Downregulation of renal aquaporins in response to unilateralureteral obstructio - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:37 |只看该作者 |倒序浏览 |打印

作者：ChunlingLi, WeidongWang, Mark A.Knepper, SørenNielsen,  JørgenFrøkiær,作者单位：1 The Water and Salt Research Center and Institute of Anatomy, University of Aarhus, DK-8000Aarhus C; Institute of Experimental Clinical Researchand Department of Clinical Physiology, AarhusUniversity Hospital-Skejby, DK-8200 Aarhus N, Denmark; and Laboratory of Kidney and Electrolyte Metabolism,Nat



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      【摘要】& B% p3 W' D% E# r; }: a3 l
   The expression of aquaporin-2 (AQP2)is decreased in rats with bilateral ureteral obstruction (BUO) andunilateral ureteral obstruction (UUO). Therefore, theexpression of additional renal aquaporins (AQP1-4) andphosphorylated AQP2 (p-AQP2), known to play a role in urinaryconcentration, was examined in a Wistar rat model with 24 h ofUUO. In obstructed kidneys, immunoblotting revealed a significantdecrease in the expression of inner medullary AQP2 to 42 ± 4, p-AQP2 to 23 ± 5, AQP3 to 19 ± 6, AQP4 to 11 ± 5, andAQP1 to 64 ± 8% of sham levels. AQP1 expression located in theproximal tubule decreased to 74 ± 4% of sham levels( P AQP4, and p-AQP2. In contralateralnonobstructed kidneys, immunoblotting also revealed significantreductions 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 andnonobstructed kidneys for 2 h, respectively, after 24 h ofUUO. Urine collection from obstructed kidneys during 2 h afterrelease of UUO revealed a significant reduction in urine osmolality andsolute-free water reabsorption (T c H 2 O).Moreover, an increase in urine production andT c H 2 O was observed in contralateral kidneys. Toexamine whether vasopressin-independent mechanisms are involved in AQP2regulation, vasopressin-deficient Brattleboro (BB) rats with 24 hof UUO were examined. Immunoblotting revealed downregulation of AQP2,p-AQP2, AQP3, and AQP1 in obstructed kidneys and downregulation ofp-AQP2 and AQP1 in nonobstructed kidneys. In conclusion, 1 )UUO is associated with severe downregulation of AQP2, AQP3, AQP4, andAQP1; thus all of these AQPs may play important roles in the impairedurinary concentrating capacity in the obstructed kidney; 2 )the reduced levels of AQP1 in the nonobstructed kidney may contributeto the compensatory increase in urine production; and 3 )downregulation of AQPs in BB rats supports the view thatvasopressin-independent pathways may be involved in AQP2 and AQP3regulation in the obstructed kidney. : {1 e2 R5 w; C$ L
      【关键词】 collecting duct proximal tubule water channel obstructivenephropathy vasopressin Brattleboro rats
   INTRODUCTION0 s, w0 k# \% j$ V+ ^! m
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URINARY TRACT OBSTRUCTION is a serious and common clinical condition, which is associated withincreased intraluminal pressure in the ureter and renal tubules thatmay cause renal parenchymal damage through a series of direct andindirect effects ( 19 ). Obstruction of the urinarytract has marked effects on renal blood flow, glomerular filtrationrate (GFR), tubular function, and parenchymal structure ( 4, 28, 35 ). Thus abnormalities in tubular function are common inobstructive nephropathy, including a reduction in urinary concentratingcapacity, altered reabsorption of solutes and water, and impairedexcretion of hydrogen and potassium. The major sites of abnormalfunction are located to the distal segments of the nephron( 18 ).9 V3 o; y: h6 P* w# j6 H
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Production of concentrated urine depends on active solute reabsorptionin the thick ascending limb of Henle's loop to generate highosmolality in the medullary interstitium. The production ofconcentrated urine also depends on high water permeability of thecollecting duct in response to antidiuretic hormone, primarily throughincreased expression of aquaporin-2 (AQP2) and -3 (AQP3) located in thecollecting duct principal cells. Recently, we reported that rats withunilateral ureteral obstruction (UUO) had reduced levels of AQP2 and aparallel impairment of solute-free water reabsorption, demonstrating afunctional association between decreased levels of AQP2 and reducedwater reabsorption in the collecting duct ( 11 ).! P7 b( I. S9 |% g4 L4 @

The AQPs are a family of membrane proteins that function as waterchannels that play key roles in the reabsorption of water in thekidney. AQP1 is located in the proximal tubule and thin descendinglimb. AQP1 knockout studies have emphasized the importance of AQP1 influid reabsorption in both the proximal tubule and the descending thinlimb of Henle ( 1, 34 ). Water transport across the apicalmembrane 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 haveelucidated important roles of AQP2 in multiple water balance disorders.A number of conditions with acquired nephrogenic diabetes insipidus areassociated with marked downregulation of AQP2 protein, such as thoseinduced by chronic lithium treatment ( 25 ), hypokalemia ( 26 ), hypercalcemia ( 7 ), and ischemicacute renal failure (ARF) ( 14, 16, 20 ). Also, AQP2 levelsand urinary concentrating capacity were markedly reduced in response tobilateral ureteral obstruction (BUO) ( 12, 15, 21 ).

