Indomethacin Enhances Shuttling of Aquaporin-2 Despite Decreased Abundance in Rat Kidney
Soo Wan Kim*,
Joon Wan Kim,
Ki Chul Choi*,
Seong Kwon Ma*,
YoonWha Oh,
Ju-Young Jung,
Jin Kim and
JongUn Lee
Departments of *Internal Medicine and Physiology, Chonnam National University Medical School, and Department of Anatomy, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
Correspondence to Dr. JongUn Lee, Department of Physiology, Chonnam National University Medical School, 5 Hak-dong, Gwangju 501-746, Korea. Phone: +82 62 220 4262; Fax: +82 62 232 1242; E-mail: julee{at}chonnam.ac.kr
The effect of nonsteroidal antiinflammatory drugs on the regulationof aquaporin-2 (AQP2) water channels in the kidney was determined.Male Sprague-Dawley rats were injected with indomethacin (5mg/kg twice a day intraperitoneally) for 2 d. The control groupwas injected with vehicle. The expression of AQP2 proteins wasdetermined in the kidney by immunoblotting and immunohistochemistry.The expression of Gs and type VI adenylyl cyclase was determinedby immunoblotting. The activity of adenylyl cyclase complexeswas determined by stimulated accumulation of cAMP. Immunoblottingrevealed that indomethacin markedly decreased the expressionof AQP2. Accordingly, however, the ratio of AQP2 expressionin the membrane fraction versus that in the cytoplasmic fractionwas increased. The urinary excretion of AQP2 proteins also increased.Immunohistochemistry demonstrated almost exclusive apical labelingof AQP2 with scanty cytoplasmic localization along the collectingduct. The expression of Gs and adenylyl cyclase VI proteinswas decreased. The generation of cAMP provoked by arginine vasopressin,sodium fluoride, or forskolin was blunted. These results suggestthat indomethacin increases the shuttling of AQP2 while it decreasesits abundance in the collecting duct.
It has long been known that inhibition of prostaglandin synthesiswith nonsteroidal antiinflammatory drugs (NSAID) may be complicatedby various functional alterations. Among others, indomethacinenhances the antidiuretic activity of arginine vasopressin (AVP)(1). It markedly reduces urine flow with no measurable changesof systemic BP, GFR, or effective renal plasma flow (2). However,mechanisms underlying the increased urinary concentrating abilityhave not been fully understood.
Over the last decade, it has been well known that epithelialwater transport occurs through aquaporin (AQP) channels. Amongits multiple isoforms, aquaporin-2 (AQP2) is mainly expressedin the collecting duct, which plays an important role in determiningthe final urinary output of water and solutes. It is regulatedby AVP/cAMP pathway in the short and long term to increase osmoticwater reabsorption in the principal cell (3–5). Moreover,alterations of its regulation have been implicated in variouspathophysiological states mainly associated with altered urinaryconcentrating ability, such as acute ischemic renal failure(6), urinary tract obstruction (7), gentamicin-induced nephropathy(8), chronic renal failure induced by surgical renal mass reduction(9), and cisplatin-induced nephropathy (10).
It has been also demonstrated that acute oral intake of ibuprofenincreases the urinary excretion of AQP2 in healthy humans (11).In this context, an altered regulation of AQP2 may be causallyrelated with the altered urinary concentration ability associatedwith the use of NSAID. The study presented here sought to examinethe effects of NSAID on the regulation of AQP2 channels in thekidney. Rats were treated with indomethacin, and their expressionof AQP2 was determined in the kidney by Western blot analysisand immunohistochemistry. To specify the point of impairmentin AVP/cAMP pathway, the activity and expression of adenylylcyclase complexes were also determined.
Animals
Male Sprague-Dawley rats weighing 200 to 250 g were used. Ratswere given indomethacin (2.5 to 5.0 mg/kg dissolved in 10 mMNa2CO3, every 12 h intraperitoneally) for 2 d. The control groupwas injected with the solvent, i.e., Na2CO3. During the experiment,the rat was kept in a metabolic cage. The amount of food inthe control group was adjusted according to that consumed inthe experimental group. Another experimental group was injectedwith diclofenac (100 mg/kg dissolved in saline, single dose,intraperitoneally), and kept for 2 d. Its control group wasinjected with saline. The whole experimental procedure conformedto the Institutional Guidelines of Experimental Animal Careand Use.
