Altered expression of urea transporters in response to ureteral obstruction

Chunling Li,1,2 Janet D. Klein,3 Weidong Wang,1,4 Mark A. Knepper,5 Søren Nielsen,1,4 Jeff M. Sands,3 and Jørgen Frøkiær1,2,6

1The Water and Salt Research Center, University of Aarhus, DK-8000 Aarhus C; 2Institute of Experimental Clinical Research and 6Department of Clinical Physiology, Aarhus University Hospital-Skejby, DK-8200 Aarhus N; 4Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark; 3Renal Division, Department of Medicine, Emory University, Atlanta, Georgia 30322; and 5Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

Submitted 30 December 2003 ; accepted in final form 11 February 2004


    ABSTRACT
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 ABSTRACT
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 DISCUSSION
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Urea plays an important role in the urinary concentrating capacity. Renal inner medullary (IM) urea transporter expression was examined in rats with bilateral (BUO) or unilateral ureteral obstruction (UUO). BUO (24 h) was associated with markedly increased plasma urea (42.4 ± 1.0 vs. 5.2 ± 0.2 mmol/l) and a significant decease in expression of UT-A1 (28 ± 8% of sham levels), UT-A3 (45 ± 11%), and UT-B (70 ± 8%). Immunocytochemistry confirmed downregulation of UT-A1 and UT-A3 in IM collecting duct and UT-B in the descending vasa recta. Three days after release of BUO, UT-A1, UT-A3, and UT-B remained significantly downregulated (UT-A1: 37 ± 6%; UT-A3: 25 ± 6%; and UT-B: 10 ± 5% of sham levels; P < 0.05) concurrent with a persistent polyuria and a marked reduction in solute-free water reabsorption (115 ± 11 vs. 196 ± 8 µl·min–1·kg–1, P < 0.05). Moreover, 14 days after release of BUO, total UT-A1, UT-A3, and UT-B remained significantly decreased compared with sham-operated controls and urine urea remained reduced (588 ± 43 vs. 1,150 ± 94 mmol/l). Consistent with increased levels of plasma urea 24 h after onset of UUO (7.4 ± 0.3 vs. 4.8 ± 0.3 mmol/l), the protein abundance of UT-A1, UT-A3, and UT-B in IM was markedly reduced in the obstructed kidney, which was confirmed by immunocytochemistry. In the nonobstructed kidney, the expression of urea transporters did not change. In conclusion, reduced expression of UT-A1, UT-A3, and UT-B levels in both BUO and UUO rats suggests that urea transporters play important roles in the impaired urinary concentrating capacity in response to urinary tract obstruction.

collecting duct; descending vasa recta; obstructive nephropathy; urine concentrating mechanism


UREA PLAYS AN important role in the urinary concentrating mechanism (2, 27). Although urea can diffuse slowly through biological membranes, the high urea permeability across red blood cells and kidney terminal inner medullary collecting duct (IMCD) occurs through facilitated transporters (27). Carrier-mediated urea transport in the loops of Henle and collecting ducts contributes to the generation of high interstitial osmolality, which is critically important for the urinary concentrating process and the regulation of renal water excretion (29, 31). Two groups of urea transporters have been identified, namely, the renal urea transporter UT-A and the erythrocyte urea transporter UT-B. Four isoforms of the UT-A transporter are expressed in the renal medulla: UT-A1 (30), UT-A2 (39), UT-A3, and UT-A4 (9). Only a single UT-B isoform has been described (27).

UT-A1 is expressed in the IMCD and is stimulated by cAMP when expressed in Xenopus laevis oocytes (3, 5, 21, 23, 30). Immunocytochemistry demonstrates localization of UT-A1 protein in the apical region of IMCD cells, and immunoelectron microscopy confirmed that the protein is present in the apical plasma membrane as well as in cytoplasmic vesicles (21). Immunoblotting studies demonstrate that UT-A1 antibodies labeled an abundant membrane protein in rat renal inner medulla (IM) with an apparent molecular mass of 97 kDa as well as a less abundant protein with an apparent molecular mass of 117 kDa (32). UT-A2 is expressed in the thin descending limb of Henle's loop (21). UT-A2 is not stimulated by cAMP when expressed in X. laevis oocytes (5). UT-A3 is most abundant in the inner third of the IM and is present in membrane stimulated by cAMP when expressed in HEK-293 cells (9). Both UT-A1 and UT-A3 are expressed in the IMCD (33). The renal tubular localization and functional roles of UT-A4 remain undefined. A renal form of the erythrocyte urea transporter UT-B has been identified, which is expressed in the descending vasa recta of the renal medulla (29, 37).

Vasopressin administration acutely increases phosphorylation of both the 117- and 97-kDa UT-A1 proteins in rat IMCD suspensions (40). The effect of administering exogenous vasopressin varies with time: 5 days of vasopressin decreases UT-A1 protein abundance in rat IM (32) and basal urea permeability in the perfused terminal IMCD (10). However, 12 days of vasopressin administration increases UT-A1 protein abundance (12). Volume-expanded rats showed a marked decrease in the abundance of the collecting duct urea transporters UT-A1 and UT-A3 (36).