There is compelling evidence that AQP2 is regulated long term byvasopressin ( 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 AQP2levels despite high circulating levels of vasopressin, indicating vasopressin-independent regulation of AQP2 ( 8 ). Anon-vasopressin-mediated effect on AQP2 expression has also beendemonstrated in rats treated with lithium, whereby dehydration caused amuch higher increase in AQP2 levels than vasopressin treatment( 25 ). Similarly, vasopressin-independent regulation wasdemonstrated using vasopressin-deficient Brattleboro (BB) rats, whichhave much lower levels of AQP2 (~50%) than rats with normalvasopressin levels ( 6, 17, 31 ). During BUO, vasopressinlevels are elevated, whereas during unilateral obstruction of theurinary tract plasma vasopressin levels are similar to controls( 32 ). In both conditions, reduced AQP2 levels have beendemonstrated, indicating vasopressin-independent regulation of AQP2expression during urinary tract obstruction.

Water transport across the basolateral membrane of the collecting ductprincipal cells is mediated by AQP3 and AQP4. Reduced levels of AQP3expression were demonstrated in rats with BUO up to 14 days afterrelease of BUO ( 21 ). Consistent with this, it is wellknown that AQP3 knockout mice are remarkably polyuric but are able togenerate a partly concentrated urine after water deprivation( 22 ). AQP4 is the most abundant basolateral water channelin the inner medullary collecting duct (IMCD). However, AQP4 knockoutmice manifest only a mild defect in maximum urinary concentratingability ( 2 ). AQP3 appears to be regulated by vasopressin,whereas there is no evidence for long-term regulation of AQP4expression in the kidney ( 36 ). Therefore, it could be speculated that the impairment in urinary concentrating capacity afterUUO may be associated with significant changes in the expression levelof basolateral collecting duct AQP3 and AQP4.4 s- w/ Q" ^. O8 k5 q
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To further increase the understanding of the molecular mechanisminvolved in the impairment of renal water handling during urinary tractobstruction, we therefore examined 1 ) whether UUO isassociated with changes in the expression of AQPs [AQP1-4 and phosphorylated (p)-AQP2] and 2 ) whether changes in theexpression of AQPs are associated with alterations in urinaryconcentrating capacity. In addition, to examine the importance ofnon-vasopressin-mediated regulation of AQPs during UUO, the expressionlevels of AQPs were analyzed in kidneys from BB rats with UUO.

METHODS, t, j  Q3 e( E2 v. V& T. k
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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 (HarlanSprague Dawley, Indianapolis, IN). The rats were maintained on astandard rodent diet (Altromin, Lage, Germany) with free access towater. During the entire experiment, rats were kept in individualmetabolic cages, with a 12:12-h artificial light-dark cycle, atemperature of 21 ± 2°C, and humidity of 55 ± 2%. Ratswere allowed to acclimatize to the cages for 5-7 days beforesurgery. Water intake, urine output, and body weight of the rats weremonitored every 24 h during the study.

Before surgery, the rats were anesthetized with halothane (HalocarbonLaboratories), and during surgery the rats were placed on a heatedtable to maintain rectal temperature at 37-38°C. Through amidline abdominal incision, the left ureter was exposed and a 5-0 silkligature occluded the midportion of the ureter. After surgery, the ratsregained consciousness and were placed in metabolic cages. Twenty-fourhours after occlusion of the left ureter, the rats were anesthetizedand both kidneys were removed, after which the animals were killed. Ina subset of animals ( protocol 3 ), the obstruction wasreleased after 24 h by inserting a polyethylene tube (PE-35) intothe proximal left ureter. A similar catheter was inserted into theproximal right ureter to allow separate collection of urine from theleft and the right kidney. After urine was collected for at least2 h, the rats were killed. The animals were allocated to theprotocols indicated below. Age- and time-matched sham-operated controlswere prepared and were observed in parallel with each UUO group (Fig. 1 ).

Fig. 1. Diagram of the study design. Protocol 1 :the left ureter was occluded for 24 h in Wistar rats withunilateral ureteral obstruction (UUO), and matched sham-operatedcontrol Wistar rats were prepared in parallel. Protocol 2 :rats were prepared similar to in protocol 1 (UUO andsham-operated controls). The weight of the kidneys and total proteincontent in kidneys were measured. Protocol 3 : rats wereprepared similar to in protocol 1 (UUO and sham-operatedcontrols). After 24 h, the obstruction was released and urine wascollected from each ureter for 2 h. Protocol 4 : theleft ureter was occluded for 24 h in Brattleboro (BB) rats withUUO, and matched sham-operated control BB rats were prepared inparallel. n, No. of rats.% v9 W9 D; g0 I, X
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Protocol 1. Münich-Wistar rats underwent UUO for 24 h ( n = 11). The kidneys were removed and prepared separately forsemiquantitative immunoblotting ( n = 7) or forimmunocytochemistry ( n = 4). For matched sham-operatedcontrol rats, n = 10.

