Downregulation of renal aquaporins in response to unilateral ureteral obstruction

Chunling Li1,2, Weidong Wang1,3, Mark A. Knepper4, Søren Nielsen1,3, and Jørgen Frøkiær1,2,5

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The expression of aquaporin-2 (AQP2) is decreased in rats with bilateral ureteral obstruction (BUO) and unilateral ureteral obstruction (UUO). Therefore, the expression of additional renal aquaporins (AQP1-4) and phosphorylated AQP2 (p-AQP2), known to play a role in urinary concentration, was examined in a Wistar rat model with 24 h of UUO. In obstructed kidneys, immunoblotting revealed a significant decrease in the expression of inner medullary AQP2 to 42 ± 4, p-AQP2 to 23 ± 5, AQP3 to 19 ± 6, AQP4 to 11 ± 5, and AQP1 to 64 ± 8% of sham levels. AQP1 expression located in the proximal tubule decreased to 74 ± 4% of sham levels (P < 0.05). Immunocytochemistry confirmed the downregulation of AQP3, AQP4, and p-AQP2. In contralateral nonobstructed kidneys, immunoblotting also revealed significant reductions of AQP1 in the inner medulla, outer medulla, and cortex, whereas expression of AQP2, AQP3, AQP4, and p-AQP2 was unchanged. Furthermore, we collected the urine from both obstructed and nonobstructed kidneys for 2 h, respectively, after 24 h of UUO. Urine collection from obstructed kidneys during 2 h after release of UUO revealed a significant reduction in urine osmolality and solute-free water reabsorption (TcH2O). Moreover, an increase in urine production and TcH2O was observed in contralateral kidneys. To examine whether vasopressin-independent mechanisms are involved in AQP2 regulation, vasopressin-deficient Brattleboro (BB) rats with 24 h of UUO were examined. Immunoblotting revealed downregulation of AQP2, p-AQP2, AQP3, and AQP1 in obstructed kidneys and downregulation of p-AQP2 and AQP1 in nonobstructed kidneys. In conclusion, 1) UUO is associated with severe downregulation of AQP2, AQP3, AQP4, and AQP1; thus all of these AQPs may play important roles in the impaired urinary concentrating capacity in the obstructed kidney; 2) the reduced levels of AQP1 in the nonobstructed kidney may contribute to the compensatory increase in urine production; and 3) downregulation of AQPs in BB rats supports the view that vasopressin-independent pathways may be involved in AQP2 and AQP3 regulation in the obstructed kidney.

collecting duct; proximal tubule; water channel; obstructive nephropathy; vasopressin; Brattleboro rats


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

URINARY TRACT OBSTRUCTION is a serious and common clinical condition, which is associated with increased intraluminal pressure in the ureter and renal tubules that may cause renal parenchymal damage through a series of direct and indirect effects (19). Obstruction of the urinary tract has marked effects on renal blood flow, glomerular filtration rate (GFR), tubular function, and parenchymal structure (4, 28, 35). Thus abnormalities in tubular function are common in obstructive nephropathy, including a reduction in urinary concentrating capacity, altered reabsorption of solutes and water, and impaired excretion of hydrogen and potassium. The major sites of abnormal function are located to the distal segments of the nephron (18).

Production of concentrated urine depends on active solute reabsorption in the thick ascending limb of Henle's loop to generate high osmolality in the medullary interstitium. The production of concentrated urine also depends on high water permeability of the collecting duct in response to antidiuretic hormone, primarily through increased expression of aquaporin-2 (AQP2) and -3 (AQP3) located in the collecting duct principal cells. Recently, we reported that rats with unilateral ureteral obstruction (UUO) had reduced levels of AQP2 and a parallel impairment of solute-free water reabsorption, demonstrating a functional association between decreased levels of AQP2 and reduced water reabsorption in the collecting duct (11).

The AQPs are a family of membrane proteins that function as water channels that play key roles in the reabsorption of water in the kidney. AQP1 is located in the proximal tubule and thin descending limb. AQP1 knockout studies have emphasized the importance of AQP1 in fluid reabsorption in both the proximal tubule and the descending thin limb of Henle (1, 34). Water transport across the apical membrane in the collecting duct principal cells is mediated by AQP2 (30). AQP2 is the chief target for vasopressin-mediated regulation of collecting duct water permeability. Recent studies have elucidated important roles of AQP2 in multiple water balance disorders. A number of conditions with acquired nephrogenic diabetes insipidus are associated with marked downregulation of AQP2 protein, such as those induced by chronic lithium treatment (25), hypokalemia (26), hypercalcemia (7), and ischemic acute renal failure (ARF) (14, 16, 20). Also, AQP2 levels and urinary concentrating capacity were markedly reduced in response to bilateral ureteral obstruction (BUO) (12, 15, 21).

There is compelling evidence that AQP2 is regulated long term by vasopressin (9, 13, 24) but that vasopressin-independent pathways also play an important role in AQP2 regulation. During vasopressin escape, rats start to excrete water and reduce their AQP2 levels despite high circulating levels of vasopressin, indicating vasopressin-independent regulation of AQP2 (8). A non-vasopressin-mediated effect on AQP2 expression has also been demonstrated in rats treated with lithium, whereby dehydration caused a much higher increase in AQP2 levels than vasopressin treatment (25). Similarly, vasopressin-independent regulation was demonstrated using vasopressin-deficient Brattleboro (BB) rats, which have much lower levels of AQP2 (~50%) than rats with normal vasopressin levels (6, 17, 31). During BUO, vasopressin levels are elevated, whereas during unilateral obstruction of the urinary tract plasma vasopressin levels are similar to controls (32). In both conditions, reduced AQP2 levels have been demonstrated, indicating vasopressin-independent regulation of AQP2 expression during urinary tract obstruction.

