Urea transport processes are induced in rat IMCD subsegments when urine concentrating ability is reduced

Akihiko Kato and Jeff M. Sands

Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Infusing urea into low-protein-fed mammals increases urine concentration within 5-10 min. To determine which urea transporter may be responsible, we measured urea transport in perfused IMCD3 segments [inner medullary collecting duct (IMCD) segments from the deepest third of the IMCD] from low-protein-fed rats. Basal facilitated urea permeability increased 78%, whereas active urea secretion was completely inhibited. This suggests that upregulation of facilitated urea transport may mediate the rapid increase in urine concentration. Next, expression of active urea transporter(s) in perfused IMCDs was determined in rats with other causes of reduced urine concentrating ability. In untreated and water diuretic rats, IMCD1 segments showed no active urea transport, nor did IMCD2 segments from untreated or hypercalcemic rats. In IMCD1 segments from hypercalcemic rats, active urea reabsorption was induced. The induced active urea reabsorption was completely inhibited by replacing perfusate Na+ with N-methyl-D-glucamine (NMDG+). Active urea secretion was completely inhibited in IMCD3 segments from hypercalcemic rats. In contrast, water diuresis stimulated active urea secretion in IMCD2 segments. The induced active urea secretion was inhibited by phloretin, ouabain, triamterene, or replacing perfusate Na+ with NMDG+. In conclusion, the response of active urea transporters to reductions in urine concentrating ability follows two paradigms: one occurs with hypercalcemia or a low-protein diet, and the second occurs only in water diuresis.

water diuresis; low-protein diet; hypercalcemia; urine concentrating mechanism; vasopressin; inner medullary collecting duct

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

UREA TRANSPORT in the kidney acts to assist or reduce water loss under different physiological conditions (9, 11, 26, 31). Nearly 40 years ago, Levinsky and Berliner (26) showed that infusing urea into humans or dogs fed a low-protein diet increases urinary concentration within 5-10 min. Infusing urea into low-protein-fed rats also increases urine concentration within minutes by increasing urea absorption from terminal inner medullary collecting ducts (IMCD), which in turn increases water absorption (11, 31, 45). The rapid increase in urea absorption suggests that a urea transporter is upregulated in terminal IMCDs by a low-protein diet. Since urea is transported by both facilitated and active transport pathways in rat IMCDs (see Ref. 36 for a review), the first goal of this study was to determine which urea transporter may be responsible for the rapid increase in urine concentration following urea infusion.

In normal rats eating 18% protein, facilitated urea transport is expressed only in terminal IMCD subsegments (IMCD2 and IMCD3) and is stimulated acutely by vasopressin (29, 35, 40). Vasopressin-stimulated facilitated urea transport is not present in initial IMCDs (IMCD1 segments) from normal rats, but is induced in IMCD1 segments by a low-protein diet (1, 15, 17). Rat IMCD subsegments also express Na+-dependent, secondary active, urea transport. In normal rats fed 18% protein, the deepest third of the IMCD, the IMCD3, actively secretes urea via a Na+-dependent, urea countertransport pathway (20). IMCD1 segments from normal rats do not express active urea transport (17, 19, 20). However, two different active, Na+-dependent, reabsorptive urea transport processes can be induced in IMCD1 segments: low-protein-fed rats express a Na+-dependent, urea cotransport process in the apical membrane (15-17), whereas furosemide induces a Na+-dependent, urea countertransport process in the basolateral membrane (19). Active urea transport is not expressed in IMCD2 segments from normal rats, nor is it induced by a low-protein diet or furosemide (17, 19, 20). Since changes in active urea transport processes have not been fully explored, the second goal of this study was to determine which, if any, active urea transporter is induced in specific IMCD subsegments from rats with reduced urine concentrating ability.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Tissue Preparation

All animal protocols were approved by the Emory University Institutional Animal Care and Use Committee. Tubules were obtained from pathogen-free male Sprague-Dawley rats (National Cancer Institute, Frederick, MD). The rats were kept in filter-top cages with autoclaved bedding and received free access to water and a normal (18%) protein diet (NIH-31; Zeigler Brothers, Gardners, PA) unless otherwise indicated below. The kidneys were placed into chilled (17°C), isotonic, dissecting solution to isolate initial (IMCD1) or terminal (IMCD2 or IMCD3) IMCD subsegments (7, 8). Initial IMCDs were identified by dissecting between the inner-outer medullary border (identified by the absence of thick ascending limbs) and the first merger of IMCDs (20, 33). IMCD2 segments were identified by dissecting between 50 and 70% of the distance between the inner-outer medullary border (0%) and the papillary tip (100%) as measured using an eyepiece micrometer (20, 33). IMCD3 segments were dissected distal to a bifurcation occurring beyond 70% of the distance between the inner-outer medullary border and the papillary tip (20, 33).

