Decreased abundance of collecting duct urea transporters UT-A1 and UT-A3 with ECF volume expansion

Xiao-Yan Wang1, Kathleen Beutler1, Jakob Nielsen1, Søren Nielsen2, Mark A. Knepper1, and Shyama Masilamani1

1 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892; and 2 The Water and Salt Research Institute, University of Aarhus, DK-8000 Aarhus C, Denmark


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Clinical disorders of extracellular fluid (ECF) volume regulation are often associated with changes in plasma urea concentration. To investigate possible renal causes, we measured the relative abundance of the urea transporters UT-A1, UT-A2, and UT-A3 in renal medulla of rats with aldosterone-induced NaCl retention. ECF volume-expanded rats received aldosterone by osmotic minipump plus a diet containing a high level of NaCl. Control rats received the same infusion of aldosterone plus a virtually NaCl-free diet, which prevented ECF volume expansion. Preliminary measurements demonstrated transient positive Na and water balance, decreased serum urea concentration, and increased urea clearance, but no change in creatinine clearance. Immunoblotting of homogenates from inner medulla showed a marked decrease in the abundance of the collecting duct urea transporters UT-A1 and UT-A3. There were no differences in the abundance of UT-A2, aquaporin (AQP)-2, AQP-3, or AQP-4 in ECF volume-expanded rats vs. controls. Time course experiments demonstrated that changes in UT-A1 abundance paralleled the fall in serum urea concentration after the switch from a low-NaCl to a high-NaCl diet, whereas the fall in UT-A3 abundance was delayed. Candesartan administration markedly decreased the abundance of UT-A1 and UT-A3 in the renal inner medulla, which is consistent with a role for the angiotensin II type 1 receptor in urea transport regulation. The results support the view that ECF-related changes in serum urea concentration are mediated, at least in part, through altered urea transporter abundance.

aldosterone; aquaporin; angiotensin; extracellular fluid


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MEASUREMENTS OF SERUM UREA concentration are often used clinically to assess the state of extracellular fluid (ECF) volume or intravascular volume. Decreased intravascular fluid volume is often associated with a rise in serum urea concentration. For example, dehydration caused by excess water loss or inadequate water intake is often accompanied by increased urea concentrations in the serum. Such a rise is often termed "prerenal azotemia." In contrast, increased vascular volume can be associated with marked decreases in serum urea concentration, for example, in the syndrome of inappropriate antidiuresis. Although these changes in serum urea concentration are often interpreted as being indicative of changes in renal hemodynamics or glomerular filtration, the role of renal tubule urea transporters in these responses has not been evaluated.

Urea is freely filtered at the glomerulus, and the amount excreted is determined by both its rate of glomerular filtration and its reabsorption by the renal tubule. The classic studies of Shannon (23) in the 1930s revealed that urea is reabsorbed in both the proximal tubule and the distal segments. Subsequent work by Schmidt-Nielsen (22) demonstrated that the proximal reabsorption was largely constitutive, whereas the distal component was highly variable and regulated. Micropuncture and microinjection studies demonstrated that regulated transport occurs in the collecting duct system (3, 16), and isolated perfused tubule studies showed that urea transport was restricted to the terminal part of the inner medullary collecting duct (IMCD) (20). The work of Chou and colleagues (1, 2) established that urea transport in the IMCD is due to the presence of specialized urea transporter proteins. Subsequently, two urea transporters have been identified in the IMCD, namely, UT-A1 (19, 24, 25) and UT-A3 (10, 30). A third isoform, UT-A2, is expressed in the descending limb of Henle's loop (19, 24, 31, 34). Here, we use semiquantitative immunoblotting, employing antibodies that specifically recognize these urea transporter isoforms in a rat model of ECF volume expansion, to determine whether aldosterone-induced volume expansion is associated with altered abundance of these urea transporters.


    METHODS
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INTRODUCTION
METHODS
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Aldosterone-induced ECF volume expansion model. Experiments were conducted in male Sprague-Dawley rats (180-220 g; Taconic Farms, Germantown, NY). All rats were maintained in metabolism cages to allow quantitative urine collections. All rats were anesthetized with methoxyflurane (Metofane, Pitman-Moore, Mundelein, IL) and implanted subcutaneously with osmotic minipumps (model 2ML2, Alzet, Palo Alto, CA) delivering 200 µg/day of aldosterone (Sigma Chemical, St. Louis, MO) (13). The minipump infusion was sustained in all rats throughout the entire time course of each experiment. In the control group (non-volume-expanded rats), the aldosterone-treated rats were maintained on a very low level of NaCl intake (0.02 meq/day) for 4 days. In the experimental group (volume expanded), aldosterone-treated rats received a higher NaCl intake (2.0 meq/day) for 4 days. NaCl intake was maintained at these levels by ration feeding of measured amounts of a gelled mixture of food and water (see Ration-feeding procedure). The rats were euthanized by decapitation. For kidney processing, the control and experimental rats were euthanized at the same time. The kidneys were harvested for semiquantitative immunoblotting for the major renal aquaporins (AQPs) and urea transporters (see Semiquantitative immunoblotting). Serum was collected at the time of decapitation for the measurement of Na, urea, glucose, and creatinine (Monarch 2000 autoanalyzer, Instrumentation Laboratories, Lexington, MA). Serum aldosterone concentration was measured by radioimmunoassay (Coat-a-Count, Diagnostic Products, Los Angeles, CA). Urinary samples were analyzed for Na, urea, and creatinine (Monarch 2000 autoanalyzer, Instrumentation Laboratories).