Protocol 2. Münich-Wistar rats underwent UUO for 24 h ( n = 6). The kidneys were removed and prepared separately formeasuring kidney weight and total amount of protein levels. For matchedsham-operated control rats, n = 6.
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Protocol 3. Münich-Wistar rats underwent UUO for 24 h followed byrelease, and the animals were observed during the next 2 h( n = 8). Urine was collected for 2 h afterrelease of UUO. For matched sham-operated control rats, n = 8.0 z" {1 y1 e8 ?/ C
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Protocol 4. BB rats underwent UUO for 24 h ( n = 6). Thekidneys were removed and prepared separately for semiquantitativeimmunoblotting. For matched sham-operated control rats, n = 6.# A# ^% W( {$ e# F/ _
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Clearance Studies

Urine was collected during 24-h periods throughout thestudy or for 2 h after release of ligation ( protocol3 ). Clearance studies were performed during the last 24 h in protocols 1 and 4 and for 2 h after releasein protocol 3. During anesthesia and before removal of thekidneys, 2-3 ml of blood were collected into a heparinized tubefor the determination of plasma electrolytes and osmolality. The plasmaand urinary concentrations of creatinine and urea, and the plasmaconcentrations of sodium and potassium, were determined (Kodak Ektachem700XRC). The concentrations of urinary sodium and potassium weredetermined by standard flame photometry (Eppendorf FCM6341). Theosmolality of urine and plasma was measured with a vapor pressureosmometer (Osmomat 030, Gonotec). Solute-free water reabsorption(T c H 2 O) was calculated by the followingformula: T c H 2 O = [(urineosmolality)/(plasma osmolality) 1] × (urine volume).5 a8 l0 o/ E) \( C. S
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Membrane Fractionation for Immunoblotting

For removal of kidneys, rats were anesthetized with halothane.The kidney was split into cortex and outer medulla and inner medullaand frozen in liquid nitrogen. Tissue (inner medulla or cortex outermedulla) was minced finely and homogenized in 1 (inner medulla) or 8 ml(cortex outer medulla) of dissecting buffer (0.3 M sucrose, 25 mMimidazole, 1 mM EDTA, pH 7.2, and the following protease inhibitors:8.5 µM leupeptin and 1 mM phenylmethylsulfonyl fluoride) with fivestrokes of a motor-driven IKA homogenizer at 1,250 rpm. Thishomogenate was centrifuged in a Beckman L8M centrifuge at 4,000 g for 15 min at 4°C to remove whole cells, nuclei, andmitochondria. Gel samples (Laemmli sample buffer containing 2% SDS)were made from this membrane preparation.8 m( t2 P, r$ o1 U  L
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Total Protein Concentration
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After removal of kidneys, they were weighed, mincedfinely, 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 thesame component was prepared and used to determine the protein concentration for each sample. Distilled water was then added to allstandards 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 tubeswere 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 installedand the choice of the BCA method. The protein standards were determinedbefore all the other samples to make a standard curve. Measurement ofeach sample was made in duplicate, and the mean values were used.

Electrophoresis and Immunoblotting9 C& s4 U6 i- z5 M; B& G

Samples of membrane fractions from the inner medulla orcortex 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. 2 A; outer medulla cortex: Fig. 2 B ). The Coomassie-stained gel was used to ascertainidentical loading or to allow for potential correction for minordifferences in loading after scanning and densitometry of major bands(see below). The other gel was subjected to blotting. After transfer tonitrocellulose membranes by electroelution, blots were blocked with 5%milk in 80 mM Na 2 HPO 4, 20 mMNaH 2 PO 4, 100 mM NaCl, and 0.1% Tween 20, pH7.5, for 1 h and incubated with primary antibodies (see below)overnight at 4°C. After a washing as above, the blots wereincubated with horseradish peroxidase-conjugated secondary antibody(P448, diluted 1:3,000, DAKO, Glostrup Denmark). After a final washingas above, antibody binding was visualized using the enhancedchemiluminescence system (Amersham International).
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Fig. 2. Commassie-stained 12% polyacrylamide minigels. Allsamples were from Wistar rats. A : samples of membranefractions were from the inner medulla (IM) of the obstructed (OBS) andsham-operated kidneys (Sham). B : samples of membranefractions were from outer medulla plus cortex of the obstructed kidneysand sham-operated kidneys.: [2 {) `' M' c

Primary Antibodies

For semiquantitative immunoblotting and immunocytochemistry, weused previously characterized polyclonal antibodies as summarized below.
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AQP1 (CHIP serum or LL266AP). Immune serum or an affinity-purified antibody to AQP1 has previouslybeen characterized ( 37 ).

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

p-AQP2 (AN244-pp-AP). An affinity-purified rabbit polyclonal antibody to p-AQP2 haspreviously been described ( 3 ).* B7 |$ B2 X, y0 ?5 b* u- q

AQP3 (LL178AP). An affinity-purified polyclonal antibody to AQP3 has previously beencharacterized ( 10 ).
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AQP4 (LL182AP). An affinity-purified polyclonal antibody to AQP4 has previously beencharacterized ( 36 ).