Protocols

Untreated (control). Rats were given an 18% protein diet and water ad libitum.

Low-protein diet. Rats were given free access to an isocaloric low-protein (8%) diet (NIH-31M, Zeigler Brothers) and water for 3 wk. Rats fed this low-protein diet grow and maintain normal values of serum albumin, creatinine, total protein, and potassium (15, 17).

Hypercalcemia. Rats were given free access to an 18% protein diet containing vitamin D [dihydrotachysterol (DHT), 4.25 mg · kg diet-1 · day-1; Roxane Laboratories, Columbus, OH] and water for 3-4 days (25, 32).

Water diuresis. Rats were fed 10% glucose in water without additional food for 1, 3-5, or 7 days. We combined the results from rats undergoing water diuresis for 3-5 days, because there were no differences in the data obtained from these rats.

Water diuresis plus food. Rats were fed 10% glucose in water and given free access to an 18% protein diet for 3-5 days. We combined the results from rats undergoing water diuresis plus food for 3-5 days, because there were no differences in the data obtained from these rats.

Water deprivation. Rats were given free access to an 18% protein diet but no water for 2-3 days. We combined the results from rats that were water restricted for 2-3 days, because there were no differences in the data obtained from these rats.

The dissecting solution was gassed with 95% O2-5% CO2 and contained (in mM) 118 NaCl, 25 NaHCO3, 2 CaCl2, 2.5 K2HPO4, 1.2 MgSO4, 5.5 glucose, and 4 creatinine. Tubules were transferred into a bath that was continuously exchanged and bubbled with 95% O2-5% CO2 gas and perfused using standard techniques (16, 20, 33, 35). Solution and urine osmolalities were measured by vapor-pressure osmometry (model 5500; Wescor, Logan, UT).

Urea Measurement

The urea concentration in perfusate, bath, and collected fluid was measured using a continuous-flow ultramicrofluorometer as described (16, 20, 33). This assay is capable of resolving differences of 4% or greater in urea concentration (33). Urea flux (Jurea) was calculated as Jurea = CoVo - ClVl, where Co is the urea concentration in the perfusate, Cl is the urea concentration in the collected fluid, Vo is the perfusion rate per unit length of tubule, and Vl is the collection rate per unit length of tubule. On the basis of this formula, a positive value for Jurea indicates urea reabsorption, whereas a negative value indicates urea secretion.

To study facilitated urea permeability, tubules were perfused with perfusate and bath solutions whose composition was identical to the dissection solution (described above) except that 5 mM urea was added to the bath solution and 5 mM raffinose was added to the perfusate solution to create a 5 mM bath-to-lumen urea gradient without an imposed osmotic gradient (15, 17, 32, 35). Urea permeability was calculated from the urea flux as described (15, 17, 35).

To study active urea transport, tubules were perfused with the same perfusate and bath solution (except during the ion substitution studies described below). Solution composition was identical to the dissection solution (described above) except that 3 mM urea was added (16, 17, 20). To calculate Jurea, Vo is assumed to be equal to Vl, because there is no osmotic gradient across the tubule and, hence, no driving force for water reabsorption. We showed previously that the measured volume flux is 0, both with and without vasopressin added to the bath solution, under these experimental conditions (17, 20).

Effect of Vasopressin or Inhibitors on Urea Transport

The urea concentration of three to four collections was measured, after which the following compounds (Sigma Chemical, St. Louis, MO) were added: 1) 10 nM arginine vasopressin added to the bath (16, 20); 2) 250 µM phloretin added to the perfusate (16, 17, 20); 3) 1 mM ouabain added to the bath (16, 20); or 4) 100 nM triamterene added to the perfusate (20). Then, three to four additional collections were obtained. Twenty minutes were allowed between the addition of these compounds and the beginning of measurements. Next, the compound was washed out for 30 min, and three to four additional collections were obtained.

A 250 mM stock solution of phloretin was prepared in absolute ethanol and added to perfusate to achieve a final concentration of 250 µM phloretin and 0.1% ethanol (16, 20). Control collections were obtained with 0.1% ethanol added to the perfusate (16, 20).

