist. Nevertheless, increased salt and water load to the distal nephron as a result of the diuresis and natriuresis of vasopressin escape could potentially result in hypertrophy and hyperplasia of the cells present in these segments, with a corresponding increase in abundance of sodium transporters. In fact, Stanton and Kaissling (9,10,11) showed clearly that increased sodium delivery to the distal nephron results in increased transport capacity of the distal tubule, i.e., the DCT, the connecting tubule, and the cortical CD.

For these studies, we used semiquantitative immunoblotting to characterize changes in the abundances of the critical sodium transporters and channels of the kidney distal tubule throughout the time course of vasopressin escape. Our hypothesis is that the abundance of one or more of these transporters and/or channels will be altered during vasopressin escape.

	Materials and Methods

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Animals and Study Design
A slightly modified version of the "vasopressin escape" model was used as described previously (3,4,5). The study protocol is shown in Figure 1. Under light methoxyflurane (Metofane) anesthesia, osmotic minipumps (model 2001 or 2002, Alza Corporation, Palo Alto, CA) to deliver 5 ng of dDAVP/h were implanted subcutaneously in 250-g male Sprague-Dawley rats (n = 48). After 4 d of dDAVP administration, during which time they received ad libitum water and pelleted AIN-76 diet (Bio-serve, Frenchtown, NJ), the rats were divided into two treatment groups. The control rats (n = 24) continued to receive ad libitum water and AIN-76 diet while the water-loaded rats (n = 24) were given a diet composed of 0.9% agar, 72% water, 27% powdered AIN-76 diet, and 0.1% sodium saccharin. The agar was melted in boiling water and poured into a mold, and then the diet and saccharin were added and stirred to an even consistency. The diet was then chilled at 4°C to a firm, gelatin-like state. All rats continued to receive the dDAVP infusions over the entire course of the experiment. At this time, rats were housed singly in metabolic cages (Harvard Apparatus, Holliston, MA) to facilitate urine collection and individual food and water intake records. Six rats from each treatment group were killed at different time points after the water loading (or control diet) was initiated, specifically after 1, 2, 3, or 7 d.

View larger version (24K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Study design. Osmotic minipumps to administer dDAVP at 5 ng/h were implanted in male Sprague-Dawley rats (250 g, n = 48). After a 4-d baseline period, half of the rats were randomly assigned to the control group (A) and the other half to the water-loaded group (B). Control animals received dry AIN-76 diet, and water-loaded rats received gelled-agar diet (72% water) for 1 to 7 additional days. Six rats in each treatment group were killed at each time point, specifically 1, 2, 3, and 7 d after initiating the treatment period. All rats were given ad libitum access to bottled water and continued to receive the dDAVP infusion over the course of the study.

Water-loaded rats were offered 90 g of the gelled-agar diet each day. On the first day, the rats generally ate most of their offered gel diet. However, on subsequent days, they ate lesser amounts of the diet, and thus size-matched control rats were pair fed an equal amount of calories in the form of dry AIN-76 pellets on the next day. In the rats that were treated for 7 d, urine was collected at 6 h, 12 h, 24 h, and then daily from that point on. For all other rats, urine was collected daily. Osmolality (freezing point depression, Advanced Osmometer, Model 3D3, Advanced Instruments, Inc., Norwood, MA), volume, and sodium content (ion selective electrode system, EL-ISE Electrolyte System, Beckman Instruments, Inc., Brea, CA) were measured. Food and water intake was measured each day. All animals were maintained at all times under conditions and protocols approved by the Georgetown University Animal Care and Use Committee, approved by the American Association for Accreditation of Laboratory Animal Care.