Renal Functional Data and Plasma AVP Level
On the experimental day, the rats were decapitated in a consciousstate to collect the trunk blood. Serum levels of creatinineand osmolality were determined. The plasma AVP concentrationwas measured by means of a commercial RIA kit (Incstar, Stillwater,MN).
Urinary AQP2 excretion
To determine the urinary excretion of AQP2, the urine was collectedin 15 ml of ice-cold 1 M Tris-Cl (pH 6.8) containing 1 mg/mlleupeptin, 1 mM sodium azide, and 0.1 mg/ml phenylmethyl sulfonylfluoride (PMSF). The debris was removed by centrifugation at1000 x g for 5 min. The supernatant was centrifuged again at100,000 x g for 1 h at 4°C, and the resultant pellet wassuspended with 30 µl of isolation buffer (0.3 M sucrose,25 mM imidazole, 1 mM EDTA, 8.5 µM leupeptin, 1 mM PMSF,pH 7.2). The sample was stabilized by heating at 95°C for5 min in sample buffer (4% SDS, 20% glycerol, 0.125 M Tris [pH6.8], 0.2% bromophenol blue, 10% 2-mercaptoethanol) and thensubjected to AQP2 determination.
Differential Centrifugation
Differential centrifugation of the kidney was carried out asdescribed by previous investigators (12). Inner medullary homogenateswere initially centrifuged at 4000 x g for 10 min at 4°Cto remove incompletely homogenized fragments and nuclei. Thepellets were resuspended in ice-cold isolation solution withprotease inhibitors and centrifuged again at 4000 x g for 10min. The supernatants were collected and centrifuged at 17,000x g for 20 min. The pellets were retained (high-density fraction,HD), and the supernatants were then pelleted by centrifugationat 200,000 x g for 1 h (low-density fraction, LD). The expressionof AQP2 was then determined in HD and LD fractions. The shuttlingwas noted by the ratio of AQP expression in HD fraction overthat in LD fraction. An increase of HD/LD represents an enhancedshuttling.
Western Blot Analysis
Amounts of AQP2 in the kidney and the urine were determinedby Western blot analysis as described previously (7). Antibodiesused were rabbit polyclonal AQP2 (Alomone Lab; Jerusalem, Israel),Gs (Calbiochem-Novabiochem; San Diego, CA), and type VI adenylylcyclase antibodies (Santa Cruz Biochemicals; Santa Cruz, CA).Heat-shock protein (HSP) 25 and HSP70 antibodies were purchasedfrom StressGen (Victoria, BC, Canada). The bound antibody wasdetected by enhanced chemiluminescence (Amersham; Little Chalfont,Buckinghamshire, UK) on hyperfilm. Relative protein levels weredetermined by analyzing the signals of autoradiograms with atransmitter-scanning videodensitometer.
Immunohistochemistry
Kidneys were fixed in rats under anesthesia with pentobarbitalsodium (50 mg/kg, intraperitoneally) by in vivo perfusion throughthe abdominal aorta with 2% paraformaldehyde-lysine-periodatein PBS, pH 7.4. They were removed and additionally fixed byimmersion in the same fixative overnight at 4°C. Fifty-µmsections were made and processed via a horseradish peroxidasepreembedding technique. Tissues were dehydrated and embeddedin wax (polyethylene glycol 400 disterate; Polysciences, Warrington,PA). Four 4- to 6-µm-thick wax sections were preparedfor postembedding immunohistochemistry.
Before Embedding
Vibratome sections were washed three times with 50 mM NH4Clin PBS for 15 min. The sections were incubated for 3 h withPBS containing 1% BSA, 0.05% saponin, and 0.2% gelatin (solutionA), and incubated with primary antibodies. They were then incubatedovernight at 4°C in a solution of AQP2 antibodies (1:500)in PBS containing 1% bovine serum albumin (solution B). Afterseveral washes with solution A, the tissue sections were incubatedfor 2 h in peroxidase-conjugated donkey anti-rabbit IgG, Fabfragment (Jackson ImmunoResearch Lab, West Grove, PA; 1:100diluted in solution B). The sections were then rinsed, firstin solution A and subsequently in 0.05 M tris(hydroxymethyl)-aminomethane(Tris) buffer (pH 7.6). For the detection of horseradish peroxidase,the sections were incubated in 0.1% 3,3'-diaminobenzidine (browncolor) in 0.05 M Tris buffer for 5 min, after which H2O2 wasadded to a final concentration of 0.01%, and the incubationwas continued for 10 min. After washing with 0.05 M Tris bufferthree times, the sections were dehydrated in a graded seriesof ethanol and embedded in Epon 812 resin, and photographed.
