cDNA array identification of genes regulated in rat renalmedulla in response to - 《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 13:24 |只看该作者 |倒序浏览 |打印

作者：Heddwen L.Brooks, ShanaAgeloff, Tae-HwanKwon, WilliamBrandt, James M.Terris, AkhilSeth, LuisMichea, SørenNielsen, RobertFenton,  Mark A.Knepper作者单位：1 Laboratory of Kidney and Electrolyte Metabolism, NationalHeart, Lung, and Blood Institute, National Institutes of Health,Bethesda, Maryland 20892; and Department of Cell Biology,Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark

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      【摘要】2 k3 z+ W2 r( Q' l
   With the aim of identifyingpossible gene targets for direct or indirect regulation by vasopressinin the renal medulla, we have carried out cDNA array experiments ininner medullas of Brattleboro rats infused with the V 2 receptor-selective vasopressin analog desamino-Cys1, D -Arg8vasopressin (dDAVP) for 72 h. Of the 1,176 genes on the array, 137 transcripts were increased by 2-fold or more, and 10 transcripts weredecreased to 0.5-fold or less. Quantitative, real-time RT-PCRmeasurements confirmed increases seen for six selected transcripts(Wilms' tumor protein, -arrestin 2, neurofibromin, casein kinaseII, aquaporin-3, and aquaporin-4). To correlate changes in mRNAexpression with changes in protein expression, we carried outquantitative immunoblotting for 28 of the proteins whose cDNAs wereon the array. For several targets including aquaporin-2, transcriptabundance and protein abundance changes did not correlate. However, formost genes examined, changes in mRNA abundances were associated withconcomitant protein abundance changes. Targets with demonstratedincreases in both protein and mRNA abundances included neurofibromin,casein kinase II, the -subunit of the epithelial Na channel( -ENaC), 11 -hydroxysteroid dehydrogenase type 2, and c-Fos.Additional cDNA arrays revealed that several transcripts that wereincreased in abundance after 72 h of dDAVP were also increasedafter 4 h, including casein kinase II, -ENaC, aquaporin-3,UT-A, and syntaxin 2. These studies have identified several transcriptswhose abundances are regulated in the inner medulla in response toinfusion of dDAVP and that could play roles in the regulation of saltand water excretion. ' f7 d9 u7 T; }  o0 o& G  [
      【关键词】 aldosterone kidney sodium epithelia
   INTRODUCTION$ _$ h! E! R# W  x: d  `, Y, T

VASOPRESSIN IS A PEPTIDE HORMONE that controls systemic osmolality throughregulation of renal water excretion. Its main site of action in thekidney is the collecting duct, where it regulates the transport ofwater, urea, and Na ( 25 ). In collecting ductprincipal cells, vasopressin binds to a G s -coupled receptor(the V 2 receptor), which stimulates an increase inintracellular cAMP content via adenylyl cyclase. Binding of vasopressinto the V 2 receptor is also associated with intracellular calcium mobilization mediated by calcium release fromryanodine-sensitive intracellular stores via the type I ryanodinereceptor, which triggers calmodulin-dependent regulatory processeswithin the cell ( 6 ). Many of the actions of vasopressin inthe collecting duct are short-term responses that do not involveactivation of gene transcription, such as stimulation of aquaporin-2trafficking to the apical plasma membrane ( 35 ) andactivation of the urea transporter UT-A1 through phosphorylation( 58 ). However, vasopressin has clear-cut long-term actionsto alter the abundance of aquaporin-2 ( 8 ), aquaporin-3( 51 ), and the epithelial Na channel (ENaC) - and -subunits ( 11 ). These long-term actions are thought tobe associated with regulatory processes at a transcriptional level,involving either the transporter genes themselves or regulatory molecules that indirectly alter transporter protein abundance. Inaddition, in renal medulla, vasopressin may have indirect effects, owing to altered interstitial osmolality, urea concentration, or ionic strength.* w: C* Q% |# E7 _# Y  B$ {

Here, we have carried out cDNA array experiments with the aim ofidentifying possible new direct or indirect gene targets forvasopressin action in renal inner medulla. For this, we have examinedlevels of 1,176 transcripts after infusion of the V 2 receptor-selective vasopressin analog desamino-Cys1, D -Arg8vasopressin (dDAVP) into Brattleboro rats, which lack endogenouslycirculating vasopressin.; g" f: V- H4 M% i4 ^