Effect of Ion Substitution on Net Urea Transport

The urea concentration of three to four collections was measured. Next, Na+ was removed from either the perfusate or bath and replaced by N-methyl-D-glucamine (NMDG+, in equimolar concentrations), and three to four collections were obtained. After the solution was changed to return Na+, three to four additional collections were obtained (16, 20).

Volume Flux Measurement

Creatinine concentration in perfusate, bath, and collected fluid was measured using a continuous-flow ultramicrocolorimeter and used to measure volume flux (17, 20, 35). The perfusion rate (Vo) was calculated as Vo = Vl(Crl/Cro), where Cro is the creatinine concentration in the perfusate, Crl is the creatinine concentration in the collected fluid, and Vo and Vl are as defined above (see Urea Measurement). Volume flux (Jv) was calculated as Jv = Vo - Vl (17, 20).

Statistics

All data are presented as means ± SE, and n = number of rats. Data from three to four collections were averaged to obtain a single value from each experimental phase in each tubule. To test for statistical significance between two groups, Student's t-test was used. To test more than two groups, an ANOVA was used, followed by Tukey's protected t-test (39) to determine which groups were significantly different. The criterion for statistical significance was P < 0.05. Paired statistical analysis was used for the vasopressin, inhibitor, and ion substitution protocols since each tubule was used as its own control; these data are shown as dots connected by lines in Figs. 1, 4, and 7-9. Unpaired statistical analysis was used for the protocols comparing different diets and the time course study; these data are shown as bars in Figs. 2, 3, 5, 6, and 10.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of a Low-Protein Diet on Facilitated and Active Urea Transport in the IMCD3

Basal facilitated urea permeability was significantly increased in IMCD3 segments from rats fed 8% protein diet (87 ± 15 ×10-5 cm/s, n = 5) compared with rats fed 18% protein diet (49 ± 3 ×10-5 cm/s; n = 5, P < 0.01; Fig. 1). Vasopressin increased facilitated urea permeability in IMCD3 segments from rats fed 18% protein (110 ± 14 ×10-5 cm/s, P < 0.01 vs. basal value) or 8% protein (184 ± 17 ×10-5 cm/s, P < 0.01 vs. basal value, P < 0.01 vs. vasopressin-stimulated value in IMCD3 segments from rats fed 18% protein).


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Fig. 1.   Facilitated urea transport in IMCD3 segments from rats fed an 18% (solid line) or an 8% (dashed line) protein diet for 3 wk. IMCD3 segments from rats fed 8% protein had a significant increase in basal urea permeability compared with IMCD3 segments from rats fed 18% protein. Arginine vasopressin (AVP, 10 nM, added to the bath) significantly increased urea permeability in rats fed either diet. Data are means ± SE; n = 5. * P < 0.01 for basal vs. AVP values. + P < 0.01 for IMCD3 segments from rats fed 18% vs. 8% protein. See METHODS (Tissue Preparation) for complete description of the 3 subsegments of the inner medullary collecting duct (IMCD1, IMCD2, and IMCD3).

Dietary protein restriction for 3 wk significantly decreased urine osmolality (18% protein diet, 1,534 ± 142 mosmol/kgH2O, n = 6; 8% protein diet, 601 ± 121 mosmol/kgH2O, n = 6, P < 0.01). There was an increase in urinary corticosterone in rats fed 8% protein (645 ± 105 ng/day, n = 5) compared with that from rats fed 18% protein (192 ± 18 ng/day; n = 5, P < 0.01).

In IMCD3 segments from rats fed 18% protein, active urea secretion was present (-9.8 ± 2.2 pmol · mm-1 · min-1, n = 10). Active urea secretion was significantly reduced in IMCD3 segments from rats fed 8% protein (1.1 ± 2.7 pmol · mm-1 · min-1; n = 5, P < 0.01 vs. 18% protein; Fig. 2).


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Fig. 2.   Net urea flux in IMCD3 segments from rats fed an 18% or an 8% protein diet for 3 wk. A significant rate of net urea secretion was present in IMCD3 segments from rats fed 18% protein. IMCD3 segments from rats fed 8% protein had a significant reduction in net urea secretion compared with IMCD3 segments from rats fed 18% protein. Data are means ± SE; n = 10 for IMCD3 segments from rats fed 18% protein and n = 5 for IMCD3 segments from rats fed 8% protein. * P < 0.01.