After Embedding
Wax sections were processed for immunohistochemistry with theavidin-biotin-horseradish peroxidase technique (Elite VectastainABC kit; Vector Laboratories; Burlington, CA). The sectionswere dewaxed with xylene and ethanol. After rinsing in tap water,they were treated with methanolic H2O2 for 30 min to inhibitthe intrinsic peroxidase. They were then permeabilized by incubationin 0.5% Triton X-100 in PBS for 15 min, and subsequently blockedwith normal goat serum (1:10 diluted in PBS) for 15 min, andincubated with rabbit polyclonal AQP2 antibodies (1:500) overnightat 4°C. After rinsing in PBS, the sections were incubatedwith biotinylated goat anti-rabbit antibodies for 1 h at roomtemperature. After further washing, the sections were incubatedwith 0.3% avidin-biotin-peroxidase complex for 1 h at room temperature.The site of the antigen-antibody reaction was visualized by3,3'-diaminobenzidine tetrahydrochloride reaction. After dehydrationwith increasing concentrations of ethanol, the sections werecovered with glass slips and photographed.
Membrane Preparation and Adenylyl Cyclase Activity
The membrane preparation was obtained as described previously(6). Adenylyl cyclase activity was assayed by the method ofBar with a slight modification (13). AVP was used to activateV2 receptors, sodium fluoride was to stimulate adenylyl cyclasein a receptor-independent but G protein–dependent manner,and forskolin was used to directly stimulate the catalytic unitof the complex. cAMP was measured in the supernatant by equilibratedRIA. Iodinated 2'-O-monosuccinyl-adenosine 3',5'-cyclic monophosphatetyrosyl methyl ester was prepared as described by previous investigators(14). All samples in one experiment were analyzed in a singleassay. Nonspecific binding was <2.0%. The 50% intercept wasat 16.5 ± 0.8 fmol/tube (n = 10). The intra- and interassaycoefficients of variation were 5.0 ± 1.2% (n = 10) and9.6 ± 1.9% (n = 10), respectively. Results were expressedas moles of cAMP generated per milligram protein per minute.
Drugs were purchased from Sigma Chemical Company (St. Louis,MO, USA), unless stated otherwise.
Statistical Analyses
Results are expressed as mean ± SEM. The statisticalsignificance of differences between the groups was determinedby unpaired t test.
Water Balance and Other Functional Parameters Table 1 shows the functional data. There were no significantdifferences in the food and water intake between the groups.In the experimental group treated with indomethacin (5 mg/kg),the urinary flow rate significantly decreased in associationwith a positive water balance. However, there were no significantchanges in the plasma clearance of creatinine. Neither the plasmalevel of AVP nor the serum osmolality was significantly affected.
Expression of AQP2 Proteins
The expression of AQP2 proteins was determined in differentsegments of the kidney. The anti-AQP2 antibody recognized 29-kDand 35- to 50-kD bands, corresponding to nonglycosylated andglycosylated AQP2, respectively. After the treatment with indomethacin(5 mg/kg), the expression was significantly decreased in thecortex, outer medulla, and inner medulla (Figure 1). Despitethe changes in AQP proteins, there were no significant differencesin the expression of HSP25 and HSP70 between the control andexperimental groups (Figure 2). The expression of AQP2 in themembrane fraction was preserved while that in the cytoplasmicfraction markedly decreased, so that HD/LD increased significantly(Figure 3). The urinary excretion of AQP2 was slightly increased(Figure 4). A lower dose of indomethacin (2.5 mg/kg) and diclofenac(100 mg/kg, single dose) similarly decreased the expressionof AQP2 in the kidney (Figure 5).
Figure 1. Representative immunoblots and densitometric analysis of aquaporin-2 (AQP2) in the cortex (C), outer medulla (OM), and inner medulla (IM). Open columns, control; hatched columns, indomethacin treated (5 mg/kg). Mean ± SEM of six rats each. *P < 0.05, **P < 0.01, ***P < 0.001 versus control.
Figure 4. Immunoblots and densitometric analysis of aquaporin-2 (AQP2) in the inner medulla after the treatment with a lower dose indomethacin (A) and diclofenac (B). Open columns, control; hatched columns, indomethacin (2.5 mg/kg) or diclofenac treated (5 mg/kg). Mean ± SEM of six rats each. ***P < 0.001 versus control.