The above protocol was repeated for immunocytochemistry (see Immunocytochemistry).

Effect of altered NaCl intake without aldosterone infusion. A control study was also performed that was identical to the aldosterone-induced ECF volume expansion study (described in the immediately preceding subsection) except for the absence of the aldosterone infusion. In the absence of aldosterone infusion, we assume that the effect of increased NaCl intake on ECF volume would be far less than with aldosterone infusion.

Time course protocol. Aldosterone-infused rats were initially placed on the control NaCl intake (0.02 meq/day) and were switched to the experimental NaCl intake (2.0 meq/day) at day 0, whereas the control rats remained on the 0.02 meq/day NaCl intake. Rats were euthanized 1, 2, or 4 days after the switch to the experimental NaCl intake. For each time point, control and experimental rats were euthanized at the same time for kidney processing. All other aspects of the experiments were the same as above.

Candesartan protocol. All rats were maintained in metabolism cages (see Aldosterone-induced ECF volume expansion model) and were fed a ration of a gelled diet (see Ration feeding procedure) containing 0.5 meq/day NaCl. All rats were implanted with osmotic minipumps (see Aldosterone-induced ECF volume expansion model) delivering 1.0 mg · kg-1 · day-1 candesartan [an angiotensin II receptor (AT1) blocker] or vehicle for 2 days. These rats did not receive aldosterone. The rats were euthanized, and the kidneys were harvested for semiquantitative immunoblotting (see Semiquantitative immunoblotting).

Ration-feeding procedure. The intakes of NaCl, calories, and water were carefully controlled by ration feeding a fixed daily amount of a gelled diet that contained all the nutrients, NaCl, and water that a rat received per day. The baseline control NaCl intake was achieved by feeding a gelled mixture of a synthetic low-NaCl diet (Formula 53140000, Ziegler Brothers, Gardner, PA), deionized water (25 ml/15 g food), and agar (0.125 g/25 ml water). The diet for the experimental group was the same except for addition of 2 meq NaCl/15 g food before gelation. All animals received the equivalent of 15 g · 200 g body weight (BW)-1 · day-1 of rat chow, which was determined by weighing the gelled mixture. Analysis of the diet demonstrated that this protocol provided ~0.02 meq · 200 g BW-1 · day-1 of NaCl for the control group and 2.0 meq · 200 g BW-1 · day-1 for the experimental group.

Antibodies. Rabbit polyclonal antibodies to the following AQPs and urea transporters were utilized: the collecting duct AQP-2 (4), AQP-3 (6), AQP-4 (27); the collecting duct urea transporters UT-A1 (19) and UT-A3 (30); and the loop of Henle urea transporter UT-A2 (19). The antisera were affinity purified against the immunizing peptides as previously described (12, 13). Specificity of the antibodies has been demonstrated by showing unique peptide-ablatable bands on immunoblots and a unique distribution of labeling by immunocytochemistry.

Semiquantitative immunoblotting. Semiquantitative immunoblotting was utilized to compare urea transporter or water channel abundance between groups of rats. The procedure has been previously described in detail (12, 28) and is summarized briefly in the following. The right kidney was dissected to obtain the cortex, inner stripe of the outer medulla, and inner medulla. These tissues were homogenized by using a tissue homogenizer (Omni 1000 fitted with a microsawtooth generator) in ice-cold isolation solution containing 250 mM sucrose/10 mM triethanolamine (Calbiochem, La Jolla, CA) with 1 µg/ml leupeptin (Bachem California, Torrance, CA) and 0.1 mg/ml phenylmethylsulfonyl fluoride (US Biochemical, Toledo, OH). The total protein concentration was measured by using the Pierce bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL). All samples were adjusted with isolation solution to a final protein concentration of 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. For each set of samples, an initial gel was stained with Coomassie blue, as described previously (5), to confirm equal loading among samples. SDS-PAGE was performed on 12% polyacrylamide gels (Ready Gels, Bio-Rad, Hercules, CA). The proteins were electrophoretically transferred from the gel to nitrocellulose membranes. After a 30-min 5% milk block, membranes were probed overnight at 4°C with the respective primary antibodies and then exposed to secondary antibody (goat anti-rabbit IgG conjugated with horseradish peroxidase, Pierce no. 31463, diluted to 1:5,000) for 1 h at room temperature. Sites of antibody-antigen reaction were visualized by using a luminol-based enhanced chemiluminescence substrate (LumiGLO, Kirkegaard and Perry Laboratories, Gaithersburg, MD) before exposure to X-ray film (Kodak no. 165-1579). The band densities were quantitated by laser densitometry (Molecular Dynamics model PDS1-P90). The densitometry values were normalized to the mean value for the control group to facilitate comparisons.