Urinary tract obstruction is associated with long-term impairment in the ability to concentrate urine. The pathophysiology behind the loss of urinary concentrating ability is complex and involves all nephron segments including the juxtamedullary, proximal, and distal tubules, the loops of Henle, and the collecting ducts. Recent studies demonstrated that aquaporins 1–3 (AQP1–3), the water channels located at the proximal tubule, descending thin limb, collecting duct, and major renal sodium transporters located along all renal nephron segments, were severely reduced during bilateral or unilateral ureteral obstruction (BUO or UUO, respectively) and release of obstruction (6, 7, 13, 1619). These findings suggest that the reduction in the renal aquaporins and major renal sodium transporters contributes to the impairment in urinary concentrating capacity and salt wasting in response to urinary tract obstruction. Because urea is also critically important for maintaining intact urinary concentrating and diluting ability in the kidney, it is hypothesized that dysregulation of urea transporters might participate in the urinary concentrating defect in response to ureteral obstruction.

To further increase the understanding of mechanisms involved in the impairment of urinary concentrating capacity during urinary tract obstruction at the molecular level, we examined 1) whether ureteral obstruction is associated with changes in the expression of the inner medullary urea transporters (UT-A1, UT-A3, and UT-B) and 2) whether changes in the expression of these urea transporters are associated with alterations in urinary concentrating capacity.


    METHODS
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 METHODS
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Experimental animals. Studies were performed in male Münich- Wistar rats initially weighing 250 g (Møllegard Breeding Centre, Eiby, Denmark). The rats were maintained on a standard rodent diet (Altromin, Lage, Germany) with free access to water. During the entire experiment, rats were kept in individual metabolic cages with a 12:12-h artificial light-dark cycle, a temperature of 21 ± 2°C, and a humidity of 55 ± 2%. Rats were allowed to acclimatize to the cages for 3 days before surgery.

Before surgery, the rats were anesthetized with halothane (Halocarbon Laboratories), and during surgery the rats were placed on a heated table to maintain rectal temperature at 37–38°C. BUO and UUO were established as previously described (17, 19). In brief, BUO was established through a midline abdominal incision, where both ureters were exposed and a 5-mm-long piece of bisected polyethylene tubing (PE-50) was placed around the midportion of each ureter. The ureter was then occluded by tightening the tubing with a 5–0 silk suture. Twenty-four hours later, the rats were killed or the obstructed ureters were decompressed by removal of the ligature and the PE tubing. With the use of this technique, the ureters could be completely occluded for 24 h without evidence of subsequent functional impairment of ureteral function. UUO was established by tightening a 5–0 silk ligature around the midportion of the left ureter. Twenty-four hours later, the rats were killed. The experiments and experimental procedures were all approved by the board of The Institute of Experimental Clinical Research.

Rats were allocated to the protocols indicated below. Age- and time-matched sham-operated controls were prepared and observed in parallel with each BUO group and UUO group in the following protocols (Fig. 1).



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Fig. 1. Diagram of the study design. BUO, bilateral ureteral obstruction; UUO, unilateral ureteral obstruction; SHAM, sham-operated controls. n = No. of rats.

 
Protocol 1. 1) BUO for 24 h (n = 10). The two kidneys were removed and separately prepared for semiquantitative immunoblotting (n = 6) or for immunocytochemistry (n = 4). 2) Sham-operated rats (n = 9). The two kidneys were removed and separately prepared for semiquantitative immunoblotting (n = 6) or for immunocytochemistry (n = 3).

Protocol 2. 1) BUO for 24 h, followed by release and observation during the next 3 days (n = 12). The two kidneys were removed and separately prepared for semiquantitative immunoblotting. 2) Sham-operated rats (n = 13).

Protocol 3. 1) BUO for 24 h, followed by release and observation during the next 14 days (n = 8). The two kidneys were removed and separately prepared for semiquantitative immunoblotting. 2) Sham-operated rats (n = 8).

Protocol 4. 1) UUO for 24 h (n = 11). The two kidneys were removed and separately prepared for semiquantitative immunoblotting (n = 7) or for immunocytochemistry (n = 4). 2) Sham-operated rats (n = 10). The two kidneys were removed and separately prepared for semiquantitative immunoblotting (n = 7) or for immunocytochemistry (n = 3).

Clearance studies. Urine was collected and clearance studies were performed after release of BUO or during 24-h periods throughout the study of UUO. At the end of each protocol, 4 ml of blood were collected into a heparinized tube for the determination of plasma electrolytes and osmolality before the rat was killed. The plasma concentrations of sodium, potassium, creatinine, and urea and urinary concentrations of creatinine and urea were determined (Vitros 950, Johnson & Johnson). The concentrations of urinary sodium and potassium were determined by standard flame photometry (Eppendorf FCM6341). The osmolality of urine and plasma was determined by freezing-point depression (The Advanced Osmometer, model 3900, Advanced Instruments, Norwood, MA and Osmomat 030-D, Gonotec, Berlin, Germany).

Membrane fractionation for immunoblotting. For removal of kidneys, rats were anesthetized with halothane. In rats with BUO, one total kidney (TK) was kept and another kidney was split into cortex plus outer medulla (OM + C) and IM. In rats with UUO, both obstructed and nonobstructed kidneys were split into cortex plus outer medulla and IM. All of them were frozen in liquid nitrogen. Tissue (TK or IM) was minced finely and homogenized in 9 ml (TK) or 1 ml (IM) of dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, and containing the following protease inhibitors: 8.5 µM leupeptin, 1 mM phenylmethyl sulfonyl fluoride), with five strokes of a motor-driven Potter-Elvehjem homogenizer, at 1,250 rpm. This homogenate was centrifuged in a Beckman L8M centrifuge at 4,000 g for 15 min at 4°C. The supernatants were assayed for protein concentration using the method of Lowry. Gel samples (in Laemmli sample buffer containing 2% SDS) were made from this pellet.