METHODS

Brattleboro rat model. Male Brattleboro rats (180-230 g) were obtained fromHarlan-Sprague Dawley (Indianapolis, IN) and maintained in atemperature- and humidity-controlled room with a 12:12-h light-darkcycle (approved ACUC protocol 9-KE-5). All animals were given freeaccess to tap water and regular pelleted rat chow during theexperiments. Under light anesthesia (isofluorane), osmotic minipumps(model 2001, Alza, Palo Alto, CA) were implanted subcutaneously in therats to deliver 5 ng/h of the V 2 -selective vasopressinanalog dDAVP (Rhone-Poulenc Rorer, Collegeville, PA). Control ratsreceived osmotic minipumps loaded with isotonic saline. After dDAVPadministration for time periods designated below, rats were killed andthe inner medullas were isolated for RNA extraction, or cortices andinner medullas were isolated for protein analysis. In some experiments, serum was collected for determination of the aldosterone concentration by radioimmunoassay (Coat-A-Count, Diagnostic Products, Los Angeles, CA).1 A, j" b3 n! e' S8 b& v* F1 w" ]
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RNA isolation. Total RNA from rat inner medullas was isolated using Qiagen RNAeasycolumns (74104, Qiagen, Valencia, CA) according to the manufacturer'sdirections. Inner medullary tissue was initially homogenized inthe manufacturer's buffer solution, using an RNAase-free sawtooth tissue homogenizer (Omni 2000). Homogenates were then passedthrough the QiaShredder column (79654, Qiagen). RNA was treated withDNAse while bound to the RNAeasy column. Total RNA concentration wasmeasured by spectrophotometry and run on agarose gels to assess RNA quality.& [- b6 I  z: u: H- g% q9 \- m

cDNA arrays. Full documentation of cDNA array procedures and results are presentedaccording to minimum information about a microarray experiment (MIAME)guidelines ( 4 ) in the SupplementalMaterials. 1 Briefly, Clontech rat 1.2 nylonfilter arrays (7854-1, Clontech Laboratories, Palo Alto, CA) wereused for cDNA array analysis according to the manufacturer'sinstructions. For each experiment, two filters are used, one forcontrol RNA samples and one for experimental RNA. Twenty-fivemicrograms of total RNA were used for each array. For the twoBrattleboro rat experiments (72- and 4-h dDAVP infusion), RNA sampleswere pooled from the inner medullas from 3 rats, with identical amountsadded from each sample (specifically, 8 µg from each inner medulla). 33 P was used for labeling in the reverse-transcriptionreactions, and filters were hybridized overnight at 50°C. Filterswere washed at a final stringency of 0.5% SDS, 0.1× SSC at 68°C.Images were captured as TIFF images using a PhosphorImager and analyzedusing the National Institutes of Health software program pSCAN( http://mscl.cit.nih.gov ). Results were normalized to the overallintensity of the individual filters. To do this, the normalizingvariable was total hybridization signal for the whole filter (for all1,176 spots), allowing the relative dot density to be calculated foreach individual gene.# m: R# ^8 i. h6 l/ Z* n
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Northern blotting. Northern blots were run to assess relative aquaporin-2, UT-A1, andUT-A2 mRNA abundances in total RNA samples from Brattleboro rat kidneyinner medullas and cortices. Aquaporin-2 Northern blots were labeledwith a digoxigenin-labeled aquaporin-2 cDNA probe as previouslydescribed ( 12 ). UT-A Northern blots were probed with 32 P-labeled cDNA probes corresponding to the entire lengthof the UT-A1 transcript ( 14 ).8 g6 j- V9 M% k" n. E9 h