Western Blotting
Preparation of Samples. Rats were killed by decapitation and the left kidney was removed rapidly. Heparinized blood was collected, as described below, for measurement of plasma sodium, osmolality, aldosterone, and corticosterone. The kidney was homogenized using a tissue homogenizer (Tissumizer, Tekmar Company, Cincinnati, OH) fitted with a 10-mm micro-sawtooth generator in 10 ml of ice-cold membrane-isolation solution, which contained 250 mM sucrose, 10 mM triethanolamine (Sigma, St. Louis, MO), 1 µg/ml leupeptin (Bachem, Torrance, CA), and 0.1 mg/ml phenylmethylsulfonyl fluoride (US Biochemical, Toledo, OH) adjusted to pH 7.6. Protein concentration was measured on the homogenates (Pierce BCA Protein Assay Reagent Kit, Pierce, Rockford, IL). All samples were then diluted with isolation solution to a protein concentration of approximately 2 µg/µl and solubilized at 60°C for 15 min in Laemmli sample buffer. Samples were stored at -80°C until ready to run on gels.

Electrophoresis and Blotting of Membranes. Initially, 5 µg of protein from each of the samples was loaded on 12% sodium dodecyl sulfate-polyacrylamide gels (precast, BioRad, Hercules, CA) and electrophoresed. These gels were stained with Coomassie Brilliant Blue (G250; BioRad) to assess quality of protein bands and precision of the protein determinations. For immunoblotting, the electrophoresis was carried out on precast minigels of 7.5, 10, or 12% polyacrylamide. The proteins were transferred from the gels electrophoretically to nitrocellulose membranes. After a 30-min 5% milk block, membranes were probed overnight at 4°C with the desired affinity-purified polyclonal antibody. The production, purification, and characterization of these primary antibodies has been described in detail (6,7,12,13). For aquaporin-2 protein blots, we used polyclonal antibody L126. This antibody was made to the same peptide as previously described L127 (14) and gives a similar labeling pattern. For probing blots, all antibodies were diluted into a solution that contained 150 mM NaCl, 50 mM sodium phosphate, 10 mg/dl sodium azide, 50 mg/dl Tween-20, and 0.1 g/dl bovine serum albumin (pH 7.5). The secondary antibody was goat anti-rabbit IgG conjugated to horseradish peroxidase (Kirkegaard and Perry Laboratories, Gaithersburg, MD) used at a concentration of 0.10 µg/ml. Sites of antibody-antigen reaction were visualized using luminol-based enhanced chemiluminescence (Lumi-GLO, Kirkegaard and Perry Laboratories) before exposure to x-ray film (Fujifilm, Fugi Medical Supplies, Stamford, CT).

Plasma Hormone Levels
Trunk blood was collected in heparinized tubes (Vacutainer, Becton-Dickinson, Franklin Lakes, NJ) and centrifuged at 3000 x g (Sorvall RT 6000 D; Sorvall, Newton, CT), and the plasma was separated. Plasma aldosterone and corticosterone levels were measured by Coat-A-Count RIA kits purchased from Diagnostics Products Corporation (Los Angeles, CA).

Statistical Analyses
Relative intensity of the resulting immunoblot band densities was carried out by laser scanning (Scanjet 6100C, Hewlett-Packard, Palo Alto, CA) followed by analysis with NIH IMAGE software (National Institutes of Health, Bethesda, MD). The statistical significance of the effects of the various treatments on protein expression was determined by an unpaired t test of densitometry values when SD were the same or by Welch's t test when SD were significantly different (Graphpad Prism; Graphpad, San Diego, CA). The effect of vasopressin escape on hormonal status over the time course of escape was also assessed by two-way ANOVA with the variables "treatment" and "time." P < 0.05 was considered statistically significant for both tests.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Time Course of Urinary Osmolality and Volume Changes
Urine osmolality (Figure 2A) and urine excretion rate (Figure 2B) are plotted in the two treatment groups for the rats that were maintained in the study for 7 d. As previously observed (4,5), urine osmolality was significantly reduced in the water-loaded rats after 2 d and remained in the range of 1800 to 2000 mOsm/kg H₂O for the remainder of the study (Figure 2A). In contrast, in the control rats, urine osmolality remained constant at approximately 3500 mOsm/kg H₂O. Urine excretion rate (Figure 2B), as observed previously (4), began to increase by 2 d of water loading and was statistically increased relative to the control rats after 6 d.