Immunocytochemistry; n# \$ V- v- l, v6 P

The kidneys from UUO rats and sham-operated rats were fixed with3% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, by retrogradeperfusion via the abdominal aorta. For immunoperoxidase microscopy,kidney blocks containing all kidney zones were dehydrated and embeddedin paraffin. The paraffin-embedded tissues were cut at 2 µm on arotary microtome (Leica). The sections were dewaxed and rehydrated. Forimmunoperoxidase labeling, endogenous peroxidase were blocked by 0.5%H 2 O 2 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 minin a microwave oven. Nonspecific binding of immunoglobulin wasprevented by incubating the sections in 50 mM NH 4 Cl 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°Cwith primary antibodies diluted in PBS supplemented with 0.1% BSA and0.3% Triton X-100. After being rinsed with PBS supplemented with 0.1%BSA, 0.05% saponin, and 0.2% gelatin for 3 × 10 min, thesections were incubated in horseradish peroxidase-conjugated Ig (P448,diluted 1:200, DAKO) diluted in PBS supplemented with 0.1% BSA and0.3% Triton X-100. The sections were washed for 3 × 10 min,followed by incubation with diaminobenzidine for 10 min. Microscopy wascarried out using a Leica DMRE light microscope. Sections from sham,UUO-obstructed, and nonobstructed kidneys were always labeled at thesame time with the same solutions to allow for comparison.

Statistics8 |  E: c. _5 M  d- Q: ?

For densitometry of immunoblots, samples from both obstructedand nonobstructed kidneys were run on each gel with corresponding shamkidneys. Abundance of AQP1, -2, -3, -4, and p-AQP2 in the samples fromthe experimental animals was calculated as a fraction of the mean shamcontrol value for that gel. Parallel Coomassie-stained gels weresubjected to densitometry and used for correction of potential minordifferences in loading. Values are presented in the text as means ± SE. Comparisons between groups were made by unpaired t -test. P values significant.8 G) L  U, K* A5 u# [$ V' i
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RESULTS

UUO Is Associated With a Urinary Concentrating Defect
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In Münich-Wistar rats with UUO for 24 h, we determinedosmolality and sodium, potassium, creatinine, and urea concentration inplasma and urine. A highly significant increase in plasmacreatinine (46.7 ± 1.9 vs. 30.3 ± 1.1 µM) and urea(7.39 ± 0.3 vs. 4.8 ± 0.3 mM) concentration and plasmaosmolality (308 ± 1 vs. 304 ± 1 mosmol/kgH 2 O;Table 1 ) were revealed. Plasma andurinary concentrations of sodium and potassium did not changecompared with sham-operated controls (Table 1 ).3 J% o# C$ W: x. y! y2 I
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Table 1. Changes in renal function in rats subjected to 24-h UUO (protocol 1)+ `( x/ q) x$ \+ |
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In addition, we tested whether renal excretion of water and sodiumchanged 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 adecrease in urinary osmolality (333 ± 8 vs. 1,135 ± 87 mosmol/kgH 2 O) and T c H 2 O [0.20 ± 0.13 vs. 8.69 ± 1.29 µl · min 1 · kgbody 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 ),T c H 2 O (21 ± 3.1 vs. 8.69 ± 1.29 µl · min 1 · kgBW 1 ), creatinine clearance (2.42 ± 0.33 vs.1.94 ± 0.24 ml · min 1 · kgBW 1 ), and potassium excretion (1.27 ± 0.20 vs.0.62 ± 0.08 µmol/min) were significantly increased comparedwith sham-operated controls, indicating compensatory changes in thecontralateral kidney (Table 2 ).

Table 2. Changes in renal function in rats subjected to 24-h UUO followed by2 h of release and sham operation9 K! f6 G7 g2 b5 E& p) w* D$ x
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To examine the role of vasopressin in renal function during UUO, weexamined the renal excretion of water and sodium from both kidneys inBB rats during 24-h UUO (Table 3 ).Similar to Münich-Wistar rats, there were a highly significantincrease 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 tothe findings in Münich-Wistar rats, there was a highlysignificant increase in urine production from contralateralnonobstructed kidneys during the 24 h of UUO (Table 3 ).8 `5 X: p1 P4 Q( }2 @1 A7 p2 L
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Table 3. Changes in renal function in Brattleboro rats subjected to 24-h UUO(protocol 4)
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Reduced Expression of AQP2 in Obstructed Kidneys
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AQP2 is expressed in the apical plasma membrane and subapicalvesicles of collecting duct principal cells. As previously shown, theaffinity-purified anti-AQP2 antibody exclusively recognizes 29- and the35- to 50-kDa bands (Figs. 3 and 4, A and C; seeTable 5 ).Consistent with previous studies, we confirmed the decline in AQP2expression in the collecting ducts of obstructed kidneys (innermedulla: 42 ± 4 vs. 100 ± 6%; Fig. 3, A and B; outer medulla cortex: 48 ± 11 vs. 100 ± 12%, P 4, A and B; seeTable 5 ). In contrast, AQP2 levels in nonobstructed kidneys did notchange (Figs. 3 and 4, C and D; see Table 5 ).Importantly, analysis of total protein content demonstrated that totalprotein content in the kidneys did not differ significantly amongobstructed, nonobstructed, and sham kidneys (Table 4 ).& C! g3 O$ f! w+ ?) K+ |+ n0 W! p