Electrophoresis and immunoblotting. Samples of membrane fractions from TK and IM were run on 12% polyacrylamide minigels (Bio-Rad Mini Protean II). For each gel, an identical gel was run in parallel and subjected to Coomassie staining. The Coomassie-stained gel was used to verify identical loading or to allow for correction for minor differences in loading after scanning and densitometry of major bands. The other gel was subjected to Western blotting analysis. After transfer by electroelution to nitrocellulose membranes, blots were blocked with 5% milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated with primary antibodies overnight at 4°C. After being washed with PBS-T, the blots were incubated with horseradish peroxidase-conjugated secondary antibody (P448, Dako, Glostrup, Denmark, diluted 1:3,000). After a final washing as above, antibody binding was visualized using the ECL (enhanced chemiluminescence) system (Amersham International).

Primary antibodies. For semiquantitative immunoblotting and immunocytochemistry, we used previously characterized affinity-purified polyclonal antibodies to 1) UT-A1 (20, 21), 2) UT-A3 (33), and 3) UT-B (34).

Immunocytochemistry. The kidneys from BUO, UUO, and sham-operated rats were fixed by retrograde perfusion via the abdominal aorta with 3% paraformaldehyde, in 0.1 M cacodylate buffer (pH 7.4). For immunoperoxidase microscopy, kidney blocks containing all kidney zones were dehydrated and embedded in paraffin. The paraffin-embedded tissues were cut at 2 µm on a rotary microtome (Leica). The sections were deparaffinated and rehydrated. For immunoperoxidase labeling, endogenous peroxidase was blocked by 0.5% H2O2 in absolute methanol for 10 min at room temperature. To reveal antigens, sections were put in 1 mmol/l TRIS solution (pH 9.0) supplemented with 0.5 mM EGTA and heated using a microwave oven for 10 min. Nonspecific binding of immunoglobulin was prevented by incubating the sections in 50 mM NH4Cl in 30 min followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C with primary antibodies diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. After being rinsed with PBS supplemented with 0.1% BSA, 0.05% saponin, and 0.2% gelatin for 3 x 10 min, the sections were washed and then incubated with horseradish peroxidase-conjugated immunoglobulin (P448, Dako, 1:200) diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. The sections were washed for 3 x 10 min and followed by incubation with diaminobenzidine for 10 min. The microscopy was carried out using a Leica DMRE light microscope (Leica, Heidelberg, Germany).

Statistics. For densitometry of immunoblots, samples from kidneys were run on each gel with corresponding sham kidneys. Renal urea transporter labeling in the samples from the experimental animals was calculated as a fraction of the mean sham control value for that gel. Parallel Coomassie-stained gels were used for correction of minor differences in loading. Values are presented as means ± SE. Comparisons between groups were made by unpaired t-test. P values <0.05 were considered significant.


    RESULTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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BUO is associated with impaired urinary concentration and urea reabsorption. Urine and plasma osmolality were examined in rats up to 14 days after release of a 24-h period of BUO and during a period of UUO for 24 h. Release of BUO was associated with the onset of polyuria, and urine production remained significantly elevated compared with sham-operated rats until 14 days after release of BUO (47 ± 3 vs. 25 ± 3 µl·min–1·kg–1; Table 1). Consistent with this, urine osmolality was reduced (1,374 ± 89 vs. 2,227 ± 188 mosmol/kgH2O at day 14 after release of BUO). At day 3 after release of BUO, there was a decrease in solute-free water reabsorption (115 ± 11 vs. 196 ± 8 µl·min–1·kg–1) and creatinine clearance (4.5 ± 0.4 vs. 6.9 ± 0.2 ml·min–1·kg–1, P < 0.05). This indicated impaired glomerular filtration rate (GFR) and urinary concentrating capacity after release of BUO. Solute-free water reabsorption and GFR recovered to sham levels 14 days after release of obstruction (Table 1).


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Table 1. Changes in renal function in rats subjected to 24-h BUO, 24-h BUO followed by release for 3 and 14 days, and 24-h UUO

 
Plasma urea increased eightfold during the complete obstruction and recovered to sham levels after release of BUO for 3 and 14 days. Urine urea decreased at day 3 (455 ± 49 vs. 900 ± 51 mmol/l) and day 14 (588 ± 43 vs. 1,150 ± 94 mmol/l) after release of obstruction compared with sham-operated controls. Similar to the trend of creatinine clearance, urea clearance was markedly reduced (3.7 ± 0.4 vs. 5.6 ± 0.3 ml·min–1·kg–1) in rats after release of BUO for 3 days and returned to sham levels at day 14 after release of BUO (Table 1). Food intake in rats with BUO decreased significantly during the first 3 days after release of BUO and then increased markedly from day 6 to day 11 after release of BUO and recovered to sham levels at 12 days after release of BUO.