Effect of Hypercalcemia on Active Urea Transport in IMCD Subsegments

Administering DHT for 3 days increased serum calcium from 10.1 ± 0.1 to 12.3 ± 0.1 mg/dl (n = 5, P < 0.01) and decreased urine osmolality from 1,534 ± 142 to 691 ± 49 mosmol/kgH2O (P < 0.01). There was an increase in urinary corticosterone excretion in DHT-treated rats (control, 192 ± 18; DHT, 293 ± 34 ng/day, P < 0.05).

Active urea secretion was significantly decreased in IMCD3 segments from hypercalcemic rats (1.2 ± 2.4 pmol · mm-1 · min-1; n = 5; Fig. 3) compared with IMCD3 segments from untreated rats (-9.7 ± 2.2 pmol · mm-1 · min-1; n = 10, P < 0.01). In contrast, there was no active urea transport in IMCD2 segments from untreated [1.6 ± 0.8 pmol · mm-1 · min-1; P = not significant (NS) vs. 0, n = 5] or hypercalcemic (3.2 ± 1.0 pmol · mm-1 · min-1; P = NS vs. 0, n = 5) rats.


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Fig. 3.   Net urea flux in IMCD subsegments from hypercalcemic rats. In untreated rats (solid bars), no significant net urea transport is present in either IMCD1 segments or IMCD2 segments, but a significant rate of net urea secretion was present in IMCD3 segments. In hypercalcemic rats [treated with dihydrotachysterol (DHT), open bars], a significant rate of net urea reabsorption was present in IMCD1 segments, but not in IMCD2 segments or IMCD3 segments. IMCD3 segments from hypercalcemic rats had a significant reduction in net urea secretion compared with IMCD3 segments from untreated rats. A positive net urea flux indicates reabsorption, whereas a negative flux indicates secretion. Data are means ± SE; n = 5 except n = 7 for IMCD1 segments from hypercalcemic rats and n = 10 for IMCD3 segments from untreated rats. * P < 0.01.

IMCD1 segments from untreated rats did not transport urea actively (0.1 ± 0.5 pmol · mm-1 · min-1; P = NS vs. 0, n = 5). In contrast, IMCD1 segments from hypercalcemic rats actively reabsorbed urea (18.4 ± 4.6 pmol · mm-1 · min-1; n = 7, P < 0.01; Fig. 3).

Removing Na+ from the perfusate (and replacing it with NMDG+) completely inhibited active urea reabsorption in IMCD1 segments from DHT-treated rats (basal, 20.3 ± 2.4; perfusate Na+ removal, 0.9 ± 1.0 pmol · mm-1 · min-1; n = 5, P < 0.01; Fig. 4A). When perfusate Na+ was restored, active urea reabsorption returned to 10.9 ± 1.6 pmol · mm-1 · min-1 (P = NS vs. control period). In contrast, removing Na+ from the bath had no significant effect on active urea reabsorption (control, 14.7 ± 4.1; bath Na+ removal, 12.9 ± 2.1 pmol · mm-1 · min-1; n = 5, P = NS; Fig. 4B).


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Fig. 4.   Effect of removing Na+ [and replacing it with N-methyl-D-glucamine+ (NMDG+)] from perfusate or bath on net urea reabsorption in IMCD1 segments from hypercalcemic rats. A: perfusate Na+ removal significantly and reversibly inhibited net urea reabsorption. B: bath Na+ removal did not change net urea reabsorption. Data are means ± SE; n = 5. * P < 0.01.

Serum and Urine Parameters in Water Diuretic Rats

There was no significant difference in serum creatinine values between untreated rats and rats given 10% sugar water ad libitum, alone (SW) or with food (SW+F) for 3 days (Table 1). In contrast, serum urea nitrogen values were significantly reduced in both groups of water diuretic rats (Table 1). Urinary corticosterone excretion was significantly increased in both groups of water diuretic rats (Table 1).

                              
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Table 1.   Serum and urine values in water diuretic rats

Next, rats were placed in metabolic cages for 3 days, and the values obtained on day 0 were compared with those after 3 days of drinking 10% sugar water, alone or with food (Table 1). Both groups of water diuretic rats had significantly increased 24-h urine volumes and decreased urine osmolalities (Table 1). Urea clearance did not change in rats receiving sugar water alone but was significantly increased in rats receiving sugar water plus food (Table 1).