Water transport across the basolateral membrane of the collecting duct principal cells is mediated by AQP3 and AQP4. Reduced levels of AQP3 expression were demonstrated in rats with BUO up to 14 days after release of BUO (21). Consistent with this, it is well known that AQP3 knockout mice are remarkably polyuric but are able to generate a partly concentrated urine after water deprivation (22). AQP4 is the most abundant basolateral water channel in the inner medullary collecting duct (IMCD). However, AQP4 knockout mice manifest only a mild defect in maximum urinary concentrating ability (2). AQP3 appears to be regulated by vasopressin, whereas there is no evidence for long-term regulation of AQP4 expression in the kidney (36). Therefore, it could be speculated that the impairment in urinary concentrating capacity after UUO may be associated with significant changes in the expression level of basolateral collecting duct AQP3 and AQP4.

To further increase the understanding of the molecular mechanism involved in the impairment of renal water handling during urinary tract obstruction, we therefore examined 1) whether UUO is associated with changes in the expression of AQPs [AQP1-4 and phosphorylated (p)-AQP2] and 2) whether changes in the expression of AQPs are associated with alterations in urinary concentrating capacity. In addition, to examine the importance of non-vasopressin-mediated regulation of AQPs during UUO, the expression levels of AQPs were analyzed in kidneys from BB rats with UUO.


    METHODS
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INTRODUCTION
METHODS
RESULTS
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Experimental Animals

Studies were performed in male Münich-Wistar rats, initially weighing 250 g (Møllegard Breeding Centre, Eiby, Denmark), and female BB rats, initially weighing 225 g (Harlan Sprague Dawley, Indianapolis, IN). 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 humidity of 55 ± 2%. Rats were allowed to acclimatize to the cages for 5-7 days before surgery. Water intake, urine output, and body weight of the rats were monitored every 24 h during the study.

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. Through a midline abdominal incision, the left ureter was exposed and a 5-0 silk ligature occluded the midportion of the ureter. After surgery, the rats regained consciousness and were placed in metabolic cages. Twenty-four hours after occlusion of the left ureter, the rats were anesthetized and both kidneys were removed, after which the animals were killed. In a subset of animals (protocol 3), the obstruction was released after 24 h by inserting a polyethylene tube (PE-35) into the proximal left ureter. A similar catheter was inserted into the proximal right ureter to allow separate collection of urine from the left and the right kidney. After urine was collected for at least 2 h, the rats were killed. The animals were allocated to the protocols indicated below. Age- and time-matched sham-operated controls were prepared and were observed in parallel with each UUO group (Fig. 1).


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Fig. 1.   Diagram of the study design. Protocol 1: the left ureter was occluded for 24 h in Wistar rats with unilateral ureteral obstruction (UUO), and matched sham-operated control Wistar rats were prepared in parallel. Protocol 2: rats were prepared similar to in protocol 1 (UUO and sham-operated controls). The weight of the kidneys and total protein content in kidneys were measured. Protocol 3: rats were prepared similar to in protocol 1 (UUO and sham-operated controls). After 24 h, the obstruction was released and urine was collected from each ureter for 2 h. Protocol 4: the left ureter was occluded for 24 h in Brattleboro (BB) rats with UUO, and matched sham-operated control BB rats were prepared in parallel. n, No. of rats.

Protocol 1. Münich-Wistar rats underwent UUO for 24 h (n = 11). The kidneys were removed and prepared separately for semiquantitative immunoblotting (n = 7) or for immunocytochemistry (n = 4). For matched sham-operated control rats, n = 10.

Protocol 2. Münich-Wistar rats underwent UUO for 24 h (n = 6). The kidneys were removed and prepared separately for measuring kidney weight and total amount of protein levels. For matched sham-operated control rats, n = 6.

Protocol 3. Münich-Wistar rats underwent UUO for 24 h followed by release, and the animals were observed during the next 2 h (n = 8). Urine was collected for 2 h after release of UUO. For matched sham-operated control rats, n = 8.

Protocol 4. BB rats underwent UUO for 24 h (n = 6). The kidneys were removed and prepared separately for semiquantitative immunoblotting. For matched sham-operated control rats, n = 6.

Clearance Studies

Urine was collected during 24-h periods throughout the study or for 2 h after release of ligation (protocol 3). Clearance studies were performed during the last 24 h in protocols 1 and 4 and for 2 h after release in protocol 3. During anesthesia and before removal of the kidneys, 2-3 ml of blood were collected into a heparinized tube for the determination of plasma electrolytes and osmolality. The plasma and urinary concentrations of creatinine and urea, and the plasma concentrations of sodium and potassium, were determined (Kodak Ektachem 700XRC). The concentrations of urinary sodium and potassium were determined by standard flame photometry (Eppendorf FCM6341). The osmolality of urine and plasma was measured with a vapor pressure osmometer (Osmomat 030, Gonotec). Solute-free water reabsorption (TcH2O) was calculated by the following formula: TcH2O = [(urine osmolality)/(plasma osmolality) -1] × (urine volume).