BUO and release of BUO are associated with long-term downregulation of UT-A1. UT-A1 has two glycosylated forms: 97 and 117 kDa (4) in the IM. The two bands were analyzed together. In rats with BUO for 24 h, the abundance of UT-A1 proteins was significantly decreased (28 ± 8 vs. 100 ± 4% of sham levels, P < 0.05). The abundance of UT-A1 proteins remained reduced at 3 days after release of BUO (37 ± 6% of sham levels) and at 14 days after release of BUO (49 ± 8% of sham levels; Fig. 2 and Table 2). Immunohistochemistry confirmed downregulation of UT-A1 in rats with BUO for 24 h (Fig. 3). In the IMCD of BUO rats (Fig. 3B), the abundance of UT-A1 labeling was much weaker at the apical plasma membrane domains and intracellular cytoplasmic vesicles compared with that seen in the kidney from sham-operated rats (Fig. 3A).



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Fig. 2. Semiquantitative immunoblotting of membrane fractions of inner medulla (IM) from BUO, release of BUO, and sham-operated rats. AC: immunoblots were reacted with affinity-purified anti-UT-A1 antibody and revealed ~97- and 117-kDa bands. Densitometric analysis of all samples from rats with BUO and release of BUO matched with sham-operated controls revealed a persistent decrease in total UT-A1 levels from 100 ± 4% in sham-operated controls to 28 ± 8% in BUO and in rats with 24-h BUO followed by release for 3 days (BUO-3daysR) to 37 ± 6% of sham levels (100 ± 11%); the downregulation was maintained in rats with release of BUO for 14 days (BUO-14daysR) to 49 ± 8% of sham levels, *P < 0.05.

 

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Table 2. Expression of urea transporters in the inner medulla in response to 24-h BUO after release of BUO, and 24-h UUO

 


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Fig. 3. Immunocytochemical analysis of UT-A1 in the IM from sham-operated controls and rats with BUO for 24 h. A: in sham-operated rats, UT-A1 labeling was seen at the apical plasma membrane domains and intracellular cytoplasmic vesicles of inner medullary collecting duct (IMCD). B: in the IMCD of BUO rats, UT-A1 labeling (arrows) was also seen at the apical plasma membrane domains and intracellular cytoplasmic vesicles of IMCD, but it was much weaker compared with that seen in kidneys from sham-operated rats. Magnification: x650.

 
BUO and release of BUO are associated with long-term downregulation of UT-A3. UT-A3 has two glycosylated forms: 67 and 44 kDa (33). The two bands were analyzed together. In rats with 24 h of BUO, UT-A3 expression was significantly reduced (45 ± 11 vs. 100 ± 7%, P < 0.05). UT-A3 abundance remained reduced at days 3 and 14 after release of BUO (Fig. 4 and Table 2). Immunohistochemistry confirmed the reduced abundance of UT-A3 labeling in rats with BUO for 24 h. In sham-operated rats, the abundance of UT-A3 labeling is seen at the apical plasma membrane domains and intracellular cytoplasmic vesicles of the middle and tip of IMCD (Fig. 5A). The abundance of UT-A3 labeling in the IMCD of BUO rats was much weaker compared with that seen in kidneys from sham-operated rats (Fig. 5D).



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Fig. 4. Semiquantitative immunoblotting of membrane fractions of IM from BUO, release of BUO, and sham-operated rats. AC: immunoblots were reacted with affinity-purified anti-UT-A3 antibody and revealed ~67- and 44-kDa bands. Densitometric analysis of all samples from kidney in rats with 24-h BUO and release of BUO and sham-operated controls revealed a persistent decrease in the expression of UT-A3 (24-h BUO: 45 ± 11 vs. 100 ± 7%; BUO-3daysR: 25 ± 6 vs. 100 ± 4%; BUO-14daysR: 51 ± 10 vs. 100 ± 10%, *P < 0.05).

 


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Fig. 5. Immunocytochemical analysis of UT-A3 in the IM from sham-operated controls, 24-h BUO, and 24-h UUO rats. A: in sham-operated rats, UT-A3 labeling was seen at the apical plasma membrane domains and intracellular cytoplasmic vesicles of the IMCD. In the IMCD of BUO kidneys (D) and in the IMCD of UUO obstructed (OBS) kidneys (C), the abundance of UT-A3 labeling (arrows) was much weaker compared with that seen in kidneys from sham-operated rats. The abundance of UT-A3 labeling in the nonobstructed (non-OBS) kidneys of UUO rats (B) was not weak compared with sham-operated controls. Magnification: x650.

 
BUO and release of BUO are associated with long-term downregulation of UT-B. UT-B is a glycosylated protein that runs as a smear between 40 and 50 kDa (34). In rats with BUO for 24 h, the abundance of UT-B protein was significantly reduced to 70 ± 8% of sham levels. The decreased expression of UT-B persisted to 3 days after release of BUO (10 ± 5% of sham levels) and 14 days after release of BUO (51 ± 5% of sham levels, P < 0.05; Fig. 6 and Table 2). Immunohistochemistry confirmed the downregulation of UT-B abundance at the vasa recta of the IM in rats with BUO (Fig. 7D) for 24 h compared with that seen in the sham-operated controls (Fig. 7A).