Active Urea Transport in IMCD1 and IMCD2 Segments From Rats Undergoing Water Diuresis

There was no active urea transport in IMCD1 segments from untreated rats or any group of water diuretic rats (untreated, 0.1 ± 0.5, P = NS vs. 0, n = 5; SW, 0.5 ± 2.7, P = NS vs. 0, n = 5; SW+F, -0.1 ± 1.5 pmol · mm-1 · min-1, P = NS vs. 0, n = 5; Fig. 5, left). IMCD1 segments from water-restricted rats also did not transport urea actively (-0.3 ± 2.0 pmol · mm-1 · min-1; P = NS vs. 0, n = 5).


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Fig. 5.   Net urea flux in IMCD subsegments from diuretic or antidiuretic rats. In untreated rats (solid bars), no significant net urea transport is present in either IMCD1 segments or IMCD2 segments. In rats made water diuretic for 3-5 days by feeding them sugar water alone (open bars) or sugar water plus 18% protein diet ad libitum (hatched bars), a significant rate of net urea secretion was present in IMCD2 segments, but not in IMCD1 segments. In rats deprived of water for 2-3 days (cross-hatched bars), no significant net urea transport is present in either IMCD1 segments or IMCD2 segments. A positive net urea flux indicates reabsorption, whereas a negative flux indicates secretion. Data are means ± SE; n = 5. * P < 0.01.

There was no active urea transport in IMCD2 segments from untreated rats (1.0 ± 0.5 pmol · mm-1 · min-1;P = NS vs. 0, n = 5). In contrast, active urea secretion was present in IMCD2 segments from both groups of rats undergoing water diuresis for 3-5 days (SW, -5.5 ± 2.1, n = 5, P < 0.01 vs. untreated rats; SW+F, -7.5 ± 2.8 pmol · mm-1 · min-1, n = 5, P < 0.01 vs. untreated rats; Fig. 5, right). IMCD2 segments from water-restricted rats did not transport urea actively (1.3 ± 2.2 pmol · mm-1 · min-1; P = NS vs. 0, n = 5).

Next, we examined the time course for expression of active urea secretion in IMCD2 segments from rats given sugar water but no food. IMCD2 segments displayed active urea secretion after 1 day of water diuresis (-7.4 ± 3.9 pmol · mm-1 · min-1; n = 5, P < 0.05 vs. day 0) and at 7 days (-12.7 ± 5.6 pmol · mm-1 · min-1;n = 7, P < 0.01 vs. day 0; Fig. 6). There were no significant differences in the values for active urea secretion between days 1 and 7.


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Fig. 6.   Time course for changes in net urea secretion. Compared with IMCD2 segments from untreated rats (0 days, n = 5), net urea secretion was significantly increased in IMCD2 segments from rats fed sugar water but no food for 1 day (n = 5), 3-5 days (n = 16), and 7 days (n = 7). Data are means ± SE. * P < 0.05.

Characteristics of Active Urea Secretion in IMCD2 Segments from Rats Given Sugar Water But No Food for 3-5 Days

Vasopressin increased active urea secretion in IMCD2 segments from -6.4 ± 1.9 to -14.4 ± 4.5 pmol · mm-1 · min-1 (n = 6, P < 0.05; Fig. 7A). There was no significant fluid flux in IMCD2 segments when vasopressin was present (-0.03 ± 0.59 nl · mm-1 · min-1; n = 4, P = NS vs. 0; Table 2).


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Fig. 7.   Effect of adding AVP (10 nM) or ouabain (1 mM) to the bath on net urea secretion in IMCD2 segments from rats fed sugar water but no food for 3-5 days. A: AVP significantly increased net urea secretion (n = 6). B: ouabain significantly and reversibly inhibited net urea secretion (n = 8). Data are means ± SE. * P < 0.05.

                              
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Table 2.   Volume flux in IMCD2 tubules from water diuretic rats with vasopressin

Ouabain decreased active urea secretion from -8.0 ± 3.2 to -2.5 ± 2.1 pmol · mm-1 · min-1 (n = 8, P < 0.01; Fig. 7B). When ouabain was washed out of the bath, active urea secretion was returned to -7.2 ± 3.0 pmol · mm-1 · min-1 (n = 8, P = NS vs. control period).

Phloretin inhibited active urea secretion from -8.2 ± 2.7 to -2.8 ± 0.9 pmol · mm-1 · min-1 (n = 6, P < 0.01; Fig. 8A). When phloretin was removed, active urea secretion returned to -7.8 ± 0.8 pmol · mm-1 · min-1 (n = 5, P = NS vs. control period).