View larger version (17K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Time course for changes in urinary osmolality (A) and volume (B) during vasopressin escape. All rats were killed after 7 d of treatment (control: dry diet plus dDAVP infusion; water-loaded: gelled agar diet plus dDAVP infusion). Urine osmolality was significantly reduced in the water-loaded rats after 2 d and remained significantly depressed for the duration of the experiment. Urinary volume began to increase in the water-loaded rats at 2 d and was significantly elevated after 6 d of water loading.

Time Course of Sodium and Potassium Excretion
Urinary sodium (Figure 3A) and urinary potassium (Figure 3B) excretion rates are plotted in the same group of animals as above. The urinary sodium excretion rate began to increase in the water-loaded rats by 1 d and was significantly elevated at 2 d (Figure 3A). However, it returned to basal levels in the water-loaded rats by 3 d and was modestly yet significantly reduced at 7 d. The urinary potassium excretion rate was not significantly different between the two groups at any time point in the study (Figure 3B).

View larger version (17K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Time course for changes in urinary sodium (A) and potassium (B) during vasopressin escape. All rats were killed after 7 d of treatment (control: dry diet plus dDAVP infusion; water-loaded: gelled agar diet plus dDAVP infusion). Urinary sodium excretion began to increase in the water-loaded rats at 1 d and peaked after 2 d, when it was significantly increased relative to the controls. Urinary potassium was not significantly different in the two treatments at any time point.

Plasma Na⁺ and Osmolality
Table 1 shows plasma osmolality and sodium levels in the trunk blood collected at the time that the rats were killed. Rats were markedly hyponatremic and hypo-osmolar from the first day of water loading.

Whole-Kidney Abundance of Aquaporin-2 Is Decreased and Aquaporin-3 Is Increased
In the absence of water loading, dDAVP infusion has been shown to increase the expression of both aquaporin-2 (15,16) and aquaporin-3 proteins (16) in rat kidney. In agreement with our previous studies (4,5,17,18,19), aquaporin-2 expression was significantly decreased by the water loading despite the continual infusion of dDAVP to both groups (Figure 4A). Figure 4A shows an immunoblot of whole-kidney homogenates probed with anti—aquaporin-2 (L126). Average band density (sum of the nonglycosylated 29-kD and the glycosylated 35- to 45-kD bands) for aquaporin-2 protein was 58% in the water-loaded group, relative to the control group. Furthermore, in agreement with our previous study (4), aquaporin-3 was significantly increased by water loading in the presence of dDAVP (Figure 4B). The average band density for aquaporin-3 protein (both bands summed) in the water-loaded group was 152% of the control mean.

View larger version (77K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Effect of vasopressin escape on the abundances of aquaporins 2 and 3. Immunoblots are of whole-kidney homogenates. All rats were killed after 3 d of treatment (control: dry diet plus dDAVP infusion; water-loaded: gelled agar diet plus dDAVP infusion), and whole-kidney samples were prepared. Each lane is loaded with a sample from a different rat (n = 6 rats/treatment). Twenty µg of total protein were loaded in each lane, and the resulting immunoblots were probed with anti—aquaporin-2 polyclonal antibody (L126) (A) or anti—aquaporin-3 antibody (L178) (B). Densities for both bands associated with aquaporin-2 or aquaporin-3 were determined by laser densitometry and summed. Aquaporin-2 was significantly (P < 0.05) decreased and aquaporin-3 was significantly increased in the water-loaded rats.

Increased Abundance of the Thiazide-Sensitive NCC
Figure 5 shows immunoblotting data for whole-kidney abundance of the NCC of the DCT during vasopressin escape. Figure 5A is an immunoblot of whole-kidney homogenates probed with anti-NCC antibody (L573) (6). In Figure 5B, a summary of band densitometry values obtained at the four time points studied (1, 2, 3, and 7 d) is shown. Band density for the 165-kD band was significantly increased by water loading only at 1 and 2 d. At 3 d, it tended to be increased but was not statistically significantly elevated (P = 0.086). At 7 d, no differences were observed between the two groups for NCC abundance.