Immunocytochemistry. Kidneys were fixed by perfusion with cold PBS (pH 7.4) for 15 s via the abdominal aorta, followed by cold 4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 3 min. The kidneys were removed and postfixed for 1 h, followed by 3- to 10-min washes with 0.1 M cacodylate buffer (pH 7.4). The tissue was dehydrated in graded ethanol and left overnight in xylene. The tissue was embedded in paraffin, and 2-µm sections were cut on a rotary microtome (Leica Microsystems, Herler, Denmark). Localization of UT-A1 was carried out by using immunoperoxidase labeling as described (7). For immunoperoxidase labeling, the secondary antibody was horseradish peroxidase conjugated to goat anti-rabbit immunoglobulin (DAKO P448, Glostrup, Denmark). For immunoperoxidase labeling, counterstaining was done by using Mayer's hematoxylin. Microscopy was carried out with a Leica DMRE light microscope (Leica Microsystems).

Presentation of data and statistical analyses. Quantitative data are presented as means ± SE. Statistical comparisons were accomplished by unpaired t-test (when variances were the same) or by Mann-Whitney rank-sum test (when variances were significantly different between groups). P values <0.05 were considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
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RESULTS
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Changes in abundance of urea transporters. We compared aldosterone-treated rats on a low-NaCl diet with aldosterone-treated rats on a high-NaCl diet to test the effect of mineralocorticoid-induced ECF volume expansion on urea transporter and AQP abundance in the kidney. UT-A1 is apically located in the IMCD and has been proposed to play a major role in urea reabsorption (19). Figure 1 shows a UT-A1 immunoblot of inner medullary homogenates from control rats (aldosterone + 4-day low NaCl) compared with volume-expanded rats (aldosterone + 4-day high NaCl). As can be seen, there is a significant decrease in the band density of UT-A1 (to 28 ± 3% of control, P < 0.05) in the volume-expanded rats. This response was confirmed by immunocytochemistry (Fig. 2). Collecting ducts from the renal inner medulla showed clear decreases in UT-A1 labeling in volume expansion vs. control. There was no obvious redistribution of UT-A1 labeling in the IMCD cells.


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Fig. 1.   Immunoblot assessing UT-A1 abundance in inner medullary homogenates from control rats (aldosterone + 4-day low-NaCl diet) vs. volume-expanded rats (aldosterone + 4-day high-NaCl diet). Each lane was loaded with a sample from a different rat. Preliminary 12% SDS-polyacrylamide gels were run and stained with Coomassie blue to confirm equality of loading in each lane. Band densities were assessed by laser densitometry. Values are means ± SE normalized to the mean value for the control group. Densitometric analysis showed an increase in UT-A1 abundance in volume-expanded rats vs. control rats. IM, inner medulla. * Significantly different from control, P < 0.05.



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Fig. 2.   Immunoperoxidase labeling for UT-A1 in the renal inner medulla of control rats (aldosterone + 4-day low-NaCl diet; A) vs. volume-expanded rats (aldosterone + 4-day high-NaCl diet; B). A general decrease in anti-UT-A1 labeling was seen in inner medullary collecting duct (IMCD) cells from volume-expanded rats vs. control rats. CD, collecting ducts. Arrows, collecting duct cells.

UT-A3 is an additional urea transporter that is expressed in the IMCD (30). Figure 3 shows a UT-A3 immunoblot of inner medullary homogenates from control rats (aldosterone + 4-day low NaCl) compared with volume-expanded rats (aldosterone + 4-day high NaCl). As can be seen, there was a significant decrease in the band density of UT-A3 (to 39 ± 8% of control, P < 0.05). This response was confirmed by immunocytochemistry (Fig. 4). Collecting ducts from the renal inner medulla showed decreases in UT-A3 labeling in volume expansion vs. control. There was no obvious redistribution of UT-A3 labeling in the IMCD cells.