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Fig. 8.   Effect of adding phloretin (0.25 mM) or triamterene (100 nM) to the perfusate on net urea secretion in IMCD2 segments from rats fed sugar water but no food for 3-5 days. A: phloretin significantly and reversibly inhibited net urea secretion (n = 6). B: triamterene significantly and reversibly inhibited net urea secretion (n = 5). Data are means ± SE. * P < 0.01.

Adding triamterene decreased active urea secretion from -7.2 ± 0.3 to 1.2 ± 0.8 pmol · mm-1 · min-1 (n = 5, P < 0.01; Fig. 8B). When triamterene was removed, active urea secretion was returned to -7.3 ± 2.4 pmol · mm-1 · min-1 (P = NS vs. control period).

Removing Na+ from the perfusate (and replacing it with NMDG+) completely inhibited active urea secretion (control, -12.4 ± 3.5; perfusate Na+ removal, 1.7 ± 1.7 pmol · mm-1 · min-1; n = 5, P < 0.01; Fig. 9A). When perfusate Na+ was restored, active urea secretion returned to -9.2 ± 1.7 pmol · mm-1 · min-1 (n = 4, P = NS vs. control period). In contrast, removing Na+ from the bath had no effect on active urea secretion in IMCD2 segments (basal, -9.0 ± 2.9; bath Na+ removal, -10.3 ± 2.6 pmol · mm-1 · min-1; n = 5, P = NS, Fig. 9B).


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Fig. 9.   Effect of removing Na+ (and replacing it with NMDG+) from the perfusate or bath on net urea secretion in IMCD2 segments from rats fed sugar water but no food for 3-5 days. A: perfusate Na+ removal significantly and reversibly inhibited net urea secretion. B: bath Na+ removal did not change net urea secretion. Data are means ± SE; n = 5. * P < 0.01.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The main results of our study are that 1) basal and vasopressin-regulated facilitated urea permeabilities are upregulated in IMCD3 segments from low-protein-fed rats and 2) the expression of active urea transport pathways vary in different rat models in which urine concentrating ability is reduced (Table 3). Thus a low-protein diet, water diuresis (18), furosemide (18), and hypercalcemia (32) are all associated with upregulation of basal and vasopressin-stimulated facilitated urea permeabilities. In contrast, reduced urine concentrating ability is associated with two patterns of changes in active urea transport. One pattern occurs in hypercalcemia (Fig. 3), a low-protein diet (Fig. 10), or with furosemide (see figure 1 in Ref. 20), in these three conditions: 1) active urea secretion decreases in IMCD3 segments; 2) active urea reabsorption appears in IMCD1 segments; and 3) no active urea transport is detectable in IMCD2 segments (17, 19, 20). The second pattern is unique to water diuresis: 1) active urea secretion is upregulated in IMCD3 segments (20), and 2) active urea secretion appears in IMCD2 segments. Water diuresis is the only condition identified that induces active urea transport in IMCD2 segments (17, 19, 20).

                              
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Table 3.   Summary of changes in active urea transport processes in IMCD subsegments


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Fig. 10.   Summary of net urea flux in IMCD subsegments from rats fed an 18% or 8% protein diet for 3 wk. Data are redrawn from Ref. 16 for IMCD1 segments, Ref. 17 for IMCD2 segments, and Fig. 1 for IMCD3 segments. In rats fed 18% protein (solid bars), no significant net urea transport is present in either IMCD1 segments or IMCD2 segments, but a significant rate of net urea secretion was present in IMCD3 segments. In rats fed 8% protein (open bars), a significant rate of net urea reabsorption was present in IMCD1 segments, but not in IMCD2 segments or IMCD3 segments. IMCD3 segments from rats fed 8% protein had a significant reduction in net urea secretion compared with IMCD3 segments from rats fed 18% protein. A positive net urea flux indicates reabsorption, whereas a negative flux indicates secretion. * P < 0.01.

Low-Protein Diet

Feeding humans or rats a low-protein diet reduces urine concentrating ability and the fractional excretion of urea (9, 11, 14, 21, 30, 38). We previously showed that the mechanisms by which a low-protein diet reduces urine concentrating ability in rats include: 1) a decrease in vasopressin-stimulated osmotic water permeability and aquaporin-2 (AQP2) protein abundance in terminal IMCDs (34); 2) an increase in UT-A1 protein abundance in inner medulla (41); 3) the appearance of vasopressin-stimulated facilitated urea permeability in initial IMCDs (IMCD1 segments) (15, 17); and 4) the appearance of active urea reabsorption in IMCD1 segments (1, 15-17). The latter two changes would increase urea absorption across the initial IMCD, thus decreasing urea delivery to terminal IMCD subsegments and urine concentrating ability (6, 43).