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Fig. 6. Semiquantitative immunoblotting of membrane fractions of IM from BUO, release of BUO, and sham-operated rats. AC: immunoblots were reacted with affinity-purified anti-UT-B antibody and revealed 45- to 60-kDa smear bands. Densitometric analysis of all samples from kidney in rats with 24-h BUO and release of BUO and sham-operated controls revealed a persistent decrease in the expression of UT-B (24-h BUO: 70 ± 8 vs. 100 ± 13%; BUO-3daysR: 10 ± 5 vs. 100 ± 8%; BUO-14daysR: 51 ± 5 vs. 100 ± 17%, *P < 0.05).

 


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Fig. 7. Immunocytochemical analysis of UT-B protein in the IM of the kidneys from sham-operated rats, 24-h BUO, and obstructed kidney (OBS) and nonobstructed kidneys (non-OBS) from 24-h UUO rats. A: in sham-operated rats, UT-B labeling was seen at the vasa recta of renal IM with the anti-UT-B antibody. The staining of UT-B in the vasa recta of renal IM from nonobstructed kidneys of UUO rats (B) was not weak compared with controls. The staining of UT-B (arrows) in the vasa recta of the renal IM from BUO rats (D) and obstructed kidney of UUO rats (C) was much weaker compared with that seen in kidneys from sham-operated rats. Magnification: x650.

 
UUO for 24 h is associated with reduced abundance of UT-A1, UT-A3, and UT-B. Plasma osmolality increased significantly, consistent with an increase in plasma urea (7.4 ± 0.3 vs. 4.8 ± 0.3 mmol/l; Table 1) and plasma creatinine (not shown), whereas urine volume, urine osmolality, urine urea, and solute-free water reabsorption were unchanged compared with sham-operated controls. Furthermore, clearance of creatinine and urea decreased markedly in rats with UUO compared with sham-operated controls (Table 1).

To test whether changes in the expression of urea transporters play an important role in renal urea handling during UUO, immunoblots were performed to examine the expression of UT-A1, UT-A3, and UT-B. As seen in Fig. 8, the levels of UT-A1 protein expression in the IM of the obstructed kidney decreased to 65 ± 13%, UT-A3 to 43 ± 11%, and UT-B to 55 ± 7% of sham levels. In the nonobstructed kidneys, the abundance of UT-A1, UT-A3, and UT-B was unchanged compared with sham-operated controls (not shown). Immunocytochemistry confirmed downregulation of UT-A1, UT-A3, and UT-B in the obstructed kidney in UUO rats (Figs. 9C, 5C, and 7C), whereas the abundance of these three urea transporters did not change in the nonobstructed kidney compared with sham-operated controls (Figs. 9, A and B, 5, A and B, and 7, A and B).



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Fig. 8. Immunoblot of membrane fractions of IM or whole kidney (WK) from obstructed kidneys of UUO rats and sham-operated rats. Immunoblots were reacted with affinity-purified anti-UT-A1 (A), anti-UT-A3 (B), and anti-UT-B (C) antibodies. Densitometric analysis of all samples revealed that in the obstructed kidney the expression of UT-A1 decreased significantly to 65 ± 13% of sham levels, UT-A3 to 43 ± 11% of sham levels, and UT-B to 55 ± 7% of sham levels, *P < 0.05.

 


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Fig. 9. Immunocytochemical analysis of UT-A1 in the IM from sham-operated controls and 24-h UUO rats. A: in sham-operated rats, UT-A1 labeling was seen at the apical plasma membrane domains and intracellular cytoplasmic vesicles of IMCD. B: abundance of UT-A1 labeling in the nonobstructed kidneys of UUO rats was not weak compared with sham-operated controls. C: in the obstructed kidney of UUO rats, UT-A1 labeling (arrows) was also seen at the apical plasma membrane domains and intracellular cytoplasmic vesicles of IMCD, but it was much weaker compared with that seen in kidneys from sham-operated rats. Magnification: x650.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previously, we demonstrated that BUO, release of BUO, and UUO are associated with a significant reduction in the expression of aquaporins and major renal sodium transporters coinciding with an impaired urinary concentrating capacity. In this study, we examined the abundance of urea transporters in response to urinary tract obstruction. The expression of UT-A1, UT-A3, and UT-B was significantly decreased in the obstructed kidney from rats with BUO and UUO. After release of BUO, the reduction of UT-A1, UT-A3, and UT-B persisted up to 14 days. In the intact nonobstructed kidney of UUO, urea transporters did not change compared with sham-operated controls. This is the first study in which the abundance of urea transporters has been examined in a rat model with ureteral obstruction. The results indicate that the downregulation of urea transporters in the IMCD and descending vasa recta might play a significant role in the impairment of urinary concentrating capacity in response to urinary tract obstruction.

Functional effects of BUO, UUO, and release of BUO on urea balance. During complete BUO, plasma urea increased and recovered to sham levels at days 3 and 14 after release of BUO, whereas in rats with UUO plasma urea increased only moderately. Because plasma urea levels were normalized despite a sustained reduction in the expression of urea transporters, the present data indicate that the renal abundance of urea transporters may not be critical in the normalization of plasma urea. The delayed normalization of plasma urea levels observed in the BUO rats may be related to a delayed recovery from surgery in the BUO rats. Consistent with these findings, urea clearance was reduced at day 3 after release of BUO and during UUO. These results indicate that urinary tract obstruction is associated with an altered urea excretion and reabsorption. Accumulation of urea and/or other natriuretic factors during the interval of complete obstruction may play a dominant role in the primarily osmotic postobstructive diuresis that occurs immediately after release of obstruction, when there is a large increase in urea excretion. Urea retained during the period of obstruction is excreted after release of obstruction and acts as an osmotic agent promoting the excretion of salt and water. However, the osmotic diuresis due to increased urea excretion cannot exclusively account for the natriuresis and diuresis seen after relief of obstruction (24).