View larger version (33K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. The abundance of the thiazide-sensitive Na-Cl co-transporter (NCC) during vasopressin escape. (A) An immunoblot of whole-kidney homogenates. Rats were killed after 2 d of treatment (control: dry diet plus dDAVP infusion; water-loaded: gelled agar diet plus dDAVP infusion), and whole-kidney samples were prepared. Each lane is loaded with a sample from a different rat (n = 6 rats/treatment). Ten µg of total protein were loaded in each lane, and the resulting immunoblot was probed with anti-NCC polyclonal antibody (L573). Band density of the 165-kD band was determined by laser densitometry. (B) Summary of densitometries obtained from similar blots over the time course of escape. NCC abundance was significantly (P < 0.05) increased after 1 or 2 d of water loading.

Increase in -ENaC Abundance during Vasopressin Escape
Figure 6 shows immunoblotting data for whole-kidney abundance of the -subunit of ENaC during vasopressin escape. Figure 6A is an immunoblot of whole-kidney homogenates probed with anti—-ENaC antibody (L766) (7). In Figure 6B, a summary of band densitometry values obtained at the four time points (1, 2, 3, and 7 d) is shown. -ENaC abundance tended to be increased over the entire time course of vasopressin escape. Band density for the 85-kD band was significantly increased by water loading after 1, 2, and 7 d.

View larger version (36K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. The abundance of the -subunit of the epithelial sodium channel (ENaC) during vasopressin escape. (A) An immunoblot of whole-kidney homogenates. Rats were killed after 2 d of treatment (control: dry diet plus dDAVP infusion; water-loaded: gelled agar diet plus dDAVP infusion), and whole-kidney samples were prepared. Each lane is loaded with a sample from a different rat (n = 6 rats/treatment). Thirty µg of total protein were loaded in each lane, and the resulting immunoblot was probed with anti—-ENaC polyclonal antibody (L766). Band density of the 85-kD band was determined by laser densitometry. (B) Summary of densitometries obtained from similar blots over the time course of escape. -ENaC abundance was significantly (P < 0.05) increased after 1, 2, or 7 d of water loading.

No Change in ß-ENaC Abundance
Figure 7 shows immunoblotting data for whole-kidney abundance of the ß-subunit of ENaC during vasopressin escape. Figure 7A is an immunoblot of whole-kidney homogenates probed with anti—ß-ENaC antibody (L558) (7). In Figure 7B, a summary of band densitometry values obtained at the four time points (1, 2, 3, and 7 d) is shown. Band densities were not significantly different between the two groups at any of the time points studied.

View larger version (40K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. The abundance of the ß-subunit of the ENaC during vasopressin escape. (A) An immunoblot of whole-kidney homogenates. Rats were killed after 2 d of treatment (control: dry diet plus dDAVP infusion; water-loaded: gelled agar diet plus dDAVP infusion), and whole-kidney samples were prepared. Each lane is loaded with a sample from a different rat (n = 6 rats/treatment). Twenty µg of total protein were loaded in each lane, and the resulting immunoblot was probed with anti—ß-ENaC polyclonal antibody (L558). Band density of the 90-kD band was determined by laser densitometry. (6) Summary of densitometries obtained from similar blots over the time course of escape. ß-ENaC abundance was not significantly affected by water loading at any time point.

-ENaC Undergoes a Qualitative Change during Vasopressin Escape
Figure 8A shows an immunoblot of whole-kidney homogenates probed with anti—-ENaC antibody (L550) (7). Here we see no significant change in the intensity of the major band associated with -ENaC (85 kD). However, we observed a significant increase in appearance of two broad bands centered around 70 kD in the water-loaded rats. These bands were observed previously in rats that were infused with aldosterone or fed low-salt diets (7). Figure 8B shows densitometric values obtained for the major band (85 kD) and for the bands centered at approximately 70 kD in the water-loaded rats normalized to their respective controls. Abundance of the 70-kD form of -ENaC was significantly increased by water loading after 2 and 7 d.