Membrane Fractionation for Immunoblotting

For removal of kidneys, rats were anesthetized with halothane. The kidney was split into cortex and outer medulla and inner medulla and frozen in liquid nitrogen. Tissue (inner medulla or cortex+outer medulla) was minced finely and homogenized in 1 (inner medulla) or 8 ml (cortex+outer medulla) of dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, and the following protease inhibitors: 8.5 µM leupeptin and 1 mM phenylmethylsulfonyl fluoride) with five strokes of a motor-driven IKA homogenizer at 1,250 rpm. This homogenate was centrifuged in a Beckman L8M centrifuge at 4,000 g for 15 min at 4°C to remove whole cells, nuclei, and mitochondria. Gel samples (Laemmli sample buffer containing 2% SDS) were made from this membrane preparation.

Total Protein Concentration

After removal of kidneys, they were weighed, minced finely, and homogenized in 9 ml of dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, and the following protease inhibitors: 8.5 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride) with five strokes of a motor-driven IKA homogenizer at 1,250 rpm. One microliter of this homogenate was used to measure the total protein concentration. A Pierce BCA protein assay kit was used. A fresh set of protein standards (BSA, 2 mg/ml, 0, 1.5, 5, 10, 20, 30 µl) containing the same component was prepared and used to determine the protein concentration for each sample. Distilled water was then added to all standards and samples to ensure the same total volume in each tube (50 µl). One milliliter of BCA protein assay reagent mix solutions (reagent A/reagent B = 50:1) was added, and all tubes were incubated at 37°C for 30 min. All samples were measured at 562 nm using a Shimadzu spectrophotometer (UV-VIS 1201 Spectrophotometer, Spectrachrom, Shimadzu, Japan) with the Protein Analysis Pack installed and the choice of the BCA method. The protein standards were determined before all the other samples to make a standard curve. Measurement of each sample was made in duplicate, and the mean values were used.

Electrophoresis and Immunoblotting

Samples of membrane fractions from the inner medulla or cortex+outer medulla 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 (inner medulla: Fig. 2A; outer medulla+cortex: Fig. 2B). The Coomassie-stained gel was used to ascertain identical loading or to allow for potential correction for minor differences in loading after scanning and densitometry of major bands (see below). The other gel was subjected to blotting. After transfer to nitrocellulose membranes by electroelution, blots were blocked with 5% milk in 80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, and 0.1% Tween 20, pH 7.5, for 1 h and incubated with primary antibodies (see below) overnight at 4°C. After a washing as above, the blots were incubated with horseradish peroxidase-conjugated secondary antibody (P448, diluted 1:3,000, DAKO, Glostrup Denmark). After a final washing as above, antibody binding was visualized using the enhanced chemiluminescence system (Amersham International).


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Fig. 2.   Commassie-stained 12% polyacrylamide minigels. All samples were from Wistar rats. A: samples of membrane fractions were from the inner medulla (IM) of the obstructed (OBS) and sham-operated kidneys (Sham). B: samples of membrane fractions were from outer medulla plus cortex of the obstructed kidneys and sham-operated kidneys.

Primary Antibodies

For semiquantitative immunoblotting and immunocytochemistry, we used previously characterized polyclonal antibodies as summarized below.

AQP1 (CHIP serum or LL266AP). Immune serum or an affinity-purified antibody to AQP1 has previously been characterized (37).

AQP2 (LL127 serum or LL127AP). Immune serum or an affinity-purified antibody to AQP2 has previously been described (6, 27).

p-AQP2 (AN244-pp-AP). An affinity-purified rabbit polyclonal antibody to p-AQP2 has previously been described (3).

AQP3 (LL178AP). An affinity-purified polyclonal antibody to AQP3 has previously been characterized (10).

AQP4 (LL182AP). An affinity-purified polyclonal antibody to AQP4 has previously been characterized (36).

Immunocytochemistry

The kidneys from UUO rats and sham-operated rats were fixed with 3% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, by retrograde perfusion via the abdominal aorta. 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 dewaxed and rehydrated. For immunoperoxidase labeling, endogenous peroxidase were blocked by 0.5% H2O2 in absolute methanol for 10 min at room temperature. To reveal antigens, sections were put in 1 mM Tris solution (pH 9.0) supplemented with 0.5 mM EGTA and heated for 10 min in a microwave oven. Nonspecific binding of immunoglobulin was prevented by incubating the sections in 50 mM NH4Cl for 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 × 10 min, the sections were incubated in horseradish peroxidase-conjugated Ig (P448, diluted 1:200, DAKO) diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. The sections were washed for 3 × 10 min, followed by incubation with diaminobenzidine for 10 min. Microscopy was carried out using a Leica DMRE light microscope. Sections from sham, UUO-obstructed, and nonobstructed kidneys were always labeled at the same time with the same solutions to allow for comparison.

Statistics

For densitometry of immunoblots, samples from both obstructed and nonobstructed kidneys were run on each gel with corresponding sham kidneys. Abundance of AQP1, -2, -3, -4, and p-AQP2 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 subjected to densitometry and used for correction of potential minor differences in loading. Values are presented in the text 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
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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UUO Is Associated With a Urinary Concentrating Defect

In Münich-Wistar rats with UUO for 24 h, we determined osmolality and sodium, potassium, creatinine, and urea concentration in plasma and urine. A highly significant increase in plasma creatinine (46.7 ± 1.9 vs. 30.3 ± 1.1 µM) and urea (7.39 ± 0.3 vs. 4.8 ± 0.3 mM) concentration and plasma osmolality (308 ± 1 vs. 304 ± 1 mosmol/kgH2O; Table 1) were revealed. Plasma and urinary concentrations of sodium and potassium did not change compared with sham-operated controls (Table 1).