Reduced expression of UT-A1 and UT-A3 in the IMCD and UT-B in the descending vasa recta in rats with BUO and release of BUO. In the present study, we demonstrated that release of BUO is associated with a persistent impairment in urinary concentrating capacity lasting up to 14 days after release of obstruction. In parallel the expression of UT-A1 and UT-A3 in the IMCD and UT-B in the descending vasa recta was downregulated in response to 24 h of BUO and persisted at days 3 and 14 after release of BUO. Thus downregulation of the collecting duct UT-A1 and UT-A3 and descending vasa recta UT-B may play an important role in the polyuria and long-term impairment in urinary concentration after release of BUO. However, the significance of downregulation of urea transporters as primary determinants for the impaired urinary concentrating capacity relative to the significance of downregulation of key renal sodium transporters and aquaporins in response to obstruction of the ureter cannot be determined from the present study.

The facilitated urea transporter (UT-A) in the terminal IMCD permits very high rates of transepithelial urea transport and delivers large amounts of urea into the deepest portions of the IM to maintain a high interstitial osmolality to obtain maximum urinary concentration. In the present study, the expression of UT-A1 and UT-A3 remained significantly reduced up to 14 days after release of BUO. This finding is consistent with the results in lithium-fed rats demonstrating a marked reduction in UT-A1 protein abundance in both inner medullary tip and base associated with reduced urinary concentrating capacity (15). Conversely, in multiple other conditions associated with urinary concentrating defects such as water diuresis, furosemide diuresis, hypercalcemia, low-protein diet, and adrenalectomy, UT-A1 protein abundance is increased in the IMCD (1, 8, 11, 20, 28, 32). Recently, we confirmed that administering vasopressin to Brattleboro rats for 5 days reduces UT-A1 protein abundance, but 12 days of vasopressin significantly increased UT-A1 protein (12). Interestingly, glucocorticoid (dexamethasone treatment) decreases urea transporter UT-A1 protein abundance (20) and also inhibits transcription and expression of the UT-A urea transporter gene in the IMCD of adrenalectomized rats (22). Thus these studies demonstrate that renal regulation of UT-A1 is complex and dysregulation of UT-A1 takes place in multiple conditions. The molecular signal for the reduced expression of urea transporters in response to urinary tract obstruction remains unclear, but downregulation of the urea transporters is in line with our previous observations of downregulation of both aquaporins and sodium transporters in response to BUO (17, 18) and UUO (16, 19), suggesting that these conditions are characterized by a temporary increase in parenchymal pressure that may trigger a reduction in the abundance of all major renal proteins.

In the present study, we found a long-term reduction in the expression of UT-B. The facilitated urea transporter (UT-B) in erythrocytes permits these cells to lose urea rapidly as they traverse the ascending vasa recta, thus preventing loss of urea from the medulla and decreasing urinary concentrating ability by decreasing the efficiency of countercurrent exchange (26). Previously, it was demonstrated that UT-B helps to maintain a high medullary urea concentration and a hypertonic medulla that is required for water reabsorption (35). Thus the UT-B-mediated urea transport in vasa recta plays an important role in the urinary concentrating process. Moreover, in UT-B knockout mice, urea permeability was 45-fold lower in erythrocytes compared with erythrocytes from wild-type mice, and the capacity to concentrate urea in the urine was more severely impaired than the capacity to concentrate other solutes (38). Thus these findings support the view that a reduced expression of UT-B as demonstrated in the present study might play an important role in the impaired urinary concentrating ability associated with BUO.

In the present study, we observed a reduced food intake after release of BUO. A reduced food intake may cause a reduction in the minimum amount of urea excreted in the terminal IMCD. This may directly or indirectly modulate the abundance of UT-A1 and UT-A3.

Reduced expression of UT-A, UT-A3, and UT-B in the obstructed kidney of UUO rats. The expression of UT-A1, UT-A3, and UT-B was examined in rats with UUO for 24 h. We found a reduction in UT-A1 and UT-A3 expression in the IMCD and UT-B expression in the descending vasa recta in the obstructed kidneys, consistent with increased plasma urea and decreased clearance of urea in rats with UUO for 24 h, whereas UT-A1, UT-A3, and UT-B in the nonobstructed kidney were unchanged compared with sham-operated controls. Previously, we demonstrated a marked reduction in renal major sodium transporters and aquaporins in the obstructed kidney from the rats with UUO for 24 h. These results suggest that renal urea transporters together with key renal sodium transporters and aquaporins might play important roles in the urinary concentration defect in rats with UUO. However, the mechanisms responsible for the downregulation of urea transporters in rats with UUO remain unclear.