Fig. 3. Semiquantitative immunoblotting of membrane fractions of IM fromUUO and sham-operated Wistar rats. A and C :immunoblots reacted with anti-aquaporin-2 (AQP2) antibody revealed29- and 35- to 50-kDa AQP2 bands. B : densitometric analysisof all samples from obstructed kidneys of rats with 24-h UUO andsham-operated controls revealed a marked downregulation in theexpression of AQP2: 42 ± 4% of sham levels (100 ± 6%;* P 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 outermedulla plus cortex from UUO and sham-operated Wistar rats. A and C : immunoblots reacted with anti-AQP2antibody. B : densitometric analysis of samples fromobstructed kidneys in rats with 24-h UUO and sham-operated controlsrevealed a significant downregulation in AQP2 expression in obstructedkidneys: 48 ± 11% of sham levels (100 ± 12%;* P D : in the nonobstructedkidneys, 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)  n1 I% w! y; ^% P5 e8 c

Semiquantitive immunoblotting of p-AQP2 was performed using antibodiesthat selectively recognize p-AQP2, which is phosphorylated at theprotein kinase A phosphorylation consensus site (Ser 256 )( 3 ). As seen in Fig. 5, inthe inner medulla the abundance of p-AQP2 decreased significantly(23 ± 5 vs. 100 ± 15%, P Table 5 ). Conversely, in nonobstructed kidneys(Fig. 5, C and D; Table 6 ) p-AQP2 levels were unaltered. Thesefindings were confirmed by immunocytochemistry. In kidneys fromsham-operated controls, immunocytochemistry showed that p-AQP2 antibodylabeled the apical plasma membrane domains of collecting duct principal cells in the inner medulla (Fig. 6 E ) and inner stripe of theouter medulla (Fig. 6 F ). In obstructed kidneys, p-AQP2labeling was much weaker in the apical plasma membrane domains ofcollecting duct principle cells in the inner medulla (Fig. 6 A ) and inner stripe of the outer medulla (Fig. 6 B ) compared with sham-operated controls. In contrast, innonobstructed kidneys, the labeling density of p-AQP2 was unchangedcompared with sham-operated rats (Fig. 6, C and D ). Thus these findings indicate a decreased abundance ofAQP2 and p-AQP2 in the collecting ducts as important elements in theimpaired urinary concentrating capacity., z2 n0 l0 h& v' y' X
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Fig. 5. Semiquantitative immunoblotting of membrane fractions of IM fromUUO 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 ratswith 24-h UUO and sham-operated controls revealed a marked decrease inthe obstructed kidney: 23 ± 5% of sham levels (100 ± 15%,* P D : densitometric analysisrevealed that p-AQP2 expression did not differ between nonobstructedkidneys 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 UUOand sham-operated controls
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Table 6. Expression of aquaporins in the nonobstructed kidneys in rats with24-h UUO and sham-operated controls# ?  N) C! N- e& H8 z

Fig. 6. Immunocytochemical localization of p-AQP2 in IM and inner stripe ofouter medulla of Wistar rat kidney. A and B : inthe 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 ductprincipal cells. C and D : in the nonobstructedkidney of UUO rats, abundant labeling of p-AQP2 is seen in the apicaland subapical domains of the principal cells of IMCD ( C ) andISOM ( D ). E and F : in thesham-operated animals, abundant labeling of p-AQP2 is seen at apicalplasma membrane and subapical domains of IMCD ( E ) and ISOMprincipal cells ( F ). Magnification: ×1,100./ s- x6 }# q. n$ v/ V4 n, g

Reduced AQP3 Expression in Obstructed Kidneys

We examined the expression of AQP3 located to the basolateralmembrane of the principal cells in the collecting ducts from rats withUUO using immunoblotting and immunocytochemical analysis. Immunoblotting demonstrated that the abundance of AQP3 in the innermedulla of obstructed kidneys was dramatically reduced: 19 ± 6 vs. 100 ± 12% in controls ( P 7, A and B; Table 5 ).Consistent with this, immunocytochemistry demonstrated a much weakerlabeling of AQP3 in the basolateral membrane domains of the principalcells in the IMCDs of obstructed kidneys (Fig. 8 A ). However, in nonobstructedkidneys AQP3 expression was unchanged compared with sham-operatedcontrols (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 wasunchanged (Fig. 8 B ) compared with sham-operated controls(Fig. 8 C ).
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Fig. 7. Semiquantitative immunoblotting of membrane fractions of IM fromUUO and sham-operated Wistar rats. A and C :immunoblots reacted with affinity-purified anti-AQP3 antibody andrevealed 27- and 33- to 40-kDa bands. B : densitometricanalysis of all samples from obstructed kidneys in rats with 24-h UUOand sham-operated controls revealed a marked reduction in AQP3expression: 19 ± 6% of sham levels (100 ± 12%,* P D : densitometric analysisrevealed that AQP3 expression did not differ between nonobstructedkidneys and sham-operated controls.
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Fig. 8. Immunocytochemical localization of AQP3 in IM of Wistarrat kidney. A : in the obstructed kidney of UUO rats, reducedlabeling of AQP3 (arrows) is detected at the basolateral membranedomains of collecting duct principal cell but is much weaker comparedwith that seen in the nonobstructed kidneys ( B ) and kidneysfrom sham-operated control rats ( C ). C : insham-operated control rats, AQP3 labeling is detected at thebasolateral domains of collecting duct principal cells similar to thatfound in the nonobstructed kidney. Magnification:×1,100.: G8 i9 H4 [% W: I  h/ Z