                              
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Table 1.   Changes in renal function in rats subjected to 24-h UUO (protocol 1)

In addition, we tested whether renal excretion of water and sodium changed from both the obstructed kidney and the contralateral nonobstructed kidney in response to UUO. After 24 h of UUO, the ligation was released and urine was collected for 2 h from both kidneys (protocol 3). In the obstructed kidneys, there was a decrease in urinary osmolality (333 ± 8 vs. 1,135 ± 87 mosmol/kgH2O) and TcH2O [0.20 ± 0.13 vs. 8.69 ± 1.29 µl · min-1 · kg body wt (BW)-1; Table 2], indicating a decreased urinary concentration in the obstructed kidneys. In contrast, in nonobstructed kidneys urinary volume (7.75 ± 0.91 vs. 3.16 ± 0.26 µl · min-1 · kg-1), TcH2O (21 ± 3.1 vs. 8.69 ± 1.29 µl · min-1 · kg BW-1), creatinine clearance (2.42 ± 0.33 vs. 1.94 ± 0.24 ml · min-1 · kg BW-1), and potassium excretion (1.27 ± 0.20 vs. 0.62 ± 0.08 µmol/min) were significantly increased compared with sham-operated controls, indicating compensatory changes in the contralateral kidney (Table 2).

                              
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Table 2.   Changes in renal function in rats subjected to 24-h UUO followed by 2 h of release and sham operation

To examine the role of vasopressin in renal function during UUO, we examined the renal excretion of water and sodium from both kidneys in BB rats during 24-h UUO (Table 3). Similar to Münich-Wistar rats, there were a highly significant increase in plasma creatinine (65.7 ± 2.8 vs. 45 ± 1.5 µM) and urea (9.4 ± 0.8 vs. 5.6 ± 0.5 mM) concentration. Plasma sodium concentration decreased slightly (151 ± 1.1 vs. 155 ± 0.4 mM; Table 3). Similar to the findings in Münich-Wistar rats, there was a highly significant increase in urine production from contralateral nonobstructed kidneys during the 24 h of UUO (Table 3).

                              
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Table 3.   Changes in renal function in Brattleboro rats subjected to 24-h UUO (protocol 4)

Reduced Expression of AQP2 in Obstructed Kidneys

AQP2 is expressed in the apical plasma membrane and subapical vesicles of collecting duct principal cells. As previously shown, the affinity-purified anti-AQP2 antibody exclusively recognizes 29- and the 35- to 50-kDa bands (Figs. 3 and 4, A and C; see Table 5). Consistent with previous studies, we confirmed the decline in AQP2 expression in the collecting ducts of obstructed kidneys (inner medulla: 42 ± 4 vs. 100 ± 6%; Fig. 3, A and B; outer medulla+cortex: 48 ± 11 vs. 100 ± 12%, P < 0.05; Fig. 4, A and B; see Table 5). In contrast, AQP2 levels in nonobstructed kidneys did not change (Figs. 3 and 4, C and D; see Table 5). Importantly, analysis of total protein content demonstrated that total protein content in the kidneys did not differ significantly among obstructed, nonobstructed, and sham kidneys (Table 4).


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Fig. 3.   Semiquantitative immunoblotting of membrane fractions of IM from UUO and sham-operated Wistar rats. A and C: immunoblots reacted with anti-aquaporin-2 (AQP2) antibody revealed 29- and 35- to 50-kDa AQP2 bands. B: densitometric analysis of all samples from obstructed kidneys of rats with 24-h UUO and sham-operated controls revealed a marked downregulation in the expression of AQP2: 42 ± 4% of sham levels (100 ± 6%; *P < 0.05). D: in the nonobstructed kidneys (non-OBS), AQP2 expression did not differ from sham-operated controls. n, No. of rats.



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Fig. 4.   Semiquantitative immunoblotting of membrane fractions of outer medulla plus cortex from UUO and sham-operated Wistar rats. A and C: immunoblots reacted with anti-AQP2 antibody. B: densitometric analysis of samples from obstructed kidneys in rats with 24-h UUO and sham-operated controls revealed a significant downregulation in AQP2 expression in obstructed kidneys: 48 ± 11% of sham levels (100 ± 12%; *P < 0.05). D: in the nonobstructed kidneys, AQP2 expression did not differ from sham-operated controls. n, No. of rats.


                              
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Table 4.   Weight and protein content of kidneys from Wistar rats (protocol 2)

Semiquantitive immunoblotting of p-AQP2 was performed using antibodies that selectively recognize p-AQP2, which is phosphorylated at the protein kinase A phosphorylation consensus site (Ser256) (3). As seen in Fig. 5, in the inner medulla the abundance of p-AQP2 decreased significantly (23 ± 5 vs. 100 ± 15%, P < 0.05; Table 5). Conversely, in nonobstructed kidneys (Fig. 5, C and D; Table 6) p-AQP2 levels were unaltered. These findings were confirmed by immunocytochemistry. In kidneys from sham-operated controls, immunocytochemistry showed that p-AQP2 antibody labeled the apical plasma membrane domains of collecting duct principal cells in the inner medulla (Fig. 6E) and inner stripe of the outer medulla (Fig. 6F). In obstructed kidneys, p-AQP2 labeling was much weaker in the apical plasma membrane domains of collecting duct principle cells in the inner medulla (Fig. 6A) and inner stripe of the outer medulla (Fig. 6B) compared with sham-operated controls. In contrast, in nonobstructed kidneys, the labeling density of p-AQP2 was unchanged compared with sham-operated rats (Fig. 6, C and D). Thus these findings indicate a decreased abundance of AQP2 and p-AQP2 in the collecting ducts as important elements in the impaired urinary concentrating capacity.