In this study, we found that feeding rats a low-protein diet for 3 wk alters both active and facilitated urea transport processes in IMCD3 segments: active urea secretion is reduced, and basal and vasopressin-stimulated facilitated urea permeabilities are increased. Both of these changes in urea transport in IMCD3 segments would decrease the fractional excretion of urea by increasing urea absorption in rats fed a low-protein diet. The increase in facilitated urea permeability and upregulation of UT-A1 protein (41) in rats fed a low-protein diet may be a mechanism that allows for the rapid absorption of urea and the acute recovery of urine concentrating ability that occurs within 5-10 min after urea is infused into mammals fed a low-protein diet (26, 31, 45).

Hypercalcemia

Hypercalcemia causes polyuria and impairs urine concentrating ability in humans and experimental animals (25, 27). Tissue slice studies in rats show that hypercalcemia decreases the inner medullary content of urea and Na+ (25, 27). Studies of isolated papillae from hypercalcemic rats show reduced levels of vasopressin-stimulated diffusional water permeability (5). We showed that the mechanisms by which hypercalcemia reduces urine concentrating ability include: 1) a decrease in vasopressin-stimulated osmotic water permeability and AQP2 protein abundance in rat terminal IMCDs; 2) an increase in basal and vasopressin-stimulated facilitated urea permeability and UT-A1 protein abundance in rat terminal IMCDs; and 3) the appearance of vasopressin-stimulated facilitated urea permeability in rat initial IMCDs (IMCD1 segments) (32).

In this study, we found that IMCD1 segments from hypercalcemic rats had significant rates of active urea reabsorption that were dependent on luminal Na+ but not on bath Na+. Thus, while hypercalcemia, a low-protein diet (16), and furosemide (20) all induce Na+-dependent active urea reabsorption in IMCD1 segments, hypercalcemia and a low-protein diet induce the same transport process, whereas furosemide induces a different one (Table 4). The increase in active urea reabsorption across IMCD1 segments will decrease the delivery of urea to the deep inner medullary interstitium, thus providing an additional mechanism that contributes to the impairment of urine concentrating ability observed in hypercalcemic animals.

                              
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Table 4.   Comparison of Na+-dependent active urea transport mechanisms in IMCD subsegments

We also found that active urea secretion was decreased in IMCD3 segments from hypercalcemic rats, similar to the response of IMCD3 segments from furosemide-treated (19) and low-protein-fed rats. Thus hypercalcemia, furosemide, and a low-protein diet cause similar changes in active urea transport in IMCD1 segments and IMCD3 segments, although the mechanism for active urea reabsorption in IMCD1 segments differs between the three conditions (Table 4). The reduction of active urea secretion in IMCD3 segments, along with the increase in facilitated urea permeability (32), may be compensatory mechanisms that diminish the washout of urea from the deep inner medullary interstitium which would otherwise occur as a result of hypercalcemia-induced increases in medullary blood flow (3).

Water Diuresis

Tissue slice experiments show that inner medullary urea content exceeds urinary values during water diuresis (2, 4, 13). Microcatheterization (44) or micropuncture (23, 24, 42) studies suggest that urea is actively secreted from the medullary interstitium into the terminal portion of the rat medullary collecting duct during water diuresis. We showed that urea is actively secreted by perfusing IMCD3 segments from rats eating a normal protein diet and consuming water ad libitum (20). We also showed that active urea secretion is upregulated twofold in IMCD3 segments from rats drinking sugar water alone and upregulated fivefold in IMCD3 segments from rats drinking sugar water and eating a normal, 18% protein diet (20).

In this study, water diuresis for 1-7 days induced active urea secretion in IMCD2 segments. This new, undescribed, active urea secretory transport in IMCD2 segments has the same functional characteristics as active urea secretion in IMCD3 segments (Table 4), suggesting that water diuresis is stimulating the same active urea secretory transport mechanism in both subsegments of the rat terminal IMCD. Interestingly, active urea secretion is, in fact, specific for terminal IMCDs, since there was no active urea secretion in initial IMCDs (IMCD1 segments) from water diuretic rats. The increase in active urea secretion could explain, at least in part, the increase in urea clearance and decrease in serum urea nitrogen we measured in the water diuretic rats.