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Fig. 3.   Immunoblot assessing UT-A3 abundance in inner medullary homogenates from control rats (aldosterone + 4-day low-NaCl diet) vs. volume-expanded rats (aldosterone + 4-day high-NaCl diet). Each lane was loaded with a sample from a different rat. Values are means ± SE normalized to the mean value for the control group. The immunoblot for UT-A3 has 2 bands, 67 and 44 kDa, because of different states of glycosylation. The 67- and 44-kDa bands were analyzed together. Densitometric analysis revealed a decrease in the band density of UT-A3. * Significantly different from control, P < 0.05.



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Fig. 4.   Immunoperoxidase labeling for UT-A3 in the renal inner medulla of control rats (aldosterone + 4-day low-NaCl diet; A) vs. volume-expanded rats (aldosterone + 4-day high-NaCl diet; B). The magnification was identical in both images. A general decrease in the anti-UT-A3 labeling was seen in IMCD cells from volume-expanded rats vs. control rats.

UT-A2 is a urea transporter located chiefly in the outer medulla of the thin descending limb of Henle's loop (31). Figure 5 shows a UT-A2 immunoblot of outer medullary homogenates from control rats (aldosterone + 4-day low NaCl) compared with volume-expanded rats (aldosterone + 4-day high NaCl). As can be seen, there was no change in UT-A2 abundance.


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Fig. 5.   Immunoblot assessing UT-A2 abundance in outer medullary homogenates from control rats (aldosterone + 4-day low-NaCl diet) vs. volume-expanded rats (aldosterone + 4-day high-NaCl diet). Each lane was loaded with a sample from a different rat. Values are means ± SE normalized to the mean value for the control group. The 55-kDa band is UT-A2, and the lower band has been proposed to be UT-A4 but has not been validated. Densitometric analysis showed no difference in UT-A2 abundance in volume-expanded rats compared with control rats. OM, outer medulla. * Significantly different from control, P < 0.05.

Changes in abundance of AQPs. To determine whether other IMCD transport proteins exhibit changes in abundance with aldosterone-induced volume expansion, we carried out immunoblotting for AQP-2, AQP-3, and AQP-4 in the inner medulla (Fig. 6). AQP-2 is apically located in the IMCD (18). Figure 6A shows an AQP-2 immunoblot of inner medullary homogenates from control rats (aldosterone + 4-day low NaCl) compared with volume-expanded rats (aldosterone + 4-day high NaCl). There was no significant change. [As described before (18), AQP-2 is seen in immunoblots as two bands with an upper band, which is the glycosylated form, and a lower band, which is the nonglycosylated form.] AQP-3 and AQP-4 are located in the basolateral plasma membrane in the IMCD (6, 27). Figure 6, B and C, shows AQP-3 and AQP-4 immunoblots of inner medullary homogenates from control rats (aldosterone + 4-day low NaCl) compared with volume-expanded rats (aldosterone + 4-day high NaCl). There was no significant change in the abundance of either AQP.


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Fig. 6.   Immunoblots assessing aquaporin (AQP)-2 (A), AQP-3 (B), and AQP-4 (C) abundance in inner medullary homogenates from control rats (aldosterone + 4-day low-NaCl diet) vs. volume-expanded rats (aldosterone + 4-day high-NaCl diet). Each lane was loaded with a sample from a different rat. Values are means ± SE normalized to the mean value for the control group. There was no change in the abundance of any of the three AQPs. * Significantly different from control, P < 0.05.

Characteristics of the aldosterone-induced ECF volume expansion model. We carried out a time course study to assess changes in serum urea concentration and urinary urea clearance relative to changes in urea transporter abundance. Aldosterone-treated rats were euthanized 1, 2, and 4 days after being switched from the low-NaCl to the high-NaCl diet. Figure 7A shows the time course of Na balance after a change from the low-NaCl to the high-NaCl diet in these rats. NaCl intake significantly exceeded NaCl output on day 1, but Na balance was reestablished after day 1 as a result of mineralocorticoid escape (32). The positive Na balance seen on the first day would predict an increase in ECF volume of ~7 ml.1 Figure 7B shows the time course of water excretion in these animals. On day 1, there was a decrease in water excretion paralleling the net Na retention on day 1, which was consistent with the occurrence of ECF volume expansion. On day 4, creatinine clearance (an index of glomerular filtration rate) was not different between control (n = 5, 66.2 ± 3.4 ml/h) and volume-expanded (n = 5, 64.6 ± 8.7 ml/h) rats.


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Fig. 7.   Aldosterone-infused rats were initially placed on the control NaCl intake (0.02 meq/day) and were switched to the experimental NaCl intake (2.0 meq/day) at day 0, whereas the control rats remained on the 0.02 meq/day NaCl intake. Rats were euthanized 1, 2, or 4 days after the switch to the experimental NaCl intake. For each time point, control rats were euthanized at the same time as experimental rats for kidney processing. The day 1 point in the graphs represents an average of the values of the control rats euthanized at the corresponding 3 time points. A: time course of Na balance after a change from the low-NaCl to the high-NaCl diet. Positive Na balance was seen on the first day, but Na balance was reestablished after day 1. B: time course of water excretion after a change from the low-NaCl to the high-NaCl diet. There was a decrease in water excretion only on day 1. * Significantly different relative to corresponding control values at the same day, P < 0.05.