Vasopressin-Independent Regulation of AQP2 and AQP3 in Obstructedand Nonobstructed Kidneys, _) m; s: f* Y4 f8 k7 M: h( r
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To examine the role of vasopressin-independent regulation of AQP2and -3 in UUO, immunoblotting was performed on kidneys from BB ratswith 24-h UUO. In obstructed kidneys, immunoblotting revealed adecreased expression of AQP2 to 55 ± 7% of sham levels, p-AQP2 to 17 ± 7% of sham levels, and AQP3 to 60 ± 10% of shamlevels (Fig. 9; Table 7 ). Moreover, incontralateral nonobstructed kidneys therewas a decreased expression of p-AQP2 to 58 ± 16% ofsham levels, whereas the expression of AQP3 increased moderately to 142 ± 10% of sham levels, P 9;Table 7 ).! O& X! z) l: P

Fig. 9. Semiquantitative immunoblotting of membrane fractions of totalkidney from the obstructed kidney and nonobstructed kidney in UUO andsham-operated BB rats. A and B : immunoblotsreacted with anti-AQP2 antibody ( A and B ), p-AQP2antibody ( C and D ), anti-AQP3 antibody( E and F ), and anti-AQP1 antibody ( G and H ). (See Table 7 for densitometric analysis).* P1 k# U  b: g7 t1 A/ E

Table 7. Expression of aquaporins in Brattleboro rats in response to 24 hof UUO
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Reduced AQP4 Expression in Obstructed Kidneys+ w3 C9 ~2 u! ]

Immunoblotting demonstrated that AQP4 expression in the innermedulla of obstructed kidneys was dramatically reduced (11 ± 5 vs. 100 ± 38%, P 10, A and B; Table 5 ). However, innonobstructed kidneys AQP4 expression was unchanged compared withsham-operated controls (152 ± 32 vs. 100 ± 24%; Fig. 10, C and D; Table 6 ). AQP4 is expressed in thebasolateral membrane of renal collecting duct principal cells andis involved in water reabsorption. Immunocytochemical analysisdemonstrated that in sham-operated rats, anti-AQP4 antibody labeled thebasolateral plasma membrane domains of collecting duct principal cellsin the inner medulla (Fig. 11 E ) and inner stripe of theouter medulla (Fig. 11 F ). In obstructed kidneys,immunocytochemistry showed that the labeling of AQP4 in the IMCD (Fig. 11 A ) and in the inner stripe of outer medullary collectingducts was much weaker (Fig. 11 B ) compared with the similarrenal 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 fromUUO and sham-operated Wistar rats. A and C :immunoblots reacted with affinity-purified anti-AQP4 antibody. B : densitometric analysis of all samples from obstructedkidneys in rats with 24-h UUO and sham-operated controls revealed amarked reduction in AQP4 expression: 11 ± 5% of sham levels(100 ± 38%, * P D :densitometric analysis revealed that AQP4 expression did not differbetween nonobstructed kidneys and sham-operated controls.