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Fig. 5.   Semiquantitative immunoblotting of membrane fractions of IM from UUO and sham-operated Wistar rats. A and C: immunoblots reacted with affinity-purified phosphorylated AQP2 (p-AQP2) antibody and revealed 29- and 35- to 50-kDa bands. B: densitometric analysis of all samples from obstructed kidney in rats with 24-h UUO and sham-operated controls revealed a marked decrease in the obstructed kidney: 23 ± 5% of sham levels (100 ± 15%, *P < 0.05). D: densitometric analysis revealed that p-AQP2 expression did not differ between nonobstructed kidneys and sham-operated controls. n, No. of rats.


                              
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Table 5.   Expression of aquaporins in obstructed kidneys of rats with 24-h UUO and sham-operated controls


                              
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Table 6.   Expression of aquaporins in the nonobstructed kidneys in rats with 24-h UUO and sham-operated controls



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Fig. 6.   Immunocytochemical localization of p-AQP2 in IM and inner stripe of outer medulla of Wistar rat kidney. A and B: in the obstructed kidney of UUO rats, reduced labeling of p-AQP2 (arrows) in principal cells of the inner medullary collecting duct (IMCD; A) and of the inner stripe of outer medulla (ISOM; B) is detected in the apical parts of collecting duct principal cells. C and D: in the nonobstructed kidney of UUO rats, abundant labeling of p-AQP2 is seen in the apical and subapical domains of the principal cells of IMCD (C) and ISOM (D). E and F: in the sham-operated animals, abundant labeling of p-AQP2 is seen at apical plasma membrane and subapical domains of IMCD (E) and ISOM principal cells (F). Magnification: ×1,100.

Reduced AQP3 Expression in Obstructed Kidneys

We examined the expression of AQP3 located to the basolateral membrane of the principal cells in the collecting ducts from rats with UUO using immunoblotting and immunocytochemical analysis. Immunoblotting demonstrated that the abundance of AQP3 in the inner medulla of obstructed kidneys was dramatically reduced: 19 ± 6 vs. 100 ± 12% in controls (P < 0.05, Fig. 7, A and B; Table 5). Consistent with this, immunocytochemistry demonstrated a much weaker labeling of AQP3 in the basolateral membrane domains of the principal cells in the IMCDs of obstructed kidneys (Fig. 8A). However, in nonobstructed kidneys AQP3 expression was unchanged compared with sham-operated controls (134 ± 12 vs. 100 ± 8%; Fig. 7, C and D; Table 6). Consistent with this, immunocytochemistry showed that the labeling density of AQP3 in nonobstructed kidneys was unchanged (Fig. 8B) compared with sham-operated controls (Fig. 8C).


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Fig. 7.   Semiquantitative immunoblotting of membrane fractions of IM from UUO and sham-operated Wistar rats. A and C: immunoblots reacted with affinity-purified anti-AQP3 antibody and revealed 27- and 33- to 40-kDa bands. B: densitometric analysis of all samples from obstructed kidneys in rats with 24-h UUO and sham-operated controls revealed a marked reduction in AQP3 expression: 19 ± 6% of sham levels (100 ± 12%, *P < 0.05). D: densitometric analysis revealed that AQP3 expression did not differ between nonobstructed kidneys and sham-operated controls.



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Fig. 8.   Immunocytochemical localization of AQP3 in IM of Wistar rat kidney. A: in the obstructed kidney of UUO rats, reduced labeling of AQP3 (arrows) is detected at the basolateral membrane domains of collecting duct principal cell but is much weaker compared with that seen in the nonobstructed kidneys (B) and kidneys from sham-operated control rats (C). C: in sham-operated control rats, AQP3 labeling is detected at the basolateral domains of collecting duct principal cells similar to that found in the nonobstructed kidney. Magnification: ×1,100.

Vasopressin-Independent Regulation of AQP2 and AQP3 in Obstructed and Nonobstructed Kidneys

To examine the role of vasopressin-independent regulation of AQP2 and -3 in UUO, immunoblotting was performed on kidneys from BB rats with 24-h UUO. In obstructed kidneys, immunoblotting revealed a decreased expression of AQP2 to 55 ± 7% of sham levels, p-AQP2 to 17 ± 7% of sham levels, and AQP3 to 60 ± 10% of sham levels (Fig. 9; Table 7). Moreover, in contralateral nonobstructed kidneys there was a decreased expression of p-AQP2 to 58 ± 16% of sham levels, whereas the expression of AQP3 increased moderately to 142 ± 10% of sham levels, P < 0.05 (Fig. 9; Table 7).