Figure 8A shows the time course of changes in urea clearance, demonstrating a significant increase after the increase in NaCl intake. Figure 8B shows the time course of changes in serum urea concentration, which decreased after the increase in NaCl intake.


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Fig. 8.   Aldosterone-infused rats were initially placed on the control NaCl intake (0.02 meq/day) and were switched to the experimental NaCl intake (2.0 meq/day) at day 0, whereas the control rats remained on the 0.02 meq/day NaCl intake. Rats were euthanized 1, 2, or 4 days after the switch to the experimental NaCl intake. For each time point, control rats were euthanized at the same time as experimental rats for kidney processing. The day 1 point in the graphs represents an average of the values of the control rats euthanized at the corresponding 3 time points. A: time course of urea clearance after a change from the low-NaCl to the high-NaCl diet. There was a significant increase in urea clearance at days 1 and 2 but not at day 4. B: time course of serum urea concentration after a change from the low-NaCl to the high-NaCl diet. There was a significant decrease in serum urea at all 3 times. * Significantly different relative to corresponding control values at the same time, P < 0.05.

Time course of changes in urea transporter abundance in volume expansion. Figure 9A compares the time course of changes in UT-A1 band densities with the time course of changes in serum urea concentration. (Note that the serum urea data are the same as shown in Fig. 8B and are repeated here for convenience.) In general, there was a parallel relationship between the two curves. Figure 9B compares the time course of changes in UT-A3 band density with the time course of changes in serum urea concentration. As seen, the fall in the abundance of UT-A3 lagged behind the fall in serum urea concentration.


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Fig. 9.   Aldosterone-infused rats were initially placed on the control NaCl intake (0.02 meq/day) and were switched to the experimental NaCl intake (2.0 meq/day) at day 0, whereas the control rats remained on the 0.02 meq/day NaCl intake. Rats were euthanized 1, 2, or 4 days after the switch to the experimental NaCl intake. For each time point, control rats were euthanized at the same time as experimental rats for kidney processing. The day 1 point in the graphs represents an average of the values of the control rats euthanized on the corresponding 3 days. (Note that the serum urea data are the same as shown in Fig. 8 and are repeated here for convenience.) Values are means ± SE normalized to the mean value for the control group. A: time course of changes in UT-A1 abundance after a change from the low-NaCl to the high-NaCl diet. In general, UT-A1 abundance decreased in parallel with the decrease in serum urea concentration. B: time course of changes in UT-A3 abundance after a change from the low-NaCl to the high-NaCl diet. There was a lag in the fall of UT-A3 abundance behind the fall in serum urea concentration. * Significantly different relative to corresponding control values at the same day, P < 0.05.

Effect of altered NaCl intake without aldosterone infusion. To test whether changing the NaCl intake without a stimulus to Na retention would alter urea transporter abundance, we performed a control study in which rats received the low-NaCl diet or high-NaCl diet for 4 days but without aldosterone infusion. Figure 10 shows immunoblots, which were probed for the three urea transporters in this control study. As seen, no changes were found in the abundance of any of the urea transporter proteins when rats on the low-NaCl diet were compared with rats on the high-NaCl diet. Thus the suppression of urea transporter abundance depends on prior treatment with aldosterone, which is presumably necessary for the volume expansion to occur.


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Fig. 10.   Control study: immunoblots assessing UT-A1 (A), UT-A3 (B), and UT-A2 (C) abundance in homogenates from control rats on 4-day low-NaCl diet vs. experimental rats on 4-day high-NaCl diet (without aldosterone infusion). Each lane was loaded with a sample from a different rat. Values are means ± SE normalized to the mean value for the control group. There were no changes in any of the urea transporter protein abundance when rats on the low-NaCl diet were compared with rats on the high-NaCl diet (in the absence of aldosterone infusion). * Significantly different from control, P < 0.05.

Effect of candesartan. Mineralocorticoid-induced ECF volume expansion is associated with suppression of the renin-angiotensin system. To test whether AT1 blockade mimics the effect of volume expansion, we infused candesartan (AT1 blocker) into rats receiving 0.5 meq/day NaCl. Figure 11 shows immunoblots of inner medullary homogenates that were probed for UT-A1 and UT-A3 in control rats compared with candesartan-treated rats. As can be seen, there was a marked fall in the abundance of UT-A1 (to 35 ± 9% of control, P < 0.05) and UT-A3 (to 28 ± 2% of control, P < 0.05) in response to candesartan infusion. These results are compatible with a role of angiotensin II in the long-term regulation of collecting duct urea transporter abundance.