Figure 5. Abundances of aquaporin-2 (AQP2) in the high-density (HD) and low-density (LD) fractions of inner medullary extracts. Legend as in Figure 1.
Immunohistochemistry Figures 6 to 9 show light micrographs of kidney sections fromcontrol and indomethacin-treated rats. The AQP2 immunoreactivitywas present on the apical membrane and throughout the cytoplasmalong the entire collecting duct as well as in the connectingtubule (CNT). The treatment with indomethacin consistently decreasedthe AQP2 immunoreactivity, being most prominent in the innermedullary collecting duct (Figure 6). Moreover, almost exclusiveapical labeling with scanty cytoplasmic localization was noted(Figures 7 and 8). In CNT, the immunoreactivity was predominantlylabeled on the basolateral membrane (Figure 9).
Figure 6. Light micrographs illustrating immunostaining of aquaporin-2 (AQP2) of 50-µm-thick sections of kidneys of control (A) and indomethacin-treated (5 mg/kg) experimental (B) rats. Immunolabeling is present along the entire collecting duct from cortex to inner medulla as well as in connecting tubule in both groups. Original magnification, x16.
Figure 7. Aquaporin-2 (AQP2) localization in the cortical collecting duct. (A) AQP2 labeling is exclusively shown in the principal cell, both in the apical region and throughout the cytoplasm in the control. (B) Almost exclusive apical labeling with scanty cytoplasmic localization is noted in the indomethacin-treated tissue. Double small arrows, apical labeling of AQP2; arrowheads, AQP2-negative intercalated cells. Original magnification, x700.
Figure 8. Aquaporin-2 (AQP2) localization in the outer (A and B) and inner (C and D) medullary collecting duct. (A and C) AQP2 immunolabeling is seen in both apical membrane and supranuclear region in the principal cell of outer and inner medullary collecting duct in the control. (B and D) AQP2 labeling is seen mainly on the apical plasma membrane (double small arrows) with scanty cytoplasmic localization in the indomethacin-treated tissue. Arrowheads, AQP2-negative intercalated cells. Original magnification, x700.
Figure 9. Aquaporin-2 (AQP2) localization in the connecting tubule. (A) AQP2 labeling is seen predominantly on the apical membrane, and weakly on the basolateral membrane in the control. (B) More intense labeling in the basolateral region in the indomethacin-treated tissue. Double small arrows, basolateral AQP2 labeling; arrowheads, AQP2-negative intercalated cells. Original magnification, x700.
Expression of Gs and Adenylyl Cyclase VI and Activities of Adenylyl Cyclase Figure 10 shows immunoblots of Gs and adenylyl cyclase VI inthe inner medulla. The anti-Gs antibody recognized a doubletat 50 and 45 kD. The Gs expression was significantly decreasedafter the indomethacin treatment. The expression of type VIadenylyl cyclase, recognized as a broad band at approximately160 kD, was significantly decreased. The generation of cAMPevoked by AVP, forskolin, or sodium fluoride also decreasedsignificantly (Figure 11).
Figure 11. cAMP production in response to arginine vasopressin (AVP), sodium fluoride, and forskolin in the inner medulla. Open circles, control; solid circles, indomethacin treated (5 mg/kg). Each point represents mean ± SEM of six experiments.
The treatment with NSAID has been known to markedly reduce theurinary flow with no measurable changes of systemic BP, GFR,or effective renal plasma flow (2). It was also demonstratedin the study presented here that indomethacin did not significantlyalter the plasma creatinine clearance. However, there was adecreased urinary excretion of sodium. These changes may inpart be related with an altered regulation of natriuretic hormonesin the kidney (15), accounting for the extracellular volumeexpansion associated with the use of NSAID (16,17). However,there have been no documents related with regulation of waterchannels, although an altered sodium handling may result inan altered water handling.
In the study presented here, there was a decrease in the abundanceof AQP2 after the treatment with indomethacin, as shown by immunoblottingand immunohistochemistry. Furthermore, indomethacin diminishedthe generation of cAMP in response to forskolin and that tosodium fluoride in the kidney, along with decreased expressionof Gs and adenylyl cyclase VI. Therefore, not only Gs proteinsbut also the catalytic unit of adenylyl cyclase may have beenfunctionally impaired by indomethacin, to which the dysregulationof AQP2 channels may in part be attributable.