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Fig. 9.   Semiquantitative immunoblotting of membrane fractions of total kidney from the obstructed kidney and nonobstructed kidney in UUO and sham-operated BB rats. A and B: immunoblots reacted with anti-AQP2 antibody (A and B), p-AQP2 antibody (C and D), anti-AQP3 antibody (E and F), and anti-AQP1 antibody (G and H). (See Table 7 for densitometric analysis). * P < 0.05, significant change in band density.


                              
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Table 7.   Expression of aquaporins in Brattleboro rats in response to 24 h of UUO

Reduced AQP4 Expression in Obstructed Kidneys

Immunoblotting demonstrated that AQP4 expression in the inner medulla of obstructed kidneys was dramatically reduced (11 ± 5 vs. 100 ± 38%, P < 0.05; Fig. 10, A and B; Table 5). However, in nonobstructed kidneys AQP4 expression was unchanged compared with sham-operated controls (152 ± 32 vs. 100 ± 24%; Fig. 10, C and D; Table 6). AQP4 is expressed in the basolateral membrane of renal collecting duct principal cells and is involved in water reabsorption. Immunocytochemical analysis demonstrated that in sham-operated rats, anti-AQP4 antibody labeled the basolateral plasma membrane domains of collecting duct principal cells in the inner medulla (Fig. 11E) and inner stripe of the outer medulla (Fig. 11F). In obstructed kidneys, immunocytochemistry showed that the labeling of AQP4 in the IMCD (Fig. 11A) and in the inner stripe of outer medullary collecting ducts was much weaker (Fig. 11B) compared with the similar renal segments in sham-operated controls and contralateral kidneys (Fig. 11, C and D).


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Fig. 10.   Semiquantitative immunoblotting of membrane fractions of IM from UUO and sham-operated Wistar rats. A and C: immunoblots reacted with affinity-purified anti-AQP4 antibody. B: densitometric analysis of all samples from obstructed kidneys in rats with 24-h UUO and sham-operated controls revealed a marked reduction in AQP4 expression: 11 ± 5% of sham levels (100 ± 38%, *P < 0.05). D: densitometric analysis revealed that AQP4 expression did not differ between nonobstructed kidneys and sham-operated controls.



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Fig. 11.   Immunocytochemical localization of AQP4 in IMCD and ISOM of Wistar rat kidney. A and B: in the obstructed kidney of UUO rats, reduced labeling of AQP4 (arrows) in principal cells of IMCD (A) and of ISOM collecting duct (B) is confined to the basolateral parts of collecting duct principal cells. C and D: in the nonobstructed kidney of UUO rats, abundant labeling of AQP4 is seen in the basolateral and adjacent cytoplasmic domains of the principal cells of IMCD (C) and ISOM collecting duct (D). E and F: in the sham-operated animals, abundant labeling of AQP4 is seen in the basolateral and adjacent cytoplasmic domains of the principal cells of IMCD (E) and inner stripe of outer medullary collecting duct (F). Magnification: ×1,100.

UUO Is Associated With Reduced AQP1 Expression in Both Obstructed and Nonobstructed Kidneys

The abundance of AQP1 protein was markedly decreased in obstructed kidneys in both the inner medulla (64 ± 8 vs. 100 ± 13%; Fig. 12, A and B; Table 5) and in the outer medulla and cortex (74 ± 4 vs. 100 ± 7%; Fig. 13, A and B; Table 5). Similarly, in nonobstructed kidneys, immunoblots revealed a markedly reduced abundance of AQP1 in the inner medulla (77 ± 3 vs. 100 ± 7%; Fig. 12, C and D; Table 6) as well as in the outer medulla and cortex (77 ± 8 vs. 100 ± 5%; Fig. 13, C and D; Table 6). Thus AQP1 located in the proximal tubule and in the descending thin limb of Henle's loop may play a role in the water balance disorders associated with obstructive nephropathy. To examine whether there were any changes in the segmental or subcellular distribution of AQP1 in the proximal tubule and descending thin limb, immunocytochemical analysis was performed (not shown). The results demonstrate that there is heterogenous labeling but no overall change in the labeling intensity, as expected with a reduction of ~30% in expression determined by semiquantitative immunoblotting. Moreover, there is no change in the segmental distribution of AQP1. The labeling was found in segments 1-3 in the proximal tubule as well as in outer medullary and inner medullary segments of the descending thin limb and vasa recta. AQP1 was confined to apical and basolateral plasma membrane domains in both obstructed, nonobstructed, and control kidneys. Thus the combined results suggest that there is uniform downregulation of AQP1 with no segmental or subcellular change.


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Fig. 12.   Semiquantitative immunoblotting of membrane fractions of IM from UUO and sham-operated Wistar rats. A and C: immunoblots are reacted with affinity-purified anti-AQP1 antibody and revealed 29- and 35- to 50-kDa AQP1 bands. B: densitometric analysis from obstructed kidneys in rats with 24-h UUO revealed a significant decrease in AQP1 levels: 64 ± 8% of sham levels (100 ± 13%, *P < 0.05). D: in the nonobstructed kidneys in rats with 24-h UUO, AQP1 expression deceased markedly to 77 ± 3% of sham levels (100 ± 7%, *P < 0.05).



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Fig. 13.   Immunoblot of membrane fractions of outer medulla and cortex from UUO and sham-operated Wistar rats. A and C: immunoblots reacted with affinity-purified anti-AQP1 antibody. B: densitometric analysis from obstructed kidneys in rats with 24-h UUO revealed a moderate but significant decrease in AQP1 levels: 77 ± 3% of sham levels (100 ± 7%, *P < 0.05). D: in nonobstructed kidneys in rats with 24-h UUO, AQP1 expression deceased significantly to 77 ± 8% of sham levels (100 ± 5%, *P < 0.05).