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Fig. 11.   Candesartan study: immunoblots assessing UT-A1 and UT-A3 abundance in inner medullary homogenates from rats receiving 1.0 mg · kg-1 · day-1 candesartan (AT1 blocker) or vehicle for 2 days. Each lane was loaded with a sample from a different rat. Values are means ± SE normalized to the mean value for the control group. There was a marked fall in the abundance of UT-A1 and UT-A3 in response to candesartan infusion vs. control. * Significantly different from control, P < 0.05.


    DISCUSSION
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Clinical states associated with ECF volume expansion are often associated with decreased serum urea concentration. In the present study, serum urea concentration fell dramatically in aldosterone-treated rats in association with the transition from low-NaCl intake to high-NaCl intake. Changes in urine flow rate are known to affect fractional excretion of urea, but in this study differences in water excretion were prevented by using a ration-feeding protocol that ensured that the intake of water and nutrients was the same in both groups. To seek an explanation for the fall in serum urea concentration, we examined the abundance of the inner medullary urea transporters that mediate IMCD urea absorption in normal rats (26). Interestingly, semiquantitative immunoblotting demonstrated that the predominant effect of aldosterone-induced volume expansion was a marked decrease in the abundance of UT-A1 and UT-A3 in the inner medulla. Results from immunoperoxidase labeling of inner medullary sections also demonstrated a decrease in anti-UT-A1 labeling, with no change in the cellular distribution. These findings provide a possible explanation for the decrease in serum urea concentration: decreased transport of urea in the collecting duct would increase urea clearance.

Several factors have been previously shown to regulate urea excretion in mammals (22). Changes in urinary flow rate itself can alter urea excretion, possibly by changing urea concentration in the tubule lumen (23). As noted above, there was no sustained difference in water excretion between control and ECF volume-expanded rats in the present study. Vasopressin strongly stimulates urea transport in the IMCD (21), and a fall in plasma vasopressin levels conceivably could have contributed to the increased urea clearance seen in this study with ECF volume expansion. The effect of vasopressin in increasing urea transport occurs rapidly and is not associated with an increase in urea transporter abundance (29). The short-term effect of vasopressin on urea transport in the IMCD is not due to stimulation of trafficking of UT-A1 (9) but may be associated with direct phosphorylation of UT-A1 (14). Glucocorticoids also increase fractional urea excretion (15), in part through a decrease in UT-A1 protein abundance (17). Thus a decrease in circulating glucocorticoid levels may contribute to the changes that were seen in this study.

Studies by Hu et al. (8) have shown a marked decrease in the abundance of UT-A1 in an experimental model of chronic renal failure (5/6 renal ablation). In the present study, ECF volume expansion was found to be associated with a decrease in the abundance of UT-A1 and UT-A3. Because chronic renal failure is associated with ECF volume expansion, it is conceivable that the suppression of UT-A1 in these two studies may have had the same cause. The decrease in the abundance of UT-A1 and UT-A3 would have a potentially beneficial effect on ECF volume expansion by increasing water excretion.

It is well established that the renin-angiotensin-aldosterone system is a major regulator of ECF volume. ECF volume expansion would be expected to decrease circulating angiotensin II concentrations. Therefore, in this study, we examined whether candesartan (AT1 blocker) administration would be associated with changes in the abundance of UT-A isoforms in the kidney. Our findings demonstrate a marked decrease in the abundance of UT-A1 and UT-A3 in candesartan-treated rats compared with control rats. This suggests that angiotensin II may mediate changes in serum urea concentration by means of changes in the abundance of renal urea transporters. Previous studies by Kato et al. (11) in vasopressin-stimulated isolated, perfused tubules from rats have shown a direct, rapid increase in urea permeability with angiotensin II. In addition, angiotensin II has been demonstrated to downregulate endothelin-A and endothelin-B mRNA in isolated IMCDs (33). Thus there is precedent for angiotensin II-mediated regulation in the IMCD.

In summary, our studies have shown that aldosterone-induced volume expansion is associated with a decrease in serum urea concentration and an increase in urea clearance associated with a decrease in the abundance of UT-A1 and UT-A3 in the renal inner medulla. One factor that may play a role in this response is angiotensin II.


    ACKNOWLEDGEMENTS

This study was funded by the Intramural Budget of the National Heart, Lung, and Blood Institute (National Institutes of Health, Project Z01-HL-01282-KE to M. A. Knepper). S. Masilamani was supported by Career Transition Award K22-HL-66994. Support was obtained from the Danish Medical Research Council, Karen Elise Jensen Foundation, and Commission of the European Union (EU-TMR Program and K. A. 3.1.2 Program). The Water and Salt Research Center at the University of Aarhus is established and supported by The Danish National Research Foundation (Danmarks Grundforskningsfond).