View larger version (38K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. The abundance of the -subunit of the ENaC during vasopressin escape. (A) An immunoblot of whole-kidney homogenates. Rats were killed after 2 d of treatment (control: dry diet plus dDAVP infusion; water-loaded: gelled agar diet plus dDAVP infusion), and whole-kidney samples were prepared. Each lane is loaded with a sample from a different rat (n = 6 rats/treatment). Twenty µg of total protein were loaded in each lane, and the resulting immunoblot was probed with anti—-ENaC polyclonal antibody (L550). Band densities of the 85- and 70-kD bands were scanned by laser densitometry. (B) Summary of band densities for the 85- and 70-kD bands in the water-loaded rats (expressed as a percentage of their respective control means) over the time course of escape. Abundance of the 70 band(s) was significantly increased by water loading at 2 and 7 d. Abundance of the 85-kD did not change over the time course of escape.

Early Increase in Abundance of the NKCC2 of the TAL
Figure 9 shows immunoblotting data for whole-kidney abundance of the NKCC2 during vasopressin escape. Figure 9A is an immunoblot of whole-kidney homogenates probed with anti-NKCC2 antibody (L320) (13). In Figure 9B, a summary of band densitometry values obtained at the four time points (1, 2, 3, and 7 d) is shown. Band density for the 161-kD band was significantly increased by water loading at day two (twofold). At 3 d, it had returned to baseline and was significantly lower than controls after 7 d of water loading.

View larger version (32K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. The abundance of the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) of the thick ascending limb during vasopressin escape. (A) An immunoblot of whole-kidney homogenates. Rats were killed after 2 d of treatment (control: dry diet plus dDAVP infusion; water-loaded: gelled agar diet plus dDAVP infusion), and whole-kidney samples were prepared. Each lane is loaded with a sample from a different rat (n = 6 rats/treatment). Five µg of total protein were loaded in each lane, and the resulting immunoblot was probed with anti-NCC polyclonal antibody (L320). Band density of the 161-kD band was determined by laser densitometry. (B) Summary of densitometries obtained from similar blots over the time course of escape. NKCC2 abundance was significantly increased by water loading at 2 d and significantly decreased by water loading at 7 d.

Effect of Water Loading on Plasma Aldosterone and Corticosterone Levels
Figure 10A shows a bar graph of plasma aldosterone levels in the rats at the time that they were killed. Aldosterone was not significantly increased at any time point (unpaired t test). Furthermore, aldosterone levels were significantly decreased by water loading when values were analyzed by two-way ANOVA (treatment x time). Figure 10B shows a bar graph of plasma corticosterone levels in the rats at the time that they were killed. Corticosterone levels were not changed by water loading at any time point (unpaired t test or two-way ANOVA). However, corticosterone levels were significantly increased in both groups of rats over time (two-way ANOVA).

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 10. Plasma aldosterone (A) and corticosterone (B) levels during the time course of vasopressin escape. Unpaired t test analysis of any time point showed no significant differences between control and water-loaded rats for either hormone. Two-way ANOVA (treatment x time) showed a significant reduction (P < 0.05) in aldosterone levels in the water-loaded rats and a significant increase in corticosterone levels in both groups of rats over the time course of the experiment.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Vasopressin escape is a critical homeostatic mechanism that prevents excessive dilution of body fluids when water intake is high or moderate and vasopressin levels are inappropriately elevated. Clinically, this disorder is known as the syndrome of inappropriate antidiuretic hormone secretion, or SIADH. Physiologically, the "escape" is characterized by a sudden increase in urine volume and decrease in urine osmolality despite the continually high circulating levels of vasopressin. In several recent studies (4,5,17,18,19), we have begun to elucidate the mechanisms for vasopressin escape, in the kidney, at the molecular level. In our rat model, we have observed a dramatic decrease in the abundance of the apical water channel aquaporin-2 mRNA and protein, in the CD principal cells. These changes coincided temporally with the physiologic parameters of escape, i.e., increased urine volume and decreased urine osmolality.