Fig. 11. Immunocytochemical localization of AQP4 in IMCD and ISOM of Wistarrat kidney. A and B : in the obstructed kidney ofUUO rats, reduced labeling of AQP4 (arrows) in principal cells of IMCD( A ) and of ISOM collecting duct ( B ) is confinedto 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 adjacentcytoplasmic domains of the principal cells of IMCD ( C ) andISOM collecting duct ( D ). E and F : inthe sham-operated animals, abundant labeling of AQP4 is seen in thebasolateral and adjacent cytoplasmic domains of the principal cells ofIMCD ( E ) and inner stripe of outer medullary collecting duct( F ). Magnification: ×1,100.
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UUO Is Associated With Reduced AQP1 Expression in Both Obstructedand Nonobstructed Kidneys
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The abundance of AQP1 protein was markedly decreased in obstructedkidneys 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 theouter medulla and cortex (77 ± 8 vs. 100 ± 5%; Fig. 13, C and D; Table 6 ). Thus AQP1 located in theproximal tubule and in the descending thin limb of Henle's loop mayplay a role in the water balance disorders associated with obstructivenephropathy. To examine whether there were any changes in the segmentalor subcellular distribution of AQP1 in the proximal tubule anddescending thin limb, immunocytochemical analysis was performed (notshown). The results demonstrate that there is heterogenous labeling butno overall change in the labeling intensity, as expected with areduction of ~30% in expression determined by semiquantitativeimmunoblotting. Moreover, there is no change in the segmentaldistribution of AQP1. The labeling was found in segments1-3 in the proximal tubule as well as in outer medullary andinner medullary segments of the descending thin limb and vasa recta.AQP1 was confined to apical and basolateral plasma membrane domains inboth obstructed, nonobstructed, and control kidneys. Thus the combinedresults suggest that there is uniform downregulation of AQP1 with nosegmental or subcellular change.- ?4 q8 f* U. Q
: x/ u" C( v$ B/ _' N# A. b" M
Fig. 12. Semiquantitative immunoblotting of membrane fractions of IM fromUUO and sham-operated Wistar rats. A and C :immunoblots are reacted with affinity-purified anti-AQP1 antibody andrevealed 29- and 35- to 50-kDa AQP1 bands. B : densitometricanalysis from obstructed kidneys in rats with 24-h UUO revealed asignificant decrease in AQP1 levels: 64 ± 8% of sham levels(100 ± 13%, * P D : in thenonobstructed kidneys in rats with 24-h UUO, AQP1 expression deceasedmarkedly to 77 ± 3% of sham levels (100 ± 7%,* P
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Fig. 13. Immunoblot of membrane fractions of outer medulla and cortex fromUUO and sham-operated Wistar rats. A and C :immunoblots reacted with affinity-purified anti-AQP1 antibody. B : densitometric analysis from obstructed kidneys in ratswith 24-h UUO revealed a moderate but significant decrease in AQP1levels: 77 ± 3% of sham levels (100 ± 7%,* P D : in nonobstructed kidneys inrats with 24-h UUO, AQP1 expression deceased significantly to 77 ± 8% of sham levels (100 ± 5%, * P

Similar to the findings in Münich-Wistar rats, the abundance ofAQP1 in BB rats was reduced in both obstructed (62 ± 4 vs. 100 ± 6%, P 100 ± 2%, P 9; Table 7 ).8 ^7 }+ G3 }6 Y  o& i; h
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DISCUSSION" }8 w( w8 z' ]& A. E
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In the present study, UUO was associated with downregulation ofAQP2, p-AQP2 [phosphorylated in the PKA phosphorylation consensus site(Ser 256 ) of AQP2], AQP3, and AQP4 in collecting ductprincipal cells. Moreover, the expression of AQP1 in whole kidneys wassignificantly downregulated in both obstructed and nonobstructedkidneys. In parallel, urinary concentrating capacity was impaired inthe 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 BBrats with low levels of circulating vasopressin. In obstructed kidneys,AQP2, p-AQP2, and AQP3 were decreased, demonstrating avasopressin-independent downregulation. In obstructed and nonobstructed kidneys, a reduced level of AQP1 was a consistent finding. The resultsstrongly support the view that AQPs play important roles in the alteredregulation of water reabsorption associated with obstructivenephropathy and that vasopressin-independent mechanisms are involved inthe downregulation of AQP2 and -3.

Reduced Expression of AQP2 in Obstructed Kidneys
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Consistent with previous studies, semiquantitive immunoblotting ofobstructed kidneys of rats with 24 h UUO demonstrated that AQP2protein expression in the outer medullary and cortical collecting ductsas well as that in the IMCD is downregulated ( 11 ). In parallel, T c H 2 O was severely reduced inobstructed kidneys, demonstrating a functional association between AQP2downregulation and water reabsorption at the collecting duct level. Insupport of these findings, we demonstrated that p-AQP2 also decreasedmarkedly in obstructed kidneys. The absence of a more pronounceddownregulation of p-AQP2 than total AQP2 indicates that trafficking ofAQP2 is still present, consistent with previous reports ( 11, 12 ). This is also in accordance with previous immunocytochemicalstudies 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 apicalplasma membrane and in subapical vesicles of collecting duct principalcells in normal rats and that p-AQP2 is subjected to trafficking to theapical plasma membrane in response to vasopressin treatment( 3 ). The decreased expression of AQP2 and p-AQP2 in thepresent study indicates that both the total abundance of AQP2 and thephosphorylated fraction of AQP2 are reduced in collecting ductprincipal cells. This may play an important role in the impairment ofwater reabsorption during obstruction.

The mechanisms responsible for the downregulation of AQP2 and p-AQP2after ureteral obstruction remain unclear. The urinary concentratingprocess depends on the coordinated function of the loop of Henle andthe collecting duct. The thick ascending limb of the loop of Henlepowers the countercurrent multiplier process responsible for generationof a corticomedullary osmotic gradient, whereas the collecting ducts,under the control of vasopressin, allow variable degrees of osmoticequilibration, resulting in a variable amount of excreted water and areciprocal relationship between urinary flow and urinary osmolality.