    FOOTNOTES

1 One milliequivalent of Na ions would be distributed into enough ECF volume to reach a plasma Na concentration of 145 meq/l. Therefore, the equivalent would be ~1 meq divide  145 meq/l = 0.0069 liter.

Address for reprint requests and other correspondence: M. A. Knepper, National Institutes of Health, Rm. 6N260, Bldg. 10, 10 Center Dr., MSC 1603, Bethesda, MD 20892-1603 (E-mail: knep{at}helix.nih.gov).

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.

10.1152/ajprenal.00250.2001

Received 10 August 2001; accepted in final form 7 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Chou, CL, and Knepper MA. Inhibition of urea transport in inner medullary collecting duct by phloretin and urea analogues. Am J Physiol Renal Fluid Electrolyte Physiol 257: F359-F365, 1989[Abstract/Free Full Text].

2.   Chou, CL, Sands JM, Nonoguchi H, and Knepper MA. Concentration dependence of urea and thiourea transport in rat inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 258: F486-F494, 1990[Abstract/Free Full Text].

3.   Danielson, RA, Schmidt-Nielsen B, and Hohberger C. Micropuncture study of the regulation of urea excretion by the collecting ducts in rats on high-and low-protein diets. In: Urea and the Kidney, edited by Schmidt-Nielsen B, and Kerr DNS. Amsterdam: Excerpta Medica, 1968, p. 375-384.

4.   Digiovanni, SR, Nielsen S, Christensen EI, and Knepper MA. Regulation of collecting duct water channel expression by vasopressin in Brattleboro rat. Proc Natl Acad Sci USA 91: 8984-8988, 1994[Abstract].

5.   Ecelbarger, CA, Nielsen S, Olson BR, Murase T, Baker EA, Knepper MA, and Verbalis JG. Role of renal aquaporins in escape from vasopressin-induced antidiuresis in rat. J Clin Invest 99: 1852-1863, 1997[Abstract/Free Full Text].

6.   Ecelbarger, CA, Terris J, Frindt G, Echevarria M, Marples D, Nielsen S, and Knepper MA. Aquaporin-3 water channel localization and regulation in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 269: F663-F672, 1995[Abstract/Free Full Text].

7.   Hager, H, Kwon TH, Vinnakova AK, Masilamani S, Brooks HL, Frokiaer J, Knepper MA, and Nielsen S. Immunocytochemical and immunoelectron microscopical localization of alpha -, beta - and gamma -ENaC in rat kidney. Am J Physiol Renal Physiol 280: F1093-F1106, 2001[Abstract/Free Full Text].

8.   Hu, MC, Bankir L, Michelet S, Rousselet G, and Trinh-Trang-Tan MM. Massive reduction of urea transporters in remnant kidney and brain of uremic rats. Kidney Int 58: 1202-1210, 2000[ISI][Medline].

9.   Inoue, T, Terris J, Ecelbarger CA, Chou CL, Nielsen S, and Knepper MA. Vasopressin regulates apical targeting of aquaporin-2 but not of UT1 urea transporter in renal collecting duct. Am J Physiol Renal Physiol 276: F559-F566, 1999[Abstract/Free Full Text].

10.   Karakashian, A, Timmer RT, Klein JD, Gunn RB, Sands JM, and Bagnasco SM. Cloning and characterization of two new isoforms of the rat kidney urea transporter: UT-A3 and UT-A4. J Am Soc Nephrol 10: 230-237, 1999[Abstract/Free Full Text].

11.   Kato, A, Klein JD, Zhang C, and Sands JM. Angiotensin II increases vasopressin-stimulated facilitated urea permeability in rat terminal IMCDs. Am J Physiol Renal Physiol 279: F835-F840, 2000[Abstract/Free Full Text].

12.   Kim, GH, Ecelbarger CA, Mitchell C, Packer RK, Wade JB, and Knepper MA. Vasopressin increases Na-K-2Cl cotransporter expression in thick ascending limb of Henle's loop. Am J Physiol Renal Physiol 276: F96-F103, 1999[Abstract/Free Full Text].

13.   Kim, GH, Masilamani S, Turner R, Mitchell C, Wade JB, and Knepper MA. The thiazide-sensitive Na-Cl cotransporter is an aldosterone-induced protein. Proc Natl Acad Sci USA 95: 14552-14557, 1998[Abstract/Free Full Text].

14.   Klein, JD, Froehlich O, Zhang C, Timmer RT, Gunn RB, and Sands JM. Phosphorylation acutely regulates UT-A1 urea transporter activity in both rat tubules and cultured cells (Abstract). FASEB J 15: A853, 2001[ISI].