Despite the decrease of its total abundance, however, the shuttlingof AQP2 was rather increased. Immunohistochemistry also demonstratedthat the residuum of AQP2 immunoreactivity along with scantycytoplasmic localization was chiefly seen in the apical regionof the principal cell in the collecting duct. The urinary excretionof AQP2 was also slightly increased, being consistent with anincreased expression in the apical plasma membrane.
An increased shuttling of AQP2 associated with a decrease ofits total abundance is seemingly contradictory. However, theurinary excretion of AQP2 does not necessarily correlate withtotal AQP2 levels in the kidney (18), but closely follows conditionsknown to change AQP2 in the apical membrane (19,20). Therefore,the increase of its urinary excretion may represent an enhancedshuttling. Furthermore, the enhanced AQP2 shuttling was apparentlytranslated into a diminished urine volume. The positive waterbalance may be a reflection of decreased urinary excretion ofsodium and concomitantly enhanced shuttling of water channels.
It has been found that PGE2 does not affect AQP2 phosphorylation,but increases the retrieval of AQP2 from the membrane fractionto the intracellular fraction, thereby counteracting the actionof AVP (21). Conversely, Pedersen et al. (11) observed thatinhibition of prostaglandin synthesis by ibuprofen in humansresults in increases of urinary AQP2 excretion. A diminishedproduction of PGE2 by NSAID would thus lead to a reduction ofAQP2 endocytosis, maintaining the apical distribution of AQP2.
It has been known that both short-term and long-term regulationof AQP2 is exerted by AVP/cAMP pathway (3,5). However, the dissociationof short-term and long-term regulation of AQP2 suggests thatthe regulatory mechanisms may differ between them. Indomethacinmay differentially affect cAMP-dependent and -independent intracellularsignaling mechanisms. It is also likely that the short-termregulation of water permeability may be more complex than generallyrecognized and may involve regulation of both exocytosis andendocytosis of AQP2, possibly via multiple signaling pathways(22,23). In addition, the plasma level of AVP was not significantlyaltered by the treatment with indomethacin in our study. Therefore,the altered regulation of AQP2 cannot be attributed to changesin AVP levels, but to a direct effect of indomethacin on theadenylyl cyclase complex. Mechanisms underlying the dissociationshould be further studied.
It may be of interest to note that indomethacin caused differentialchanges in AQP2 targeting in different portions of the tubule:an exclusive apical labeling with scanty cytoplasmic localizationwas noted in the collecting duct while an enhanced labelingwas shown in the basolateral domain in CNT. It is not clearwhether basolaterally located AQP2 acts as a novel exit of waterin CNT, although AQP3 and AQP4 have been considered as mainexit in the basolateral domain of the collecting duct (24,25).The regulation of AQP2 may differ in different portions of thetubule.
Finally, our study also demonstrated that indomethacin in alower dose or diclofenac, another NSAID, similarly decreasedthe expression of AQP2 channels. In addition, there were nosignificant differences in the expression of HSP25 and HSP70between the control and experimental groups. The HSP may beinduced by exposure to heat or other stresses and play a protectiverole in minimizing the damaging effect of such stresses. A clearprotective effect of over-expression of HSP70 has been demonstratedagainst subsequent challenge with hypoxia (26,27). The diminishedexpression of AQP channels cannot be ascribed to a nonspecificeffect of indomethacin per se.
In summary, indomethacin markedly reduced the expression ofAQP2 water channels in the collecting duct along the activityof AVP/cAMP pathway. Accordingly, however, the shuttling ofAQP2 was rather enhanced. The altered regulation of AQP2 mayin part be causally related with the altered urinary concentratingability and body water balance associated with the use of NSAID.
Acknowledgments
This work was supported by research grants from Korean Societyof Nephrology (2003) and Chonnam National University MedicalResearch Center for Gene Regulation (R13-2002-013-01003-0).
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Received for publication October 7, 2003.
Accepted for publication September 1, 2004.
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Abstract
Introduction
and type VI adenylyl cyclase was determined by immunoblotting. The activity of adenylyl cyclase complexes was determined by stimulated accumulation of cAMP. Immunoblotting revealed that indomethacin markedly decreased the expression of AQP2. Accordingly, however, the ratio of AQP2 expression in the membrane fraction versus that in the cytoplasmic fraction was increased. The urinary excretion of AQP2 proteins also increased. Immunohistochemistry demonstrated almost exclusive apical labeling of AQP2 with scanty cytoplasmic localization along the collecting duct. The expression of Gs