Hyponatremia is the hallmark of SIADH and is due primarily to inappropriately regulated water reabsorption in the CD. However, the early natriuresis of vasopressin-induced water retention escape might also exacerbate the developing hyponatremia. Nevertheless, urinary sodium excretion levels return to baseline fairly rapidly even though the escape from the antidiuretic effects of vasopressin continues. This compensation, which helps to restore sodium balance, could be partly mediated by increased sodium reabsorption in the distal nephron, a portion of the tubule that "fine tunes" body sodium levels.

In the current studies, using semiquantitative immunoblotting, we tested the hypothesis that the abundances of one or more of the critical sodium transporters/channels in the distal nephron might change during vasopressin escape. The transporters/channels that we examined include the thiazide-sensitive NCC of the DCT, also known as TSC (20), the three subunits (, ß, and ) of the amiloride-sensitive ENaC of the CD, and the bumetanide-sensitive NKCC2 of the TAL, also known as BSC1 (20). Recent cloning of NCC (20), NKCC2 (20,21), and ENaC (, ß, and subunits) (22,23) from the rat has made it possible to generate peptide-derived, polyclonal antibodies in rabbits specific for each of the above proteins. Each of these antibodies is sensitive enough to detect its respective protein in native tissue. The characterization of these antibodies has been published previously (6,7,13).

Aldosterone-Like Pattern of Protein Changes
The pattern of protein changes observed, i.e., increased abundances of NCC and -ENaC, along with the qualitative change in -ENaC (the increased abundance of a dimer that runs at 70 kD) is nearly identical to what has been observed recently in studies of aldosterone effects in the rat kidney by Kim et al. (6) and Masilamani et al. (7). In those studies, they showed by immunoblotting that aldosterone infusion by osmotic minipump or feeding a low-sodium diet (0.2 mEq/200 g body wt per /d) resulted in increased abundances of NCC (6) and -ENaC (7) in rat kidney, as well as a very similar qualitative change in -ENaC expression (7). Therefore, our first prediction was that elevated aldosterone levels might be responsible for these changes in our studies. However, we found no correlation between aldosterone levels and expression of any of the proteins. In fact, aldosterone levels were significantly suppressed in the water-loaded rats. These results are not surprising and are, in fact, in agreement with what many others have observed. Several investigators have reported decreased renin activity in patients with SIADH (24,25) and in rat (26,27) and dog (28,29) models of SIADH. Likewise, Cogan et al. (30) reported decreased aldosterone levels in human patients with SIADH and in normal subjects given dDAVP followed by a water load. Nevertheless, other data are conflicting, suggesting that aldosterone levels are either elevated (31) or unchanged in patients with SIADH (24,32). Furthermore, in our current studies, corticosterone levels were not significantly elevated by water loading, suggesting that this hormone was not replacing aldosterone at the mineralocorticoid receptor and perhaps in some way was overwhelming the protective capacity of 11-ß-dehydroxysteroid dehydrogenase-2 (11-ß-HSD-2), the enzyme responsible for cortisol degradation in these cells. Nevertheless, because the pattern is so intriguingly similar to what has been observed with high aldosterone levels, we suggest that these mineralocorticoid-like effects may be the result of several potential possibilities. First, there could be an increase in mineralocorticoid receptor number or activity during vasopressin escape. Second, the activity or abundance of 11-ß-HSD-2 in these cells may be decreased. Regulation of the activity of this enzyme has been demonstrated in acid-loaded rats (33) or in highly stressed rats (34). Third, another steroid or steroid-like hormone (other than aldosterone or corticosterone) that has activity at mineralocorticoid receptors may be released during vasopressin escape. Finally, it is possible that there is in these animals an unusual diurnal pattern to the aldosterone release that could cause us to assume inappropriately that these animals have low aldosterone levels when in fact they might vary from low to high levels based on fluctuations in extracellular volume status or time of day. Diurnal patterns of aldosterone release are well known (35) and are found to be disrupted in many disease states (36). However, it is also possible that these protein changes are only coincidentally similar to what was previously observed in rats with high aldosterone levels (either endogenously or exogenously) and that the mechanism(s) for their regulation is completely independent of the mineralocorticoid receptor.