15.   Knepper, MA, Danielson RA, Saidel GM, and Johnston KH. Effects of dietary protein restriction and glucocorticoid administration on urea excretion in rats. Kidney Int 8: 303-315, 1975[ISI][Medline].

16.   Lassiter, WE, Gottschalk CW, and Mylle M. Micropuncture study of net transtubular movement of water and urea in nondiuretic mammalian kidney. Am J Physiol 200: 1139-1146, 1961[ISI].

17.   Naruse, M, Klein JD, Ashkar ZM, Jacobs JD, and Sands JM. Glucocorticoids downregulate the vasopressin-regulated urea transporter in rat terminal inner medullary collecting ducts. J Am Soc Nephrol 8: 517-523, 1997[Abstract].

18.   Nielsen, S, Digiovanni SR, Christensen EI, Knepper MA, and Harris HW. Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney. Proc Natl Acad Sci USA 90: 11663-11667, 1993[Abstract].

19.   Nielsen, S, Terris J, Smith CP, Hediger MA, Ecelbarger CA, and Knepper MA. Cellular and subcellular localization of the vasopressin-regulated urea transporter in rat kidney. Proc Natl Acad Sci USA 93: 5495-5500, 1996[Abstract/Free Full Text].

20.   Sands, JM, and Knepper MA. Urea permeability of mammalian inner medullary collecting duct system and papillary surface epithelium. J Clin Invest 79: 138-147, 1987[ISI][Medline].

21.   Sands, JM, Nonoguchi H, and Knepper MA. Vasopressin effects on urea and H2O transport in inner medullary collecting duct subsegments. Am J Physiol Renal Fluid Electrolyte Physiol 253: F823-F832, 1987[Abstract/Free Full Text].

22.   Schmidt-Nielsen, B. Urea excretion in mammals. Physiol Rev 38: 139-168, 1958[Free Full Text].

23.   Shannon, JA. Glomerular filtration and urea excretion in relation to urine flow in the dog. Am J Physiol 117: 206-225, 1936.

24.   Shayakul, C, Knepper MA, Smith CP, Digiovanni SR, and Hediger MA. Segmental localization of urea transporter mRNAs in rat kidney. Am J Physiol Renal Physiol 272: F654-F660, 1997[Abstract/Free Full Text].

25.   Shayakul, C, Steel A, and Hediger MA. Molecular cloning and characterization of the vasopressin-regulated urea transporter of rat kidney collecting ducts. J Clin Invest 98: 2580-2587, 1996[Abstract/Free Full Text].

26.   Star, RA, and Knepper MA. The vasopressin-regulated urea transporter in renal inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 259: F393-F401, 1990[Abstract/Free Full Text].

27.   Terris, J, Ecelbarger CA, Marples D, Knepper MA, and Nielsen S. Distribution of aquaporin-4 water channel expression within rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 269: F775-F785, 1995[Abstract/Free Full Text].

28.   Terris, J, Ecelbarger CA, Nielsen S, and Knepper MA. Long-term regulation of four renal aquaporins in rat. Am J Physiol Renal Fluid Electrolyte Physiol 271: F414-F422, 1996[Abstract/Free Full Text].

29.   Terris, J, Ecelbarger CA, Sands JM, and Knepper MA. Long-term regulation of renal urea transporter protein expression in rat. J Am Soc Nephrol 9: 729-736, 1998[Abstract].

30.   Terris, JM, Knepper MA, and Wade JB. UT-A3: localization and characterization of an additional urea transporter isoform in the IMCD. Am J Physiol Renal Physiol 280: F325-F332, 2001[Abstract/Free Full Text].

31.   Wade, JB, Lee AJ, Liu J, Ecelbarger CA, Mitchell C, Bradford AD, Terris J, Kim GH, and Knepper MA. UT-A2: a 55 kDa urea transporter in thin descending limb whose abundance is regulated by vasopressin. Am J Physiol Renal Physiol 278: F52-F62, 2000[Abstract/Free Full Text].

32.   Wang, XY, Masilamani S, Nielsen J, Kwon TH, Brooks HL, Nielsen S, and Knepper MA. The renal thiazide-sensitive Na-Cl cotransporter as mediator of the aldosterone-escape phenomenon. J Clin Invest 108: 215-222, 2001[Abstract/Free Full Text].

33.   Wong, NL, and Tsui JK. Angiotensin regulates endothelin-B receptor in rat inner medullary collecting duct. Metabolism 50: 661-666, 2001[ISI][Medline].

34.   You, G, Smith CP, Kanai Y, Lee WS, Stelzner M, and Hediger MA. Cloning and characterization of the vasopressin-regulated urea transporter. Nature 365: 844-847, 1993[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 282(4):F577-F584