Ureteral occlusion induces a complex series of hormonal changes in the obstructed kidney (14), which may influence the functional changes in the contralateral kidney. During unilateral obstruction of the urinary tract, plasma vasopressin levels are similar to controls (25). Thus vasopressin is unlikely to be involved in the downregulation of urea transporters in the obstructed kidneys. Further studies are needed to address the mechanisms of regulating urea transporters in response to urinary tract obstruction.

Summary. In conclusion, the expression of UT-A1, UT-A3, and UT-B proteins was reduced during BUO and UUO and after release of BUO, suggesting that reduction of urea transporters in the IMCD and descending vasa recta may contribute to the impairment in urinary concentrating capacity associated with obstructive nephropathy. These results may indicate that downregulation of urea transport is part of the molecular mechanism contributing to the urinary concentrating defect in response to urinary tract obstruction. Further studies are needed to examine the possible roles of hormonal mediators and other factors involved in the dysregulation of urea transporters in response to ureteral obstruction.


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 METHODS
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The Water and Salt Research Centre at the University of Aarhus is established and supported by the Danish National Research Foundation (Danmarks Grundforskningsfond). Support for this study was provided by The Karen Elise Jensen Foundation, The Human Frontier Science Program, The Novo Nordisk Foundation, The Commission of the European Union (EU-Aquaplugs and EU Action Programs), The Danish Medical Research Council, The University of Aarhus Research Foundation, The Danish Research Academy, The University of Aarhus, the intramural budget of the National Heart, Lung, and Blood Institute, National Institutes of Health (NIH), and NIH Grants DK-41707 and DK-63657.


    ACKNOWLEDGMENTS
 
The authors thank G. Kall, I. M. Paulsen, D. Wulff, M. Vistisen, H. Høyer, Z. Nikrozi, L. V. Holbech, M. Pedersen, and I. M. Jalk for expert technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. Frøkiær, The Water and Salt Research Center/Institute of Experimental Clinical Research, Aarhus Univ. Hospital-Skejby, DK-8200 Aarhus, Denmark (E-mail: JF{at}IEKF.AU.DK).

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.