Potential Role of Sodium Load
An additional factor that might contribute to increased sodium transporter expression in the distal nephron during vasopressin escape is an increased sodium load delivered to the distal nephron as a result of extracellular fluid volume expansion in the water-loaded rats. Micropuncture studies by Stanton and Kaissling (9,10,11,37) demonstrated that chronically increased sodium delivery to the distal tubule, i.e., the DCT through the cortical CD, as a result of either furosemide treatment (9,10,11) or a high-sodium diet (37), will enhance the transport capacity of these segments. These adaptations seem to be mediated by ultrastructural changes in the cells that enhance transporting capacity, e.g., increased cell volume, basolateral membrane area, and mitochondrial volume (9,10,11). Additional studies (38) showed that cellular hyperplasia also likely plays a role in the enhanced NaCl transporting capacity of these cells. Nevertheless, Fanestil et al. (39) reported no increase in thiazide receptor binding (a measure of active number of NCC molecules) in normotensive rats that were fed a high-sodium diet. However, as one would expect, the influence of dietary sodium loading does not seem to be as strong as that of furosemide. In fact, Ellison et al. (40) reported that increased dietary NaCl does not increase distal (post-macula densa) sodium delivery. It is likely, during the transient natriuretic phase of vasopressin escape, that the sodium load delivered to the distal nephron is in between what is observed with a high-sodium diet and furosemide administration. Thus, how it might affect NCC abundance is unclear. Additional studies to examine the effects of furosemide administration on NCC abundance should shed some light on this issue.

Nonetheless, an increase in abundance of NKCC2 was observed at 2 d of water loading in the water-loaded rats (Figure 9). We hypothesize that the natriuresis that is evident after 1 and 2 d of water loading may be at least partially responsible for increasing NKCC2 abundance. We previously reported (41,42) that NKCC2 abundance is increased either by offering rats isotonic saline to drink in lieu of plain water (41) or by increasing sodium content of the diet (42). This is in agreement with earlier studies by Landwehr, Klose, and Giebisch (43) that demonstrated increased TAL sodium reabsorption in the rat during isotonic saline infusion, as assessed by micropuncture. The increase in NKCC2 abundance that we observe, however, is very transient and seems to revert when the natriuretic phase of vasopressin escape has subsided (Figure 3A).

A Role for Downregulation of the Vasopressin V2 Receptor/Vasopressin Resistance ?
Several groups (44,45) along with ourselves (19) have demonstrated that V2 receptors desensitize after chronic exposure to high levels of either vasopressin or its analogues, e.g., we observed decreased binding of a radiolabeled V2-receptor selective analogue of vasopressin (19) in the inner medulla from rats chronically treated with dDAVP relative to untreated control rats. Furthermore, studies by our group (5,18,19) and others (46) demonstrated an additional downregulation of V2 binding (19,46), mRNA expression (18,46), and cyclic AMP production (5) in rats undergoing vasopressin escape (dDAVP treatment plus water load) relative to their dDAVP-treated, non—water-loaded controls. We have shown clearly that the abundances of aquaporin-2, aquaporin-3, NKCC2, and ß- and -ENaC are increased by infusion of dDAVP or water restriction (13,15,16,47). However, during vasopressin escape, we see a marked, consistent downregulation of only aquaporin-2 protein and mRNA. We observed no changes in the overall abundances of ß- or -ENaC and increased aquaporin-3 protein. Thus, it seems that vasopressin resistance of the CD is not important in regulating the abundance of these other CD proteins during vasopressin escape. Nevertheless, the eventual significant reduction in NKCC2 abundance at 7 d hypothetically could be explained by this relative decrease in vasopressin sensitivity of the V2 receptor. V2 receptors are found to be expressed in the TAL of rats (48,49), as well as in the CD. However, it is unclear whether the V2 receptors of the TAL have a similar relative resistance to vasopressin during vasopressin escape as has been observed in the inner medullary CD.