It is well established that AQP2 is regulated, both short term and longterm, by vasopressin ( 29, 30 ). However, mechanisms otherthan vasopressin also seem to be involved in long-term regulation ofAQP2 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 AQP2and AQP3 in rats with BUO up to 14 days after release of theobstruction ( 12, 15, 21 ), suggesting thatvasopressin-independent signal pathways may be involved in theregulation of AQP2 in obstructive nephropathy. The results of thepresent study showed that AQP2 and p-AQP2 levels were markedlydecreased in the obstructed kidneys of BB rats with UUO. Consistentwith previous studies demonstrating that vasopressin-independentpathways can modify AQP2 expression ( 8, 9, 38 ), thepresent findings demonstrate that vasopressin-independent pathways areinvolved in AQP2 (and AQP3; see below) dysregulation in response to UUO.. i6 o8 _2 P9 _3 o

The present study revealed unaltered expression levels of AQP2 andp-AQP2 in nonobstructed kidneys. This finding is consistent with theabsence of a major reduction in AQP2 abundance in nonobstructed kidneys, which we demonstrated previously ( 11 ). Themarkedly reduced abundance of p-AQP2 in nonobstructed kidneys of BBrats 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 inT c H 2 O in the nonobstructed kidneys of B ratscompared with sham-operated control rats. It should be emphasized thatthis may not be a general response in a nonobstructed kidney. Thus UUOmay be associated with unchanged or slightly reduced AQP2 abundance ina nonobstructed kidney.1 G1 O% X8 T) {& x0 y
/ \5 s8 y2 v7 i  B
Reduced Expression of AQP3 and AQP4 in Obstructed Kidneys' s  M3 S. E2 C0 U

AQP3 and AQP4 represent exit pathways of water through thebasolateral 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 onlypartially concentrated urine after water deprivation ( 22, 39 ). AQP4 is responsible for the majority of basolateralmembrane water movement in the IMCD, but AQP4 knockout mice onlydemonstrate a very mild defect in urinary concentrating ability( 2 ). The AQP3/AQP4 double-knockout mice had a greaterimpairment of urinary concentrating ability than did the AQP3single-knockout mice ( 22 ). Similar to the findings inWistar rats, AQP3 abundance was also reduced in BB rats, demonstratingthat vasopressin-independent mechanisms are involved in AQP3regulation. Thus our results demonstrated downregulation of AQP3 and -4 together with a parallel impairment of urine-concentrating capacityafter UUO, suggesting that downregulation of AQP3 and AQP4 may playimportant roles in urinary concentrating capacity.! n9 c2 ^3 n" E1 x
! e* y- f1 T9 ?9 ^9 s. @% \
Reduced AQP1 Expression in Both Obstructed and NonobstructedKidneys; V% {2 N* {& P9 w0 Z/ C: }% \6 T  ~

In the present studies, AQP1, expressed in the proximal tubule anddescending thin limb of Henle's loop, was moderately decreased in bothobstructed and nonobstructed kidneys compared with sham-operated controls. Recently, it was reported that AQP1 knockout mice have asevere urinary concentrating defect, decreased transepithelial waterpermeability in proximal tubule and descending thin limb of Henle'sloop, and defective fluid absorption, indicating that AQP1 plays avital role in the countercurrent multiplier mechanism by allowingefficient osmotic water equilibration ( 1, 23, 34 ). Changesin proximal tubule fluid transport may have significant effects on theurinary concentrating mechanism by altering flow rates of tubule fluiddelivery to the thick ascending limbs and collecting ducts. Therefore,impairment of proximal tubule function, demonstrated by a decline inAQP1 protein abundance, could contribute to the changes in urinary flow./ f  z2 X+ S6 }( ]

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

Ureteral occlusion induces a complex series of hormonal changes in theobstructed kidney ( 18 ), which may influence the functional changes in the contralateral kidney. Similarly, it has beendemonstrated that renorenal reflexes are also important for themodulation of urinary output in the contralateral kidney during UUO( 5 ). It is likely that several mechanisms are involved atthe same time and that the role of such mechanisms in the regulation of channels and transporters in the nonobstructed kidney are at present unknown.
) R/ T. f" Y) S$ R" J( {# U& c
The importance of local factors in the dysregulation of AQPs inresponse to obstruction was addressed by examining the effect of UUO onthe 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 tocompensate 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 kidneysand unchanged expression levels of AQP2, -3, and -4 in thenonobstructed kidneys of the same animals are consistent with thisview, which emphasizes that local, intrarenal factors play a major rolein the induction of downregulation. However, systemic factors alsoappear to be involved, because there was a significant decrease in AQP1protein levels in nonobstructed kidneys compared with sham-operatedcontrols in both Wistar and BB rats. Interestingly, a similar reductionin 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 caninduce renal failure. Because all the transporters we examined in the obstructed kidney are downregulated, it may be speculated thatobstruction induces a general downregulation of all tubular transportproteins. This will be addressed in future studies.
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Summary

The present study demonstrated that UUO in rats is associated withdownregulation of renal AQP2, p-AQP2, AQP3, AQP4, and AQP1, consistentwith an impaired urinary concentrating capacity in the obstructedkidney. The reduction of these AQPs in UUO rats may, at least in part,contribute to the impaired water metabolism and may reflect reductionsof net reabsorption of water at several nephron sites in the obstructedkidney, 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 contralateralkidney may contribute to the compensatory increase in urinary flow.5 k2 L2 @5 ^4 C9 A" p5 D& l
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ACKNOWLEDGEMENTS8 P  E& |( B0 Z5 {; f) q- l6 P

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.- F& E* |+ c8 W8 R4 N
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