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 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ashkar ZM, Martial S, Isozaki T, Price SR, and Sands JM. Urea transport in initial IMCD of rats fed a low-protein diet: functional properties and mRNA abundance. Am J Physiol Renal Fluid Electrolyte Physiol 268: F1218–F1223, 1995.[Abstract/Free Full Text]
  2. Bagnasco SM. How renal cells handle urea. Cell Physiol Biochem 10: 379–384, 2000.[CrossRef][ISI][Medline]
  3. Bagnasco SM, Peng T, Janech MG, Karakashian A, and Sands JM. Cloning and characterization of the human urea transporter UT-A1 and mapping of the human Slc14a2 gene. Am J Physiol Renal Physiol 281: F400–F406, 2001.[Abstract/Free Full Text]
  4. Bradford AD, Terris JM, Ecelbarger CA, Klein JD, Sands JM, Chou CL, and Knepper MA. 97- And 117-kDa forms of collecting duct urea transporter UT-A1 are due to different states of glycosylation. Am J Physiol Renal Physiol 281: F133–F143, 2001.[Abstract/Free Full Text]
  5. Fenton RA, Cottingham CA, Stewart GS, Howorth A, Hewitt JA, and Smith CP. Structure and characterization of the mouse UT-A gene (Slc14a2). Am J Physiol Renal Physiol 282: F630–F638, 2002.[Abstract/Free Full Text]
  6. Frøkiær J, Christensen BM, Marples D, Djurhuus JC, Jensen UB, Knepper MA, and Nielsen S. Downregulation of aquaporin-2 parallels changes in renal water excretion in unilateral ureteral obstruction. Am J Physiol Renal Physiol 273: F213–F223, 1997.[Abstract/Free Full Text]
  7. Frøkiær J, Marples D, Knepper MA, and Nielsen S. Bilateral ureteral obstruction downregulates expression of vasopressin-sensitive AQP-2 water channel in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 270: F657–F668, 1996.[Abstract/Free Full Text]
  8. Isozaki T, Verlander JW, and Sands JM. Low protein diet alters urea transport and cell structure in rat initial inner medullary collecting duct. J Clin Invest 92: 2448–2457, 1993.[ISI][Medline]
  9. 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]
  10. Kato A, Naruse M, Knepper MA, and Sands JM. Long-term regulation of inner medullary collecting duct urea transport in rat. J Am Soc Nephrol 9: 737–745, 1998.[Abstract]
  11. Kato A and Sands JM. Urea transport processes are induced in rat IMCD subsegments when urine concentrating ability is reduced. Am J Physiol Renal Physiol 276: F62–F71, 1999.[Abstract/Free Full Text]
  12. Kim D, Sands JM, and Klein JD. Role of vasopressin in diabetes mellitus-induced changes in medullary transport proteins involved in urine concentration in Brattleboro rats. Am J Physiol Renal Physiol 286: F760–F766, 2004.[Abstract/Free Full Text]
  13. Kim SW, Cho SH, Oh BS, Yeum CH, Choi KC, Ahn KY, and Lee J. Diminished renal expression of aquaporin water channels in rats with experimental bilateral ureteral obstruction. J Am Soc Nephrol 12: 2019–2028, 2001.[Abstract/Free Full Text]
  14. Klahr S. Pathophysiology of obstructive nephropathy. A 1991 update. Semin Nephrol 11: 156–168, 1991.[ISI][Medline]
  15. Klein JD, Gunn RB, Roberts BR, and Sands JM. Downregulation of urea transporters in the renal inner medulla of lithium-fed rats. Kidney Int 61: 995–1002, 2002.[CrossRef][ISI][Medline]
  16. Li C, Wang W, Knepper MA, Nielsen S, and Frøkiær J. Downregulation of renal aquaporins in response to unilateral ureteral obstruction. Am J Physiol Renal Physiol 284: F1066–F1079, 2003.[Abstract/Free Full Text]
  17. Li C, Wang W, Kwon TH, Isikay L, Wen JG, Marples D, Djurhuus JC, Stockwell A, Knepper MA, Nielsen S, and Frøkiær J. Downregulation of AQP1, -2, and -3 after ureteral obstruction is associated with a long-term urine-concentrating defect. Am J Physiol Renal Physiol 281: F163–F171, 2001.[Abstract/Free Full Text]
  18. Li C, Wang W, Kwon TH, Knepper MA, Nielsen S, and Frøkiær J. Altered expression of major renal Na transporters in rats with bilateral ureteral obstruction and release of obstruction. Am J Physiol Renal Physiol 285: F889–F901, 2003.[Abstract/Free Full Text]
  19. Li C, Wang W, Kwon TH, Knepper MA, Nielsen S, and Frøkiær J. Altered expression of major renal Na transporters in rats with unilateral ureteral obstruction. Am J Physiol Renal Physiol 284: F155–F166, 2003.[Abstract/Free Full Text]
  20. 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]
  21. 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]
  22. Peng T, Sands JM, and Bagnasco SM. Glucocorticoids inhibit transcription and expression of the UT-A urea transporter gene. Am J Physiol Renal Physiol 282: F853–F858, 2002.[Abstract/Free Full Text]
  23. Promeneur D, Rousselet G, Bankir L, Bailly P, Cartron JP, Ripoche P, and Trinh-Trang TM. Evidence for distinct vascular and tubular urea transporters in the rat kidney. J Am Soc Nephrol 7: 852–860, 1996.[Abstract]
  24. Purkerson ML and Klahr S. Protein intake conditions the diuresis seen after relief of bilateral ureteral obstruction in the rat. Proc Soc Exp Biol Med 177: 62–68, 1984.[Abstract]
  25. Reyes AA, Robertson G, and Klahr S. Role of vasopressin in rats with bilateral ureteral obstruction. Proc Soc Exp Biol Med 197: 49–55, 1991.[Abstract]
  26. Sands JM. Regulation of renal urea transporters. J Am Soc Nephrol 10: 635–646, 1999.[Abstract/Free Full Text]
  27. Sands JM. Molecular approaches to urea transporters. J Am Soc Nephrol 13: 2795–2806, 2002.[Abstract/Free Full Text]
  28. Sands JM, Flores FX, Kato A, Baum MA, Brown EM, Ward DT, Hebert SC, and Harris HW. Vasopressin-elicited water and urea permeabilities are altered in IMCD in hypercalcemic rats. Am J Physiol Renal Physiol 274: F978–F985, 1998.[Abstract/Free Full Text]
  29. Sands JM, Timmer RT, and Gunn RB. Urea transporters in kidney and erythrocytes. Am J Physiol Renal Physiol 273: F321–F339, 1997.[Abstract/Free Full Text]
  30. 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]
  31. Star RA. Apical membrane limits urea permeation across the rat inner medullary collecting duct. J Clin Invest 86: 1172–1178, 1990.[ISI][Medline]
  32. 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]
  33. 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]
  34. Timmer RT, Klein JD, Bagnasco SM, Doran JJ, Verlander JW, Gunn RB, and Sands JM. Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues. Am J Physiol Cell Physiol 281: C1318–C1325, 2001.[Abstract/Free Full Text]
  35. Trinh-Trang-Tan MM, Lasbennes F, Gane P, Roudier N, Ripoche P, Cartron JP, and Bailly P. UT-B1 proteins in rat: tissue distribution and regulation by antidiuretic hormone in kidney. Am J Physiol Renal Physiol 283: F912–F922, 2002.[Abstract/Free Full Text]
  36. Wang XY, Beutler K, Nielsen J, Nielsen S, Knepper MA, and Masilamani S. Decreased abundance of collecting duct urea transporters UT-A1 and UT-A3 with ECF volume expansion. Am J Physiol Renal Physiol 282: F577–F584, 2002.[Abstract/Free Full Text]
  37. Xu Y, Olives B, Bailly P, Fischer E, Ripoche P, Ronco P, Cartron JP, and Rondeau E. Endothelial cells of the kidney vasa recta express the urea transporter HUT11. Kidney Int 51: 138–146, 1997.[ISI][Medline]
  38. Yang B, Bankir L, Gillespie A, Epstein CJ, and Verkman AS. Urea-selective concentrating defect in transgenic mice lacking urea transporter UT-B. J Biol Chem 277: 10633–10637, 2002.[Abstract/Free Full Text]
  39. 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.[CrossRef][ISI][Medline]
  40. Zhang C, Sands JM, and Klein JD. Vasopressin rapidly increases phosphorylation of UT-A1 urea transporter in rat IMCDs through PKA. Am J Physiol Renal Physiol 282: F85–F90, 2002.[Abstract/Free Full Text]