Primary Signal(s) for Vasopressin Escape
The primary signal that is responsible for vasopressin escape remains elusive. It seems likely that the transient volume expansion that occurs in the first 1 to 2 d of water loading may be critical. Early studies by Hall et al. (8) and Cowley et al. (28) using dogs in which renal artery pressure was servocontrolled strongly suggest that increased renal artery pressure is necessary for both the diuresis and the natriuresis of vasopressin escape. Increased prostaglandin E₂ release during volume expansion has been demonstrated (17,26,50), but it alone does not seem to be responsible for the diuresis of vasopressin escape (17,26). Increased nitric oxide (NO) production also has been implicated as a potential important mediator of vasopressin escape, because NO synthase inhibition by L-omega-nitro-L-arginine methyl ester (L-NAME; a nonselective inhibitor of NO synthases) when combined with inhibition of cyclooxygenases by indomethacin has been reported to antagonize the diuresis of escape (17). Furthermore, this inhibition of escape coincided with a relative increase in aquaporin-2 abundance. Nevertheless, little is understood about the regulation of renal transporters by NO or NO suppression. It may be that the same primary signal that eventually suppresses aquaporin-2 expression also is effective in increasing the abundance of -ENaC and NCC. Conversely, the signal or signals that mediate the upregulation of sodium transporters in the distal nephron during vasopressin escape may be totally independent from those that mediate the downregulation of aquaporin-2 protein. All of these possibilities will require further studies using this and similar models of vasopressin escape.

Physiologic Relevance
Changes in abundance are by no means the only way in which transporters or transport capacity of membranes is regulated. Nevertheless, the changes observed in the DCT and CD, i.e., increased abundances of NCC and -ENaC and the qualitative change in -ENaC, would be predicted to increase sodium chloride reabsorption in these segments rather than facilitate natriuresis. This is based primarily on the fact that aldosterone dramatically increases sodium chloride transport capacity in these cells (51,52,53) and our observation that the pattern of protein changes in this model of vasopressin escape is similar to what is seen with aldosterone infusion or feeding a low-salt diet (6,7). Thus, the changes in distal sodium transporters that we have observed are more likely to represent adaptive responses that allow for the conservation of sodium in response to increased sodium load delivered to the distal tubule and/or other factors that arise from perhaps hyponatremia that activate mineralocorticoid-like mechanisms. We speculate that decreased proximal tubule sodium reabsorption is the root of the natriuresis, thereby resulting in increased distal tubule sodium delivery. The extent to which such changes in distal tubular sodium reabsorptive capacity can compensate for a proximally generated natriuresis depends on the relative magnitude of the induced natriuresis. However, even if they were insufficient to reverse the natriuresis, they would be predicted to blunt sodium losses and ameliorate the hyponatremia and, therefore, may play a role in attenuating the natriuresis that peaks very early in the vasopressin-escape phenomenon. Further work is needed to address the specific mechanisms that are responsible for the observed distal tubule protein changes, as well as the physiologic significance of these effects.

	Acknowledgments

 
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases grant nos. DK38094 (J.V., C.E.) and K01 #DK02672-01 (C.E.); the George E. Schriener, M.D. Young Investigator Grant of the National Kidney Foundation (C.E.) at Georgetown University; and the intramural budget of the National Heart, Lung, and Blood Institute (M.K.).

	Footnotes

 
Dr. Jeff M. Sands served as guest editor and supervised the review and final disposition of this manuscript.

	References

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