Similar to the findings in Münich-Wistar rats, the abundance of AQP1 in BB rats was reduced in both obstructed (62 ± 4 vs. 100 ± 6%, P < 0.05) and the nonobstructed kidneys (75 ± 5 vs. 100 ± 2%, P < 0.05; Fig. 9; Table 7).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, UUO was associated with downregulation of AQP2, p-AQP2 [phosphorylated in the PKA phosphorylation consensus site (Ser256) of AQP2], AQP3, and AQP4 in collecting duct principal cells. Moreover, the expression of AQP1 in whole kidneys was significantly downregulated in both obstructed and nonobstructed kidneys. In parallel, urinary concentrating capacity was impaired in the obstructed kidney. In contrast, the expression levels of p-AQP2, AQP3, and AQP4 in nonobstructed kidneys did not change. Furthermore, we examined the expression levels of AQPs in response to 24-h UUO in BB rats with low levels of circulating vasopressin. In obstructed kidneys, AQP2, p-AQP2, and AQP3 were decreased, demonstrating a vasopressin-independent downregulation. In obstructed and nonobstructed kidneys, a reduced level of AQP1 was a consistent finding. The results strongly support the view that AQPs play important roles in the altered regulation of water reabsorption associated with obstructive nephropathy and that vasopressin-independent mechanisms are involved in the downregulation of AQP2 and -3.

Reduced Expression of AQP2 in Obstructed Kidneys

Consistent with previous studies, semiquantitive immunoblotting of obstructed kidneys of rats with 24 h UUO demonstrated that AQP2 protein expression in the outer medullary and cortical collecting ducts as well as that in the IMCD is downregulated (11). In parallel, TcH2O was severely reduced in obstructed kidneys, demonstrating a functional association between AQP2 downregulation and water reabsorption at the collecting duct level. In support of these findings, we demonstrated that p-AQP2 also decreased markedly in obstructed kidneys. The absence of a more pronounced downregulation of p-AQP2 than total AQP2 indicates that trafficking of AQP2 is still present, consistent with previous reports (11, 12). This is also in accordance with previous immunocytochemical studies demonstrating a weaker labeling of p-AQP2 in the apical plasma membrane and subapical vesicle domains of collecting duct principal cells (3).

Recent studies suggested that p-AQP2 is present in both the apical plasma membrane and in subapical vesicles of collecting duct principal cells in normal rats and that p-AQP2 is subjected to trafficking to the apical plasma membrane in response to vasopressin treatment (3). The decreased expression of AQP2 and p-AQP2 in the present study indicates that both the total abundance of AQP2 and the phosphorylated fraction of AQP2 are reduced in collecting duct principal cells. This may play an important role in the impairment of water reabsorption during obstruction.

The mechanisms responsible for the downregulation of AQP2 and p-AQP2 after ureteral obstruction remain unclear. The urinary concentrating process depends on the coordinated function of the loop of Henle and the collecting duct. The thick ascending limb of the loop of Henle powers the countercurrent multiplier process responsible for generation of a corticomedullary osmotic gradient, whereas the collecting ducts, under the control of vasopressin, allow variable degrees of osmotic equilibration, resulting in a variable amount of excreted water and a reciprocal relationship between urinary flow and urinary osmolality.

It is well established that AQP2 is regulated, both short term and long term, by vasopressin (29, 30). However, mechanisms other than vasopressin also seem to be involved in long-term regulation of AQP2 expression. Rats with BUO have significantly higher plasma vasopressin values than sham-operated rats (32). However, our laboratory and others have demonstrated the downregulation of AQP2 and AQP3 in rats with BUO up to 14 days after release of the obstruction (12, 15, 21), suggesting that vasopressin-independent signal pathways may be involved in the regulation of AQP2 in obstructive nephropathy. The results of the present study showed that AQP2 and p-AQP2 levels were markedly decreased in the obstructed kidneys of BB rats with UUO. Consistent with previous studies demonstrating that vasopressin-independent pathways can modify AQP2 expression (8, 9, 38), the present findings demonstrate that vasopressin-independent pathways are involved in AQP2 (and AQP3; see below) dysregulation in response to UUO.

The present study revealed unaltered expression levels of AQP2 and p-AQP2 in nonobstructed kidneys. This finding is consistent with the absence of a major reduction in AQP2 abundance in nonobstructed kidneys, which we demonstrated previously (11). The markedly reduced abundance of p-AQP2 in nonobstructed kidneys of BB rats compared with the p-AQP2 levels maintained in the nonobstructed kidneys of Münich-Wistar rats may suggest that intact vasopressin levels are essential for the maintenance of p-AQP2 levels after UUO. This is further supported by a significant reduction in TcH2O in the nonobstructed kidneys of B rats compared with sham-operated control rats. It should be emphasized that this may not be a general response in a nonobstructed kidney. Thus UUO may be associated with unchanged or slightly reduced AQP2 abundance in a nonobstructed kidney.