Summary and Perspective

We found that a low-protein diet increases basal and vasopressin-stimulated facilitated urea permeabilities in IMCD3 segments. Thus expression of basal and vasopressin-stimulated facilitated urea permeabilities are similar in IMCD3 segments from all of the rat models in which urine concentrating ability is reduced (low-protein diet, water diuresis, hypercalcemia, or furosemide-treatment; see Refs. 18 and 32). We speculate that upregulation of facilitated urea permeability may be the mechanism by which urine concentrating ability is rapidly restored when urea is infused into humans, dogs, or rats (26, 31, 45).

We also found that a low-protein diet or hypercalcemia decreases active urea secretion in IMCD3 segments and induces active urea reabsorption in IMCD1 segments. Active urea reabsorption in IMCD1 segments from hypercalcemic rats is dependent on luminal Na+, similar to the Na+-dependent active urea reabsorptive transport process in IMCD1 segments from rats fed a low-protein diet (16). In normal rats, active urea secretion in IMCD3 segments, accompanied by sodium reabsorption (sodium/urea countertransport), could contribute to urinary concentration by increasing the interstitial sodium concentration in the deep inner medulla. Thus sodium/urea countertransport in IMCD3 segments could be a mechanism whereby the urea gradient generated by active NaCl absorption in thick ascending limbs is used to augment the sodium concentration in the deepest portions of the inner medullary interstitium. The decrease in active urea secretion in low-protein or hypercalcemic rats would therefore reduce urine concentrating ability by eliminating this mechanism.

Last, we found that water diuresis induces active urea secretion in IMCD2 segments. This previously unrecognized urea secretory process is a secondary active, phloretin-inhibitable, triamterene-inhibitable, luminal Na+-dependent, urea transport process. The mechanism by which triamterene inhibits active urea secretion cannot be determined until the cotransporter is cloned. However, triamterene could act directly on the sodium site of the cotransporter, similar to its effect on the sodium-proton exchanger, or indirectly via an effect on sodium channels (10, 12, 37).

The expression of active urea transport pathways follows two general paradigms in these different rat models in which urine concentrating ability is reduced (Table 3): one paradigm occurs in hypercalcemic rats, rats fed a low-protein diet (16, 17), or in furosemide-treated rats (19); the other occurs only in water diuretic rats (20). We speculate that both paradigms limit urine concentrating ability by decreasing urea delivery into the inner medullary tip (or papilla). In the first paradigm, induction of active urea reabsorption in IMCD1 segments will increase urea absorption into the inner medullary base, thereby decreasing urea delivery to terminal IMCDs. The inhibition of sodium/urea countertransport in IMCD3 segments would tend to limit the loss of urea from the inner medullary tip and may also be a mechanism that contributes to the decrease in inner medullary sodium content (14, 27). In the second paradigm, upregulation of active urea secretion in terminal IMCD subsegments will decrease the urea content in the papilla. Thus active urea transport mechanisms are altered so that papillary urea content is reduced in rats with reduced urine concentrating ability. Future studies will be needed to determine why different active urea transporters are upregulated or downregulated in these different physiological conditions. Overall, these findings suggest that changes in active and facilitated urea transport processes in each of the three subsegments of the IMCD, along with changes in osmotic water permeability (22, 28, 32, 34), contribute to the reduction of urinary concentrating ability during water diuresis, hypercalcemia, dietary protein restriction, and furosemide diuresis.

    ACKNOWLEDGEMENTS

We thank Dr. William E. Mitch (Emory University) for critical reading of this manuscript.

    FOOTNOTES

Portions of this work have been published in abstract form (J. Am. Soc. Nephrol. 8: 20, 1997; and J. Am. Soc. Nephrol. 9: 20A-21A, 1998) and presented at the 30th and 31st Annual Meetings of the American Society of Nephrology, November 2-5, 1997, San Antonio, TX, and October 25-28, 1998, Philadelphia, PA.

This work was supported by American Heart Association Grant-in-Aid 96006090 and by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-41707 and P01-DK-50268.

Present address of A. Kato: First Department of Medicine, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-31, Japan.

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.

Address for reprint requests: J. M. Sands, Emory Univ. School of Medicine, Renal Division, WMRB Rm. 338, 1639 Pierce Drive, NE, Atlanta, GA 30322.

Received 20 July 1998; accepted in final form 17 September 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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