Reduced Expression of AQP3 and AQP4 in Obstructed Kidneys

AQP3 and AQP4 represent exit pathways of water through the basolateral membrane in collecting duct principal cells. Immunoblotting and immunocytochemistry demonstrated that both AQP3 and AQP4 decreased significantly in obstructed kidneys compared with sham-operated controls. It is well known that the kidney deletion of AQP3 produced marked polyuria and that AQP3 knockout mice are able to generate only partially concentrated urine after water deprivation (22, 39). AQP4 is responsible for the majority of basolateral membrane water movement in the IMCD, but AQP4 knockout mice only demonstrate a very mild defect in urinary concentrating ability (2). The AQP3/AQP4 double-knockout mice had a greater impairment of urinary concentrating ability than did the AQP3 single-knockout mice (22). Similar to the findings in Wistar rats, AQP3 abundance was also reduced in BB rats, demonstrating that vasopressin-independent mechanisms are involved in AQP3 regulation. Thus our results demonstrated downregulation of AQP3 and -4 together with a parallel impairment of urine-concentrating capacity after UUO, suggesting that downregulation of AQP3 and AQP4 may play important roles in urinary concentrating capacity.

Reduced AQP1 Expression in Both Obstructed and Nonobstructed Kidneys

In the present studies, AQP1, expressed in the proximal tubule and descending thin limb of Henle's loop, was moderately decreased in both obstructed and nonobstructed kidneys compared with sham-operated controls. Recently, it was reported that AQP1 knockout mice have a severe urinary concentrating defect, decreased transepithelial water permeability in proximal tubule and descending thin limb of Henle's loop, and defective fluid absorption, indicating that AQP1 plays a vital role in the countercurrent multiplier mechanism by allowing efficient osmotic water equilibration (1, 23, 34). Changes in proximal tubule fluid transport may have significant effects on the urinary concentrating mechanism by altering flow rates of tubule fluid delivery to the thick ascending limbs and collecting ducts. Therefore, impairment of proximal tubule function, demonstrated by a decline in AQP1 protein abundance, could contribute to the changes in urinary flow.

Consistent with previous studies (4, 19), creatinine clearance evidence was found for a severe reduction in GFR in obstructed kidneys immediately after release of the obstruction. This may in part explain the relatively low production of urine despite the markedly reduced abundance of AQP1-4 in obstructed kidneys. Furthermore, it may be hypothesized that an increased delivery of NaCl at the macula densa may be associated with a resetting of the tubuloglomerular feedback response, similar to what has been suggested in AQP1 knockout mice (33), thus further reducing GFR after release of the obstruction.

Ureteral occlusion induces a complex series of hormonal changes in the obstructed kidney (18), which may influence the functional changes in the contralateral kidney. Similarly, it has been demonstrated that renorenal reflexes are also important for the modulation of urinary output in the contralateral kidney during UUO (5). It is likely that several mechanisms are involved at the same time and that the role of such mechanisms in the regulation of channels and transporters in the nonobstructed kidney are at present unknown.

The importance of local factors in the dysregulation of AQPs in response to obstruction was addressed by examining the effect of UUO on the expression of AQP1-4 in the contralateral nonobstructed kidney. The contralateral kidney demonstrated increased reabsorption and excretion of water and solutes during UUO, which takes place to compensate for the impaired excretion from the obstructed kidney. Unlike local factors, systemic factors would likely produce effects that operate simultaneously. The very extensive downregulation of AQP2, p-AQP2, AQP3, AQP4, and AQP1 protein found in the obstructed kidneys and unchanged expression levels of AQP2, -3, and -4 in the nonobstructed kidneys of the same animals are consistent with this view, which emphasizes that local, intrarenal factors play a major role in the induction of downregulation. However, systemic factors also appear to be involved, because there was a significant decrease in AQP1 protein levels in nonobstructed kidneys compared with sham-operated controls in both Wistar and BB rats. Interestingly, a similar reduction in the abundance of AQPs was observed in ischemia-induced ARF (16), suggesting that a common signal transduction pathway may be involved in these two animal experiments, both of which can induce renal failure. Because all the transporters we examined in the obstructed kidney are downregulated, it may be speculated that obstruction induces a general downregulation of all tubular transport proteins. This will be addressed in future studies.

Summary

The present study demonstrated that UUO in rats is associated with downregulation of renal AQP2, p-AQP2, AQP3, AQP4, and AQP1, consistent with an impaired urinary concentrating capacity in the obstructed kidney. The reduction of these AQPs in UUO rats may, at least in part, contribute to the impaired water metabolism and may reflect reductions of net reabsorption of water at several nephron sites in the obstructed kidney, including the proximal straight tubule, descending thin limb, the inner stripe of outer medullary collecting duct, and the IMCD. Unaltered levels of AQPs and decreased AQP1 levels in the contralateral kidney may contribute to the compensatory increase in urinary flow.


    ACKNOWLEDGEMENTS

The authors thank Gitte Kall, Inger Merete Paulsen, Dorte Wulff, Mette Vistisen, Helle Høyer, Zhile Nikrozi, Lotte Valentin Holbech, Merete Pedersen, and Ida Maria Jalk for expert technical assistance.


    FOOTNOTES

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, and the intramural budget of the National Heart, Lung, and Blood Institute.

Address for reprint requests and other correspondence: J. Frøkiær, The Water and Salt Research Ctr., 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.

First published January 7, 2003;10.1152/ajprenal.00090.2002

Received 7 March 2002; accepted in final form 2 January 2003.


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DISCUSSION
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Am J Physiol Renal Fluid Electrolyte Physiol 284(5):F1066-F1079
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