Real-time RT-PCR. Quantitative, real-time RT-PCR was used to validate selected arrayresults as previously described ( 5, 40 ). DNase-treated (Ambion) total RNA (1 µg) from rat kidney inner medulla samples fromcontrol or dDAVP-infused rats (5 vs. 5) was reverse transcribed usingoligo-dT and Superscript II reverse transcriptase (Invitrogen) following the manufacturer's recommended protocol. RT-negative controls were performed to assess the presence of possible genomic contamination of RNA samples. PCR primers were designed to amplify targets between 80 and 150 bp in length, with minimal secondary structure. Sequences of specific primer pairs are listed in the Supplemental Materials. Real-time PCR was performed on an ABI Prism7900HT system, using 1 µl of a 1:100 dilution of the original RTreaction product, 18 pmol (each) of gene-specific primers, and theQuantitect SYBR green PCR kit (Qiagen) according to the manufacturer'sprotocol. Specificity of the amplified product was determined usingmelting curve analysis ( 5 ). Relative quantitation of geneexpression was determined using the comparative C T method, with validation experiments performed to determine that amplification efficiencies were equal between control and experimental groups ( 5 ) as outlined at http://docs.appliedbiosystems.com/pebiodocs/04303859. pdf. Allexperiments were repeated at least twice, on separate days, to validate results.
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Antibodies. The study utilized affinity-purified rabbit polyclonal antibodiesproduced in our laboratory recognizing UT-A1, UT-A2, -ENaC, -ENaC, -ENaC, synaptotagmin 5, syntaxin-2, syntaxin-3,syntaxin-4, vesicle-associated membrane protein (VAMP2), renin,aquaporin-1, aquaporin-2, aquaporin-3, and the Na-K-Cl cotransportertype 2 (NKCC2) ( 8, 13, 28-30, 36, 37, 51 ). Additionalantibodies were commercially obtained: mouse monoclonal antibodies toNa-K-ATPase 1 -subunit (05-369, UpstateBiotechnology, Lake Placid, NY), casein kinase II (sc-12739, SantaCruz Biotechnology, Santa Cruz, CA), calbindin D (C8666, Sigma, St.Louis, MO) and -arrestin 2 (sc-13140, Santa Cruz Biotechnology); asheep polyclonal antibody to 11 -hydroxysteroid dehydrogenase type 2 (11 -HSD2; AB1296, Chemicon, Temecula, CA); goat polyclonalantibodies to the Wilms' tumor protein (WT1; sc-15422, Santa CruzBiotechnology) and CD5 (sc-6984, Santa Cruz Biotechnology) and rabbitpolyclonal antibodies to neurofibromin (sc-68, Santa CruzBiotechnology), the endothelin B receptor (AER-002, Alomone Labs, Jerusalem, Israel), endothelial nitric oxide synthase (eNOS; 160880, Cayman Chemical, Ann Arbor, MI), neuronal (n)NOS (160870, Cayman), -actin (A2066, Sigma), c-Fos (06-341, Upstate) andc-Jun (KAP-TF102E, StressGen Biotechnologies, Victoria, BC, Canada); and a phospho-specific rabbit antibody to c-Jun phosphorylated at Ser73(06-659, Upstate).

Protein sample preparation, SDS-PAGE electrophoresis, andimmunoblotting. Kidneys were dissected into regions and homogenized in ice-coldisolation solution (250 mM sucrose, 10 mM triethanolamine, pH 7.6, containing 1 mg/ml leupeptin, 0.1 mg/ml phenylmethylsulfonyl fluoride)using a tissue homogenizer (Omni 1000 fitted with a microsawtoothgenerator) at maximum speed for three 15-s intervals. Total proteinconcentrations were measured (BCA kit, Pierce, Rockford, IL), and thesamples were solubilized in Laemmli sample buffer at 60°C for 15 min.8 W" d+ J/ ?$ k3 U
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Semiquantitative immunoblotting was carried out as previously described( 22, 51 ) to assess the relative abundances of individualproteins in the dDAVP-treated Brattleboro rats compared with controlBrattleboro rats. To confirm that protein loading of the gels wasequal, preliminary 12% polyacrylamide gels were stained with Coomassieblue, as previously described ( 51 ).& J% O  V" z* c+ c

Proteins were separated on 10 or 12% polyacrylamide gels by SDS-PAGEand transferred to nitrocellulose membranes electrophoretically (Bio-Rad Mini Trans-Blot Cell). Membranes were blocked for 1 h atroom temperature with 5% nonfat dry milk and probed overnight at 4°Cwith the appropriate affinity-purified polyclonal antibody. Membraneswere washed and exposed to one of the following horseradish peroxidase-labeled secondary antibodies for 1 h at roomtemperature: donkey anti-sheep IgG (1713-035-147, diluted to1:5,000, Jackson Laboratories), goat anti-rabbit IgG (31463, diluted to1:5,000, Pierce), mouse anti-goat IgG (31400, diluted 1:5,000, Pierce), or rabbit anti-mouse IgG (31450, diluted to 1:5,000, Pierce). After awashing, bands were visualized using a luminol-based enhanced chemiluminescence substrate (LumiGLO, Kirkegaard and PerryLaboratories, Gaithersburg, MD). Band densities were determined bylaser densitometry (Personal Densitometer SI, Molecular Dynamics, SanJose, CA).& a! r) G. I% ^' h4 U# |
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Immunocytochemistry. Control and dDAVP-treated Brattleboro rats were prepared asdescribed above. The kidneys were fixed by perfusion with cold PBS (pH7.4) for 15 s via the abdominal aorta, followed by cold 4%paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 3 min. Thekidneys were removed and postfixed for 1 h, followed by 3 × 10-min washes with 0.1 M cacodylate buffer (pH 7.4). The tissue wasdehydrated in graded ethanol and left overnight in xylene. The tissuewas embedded in paraffin and 2-µm sections were cut on a rotarymicrotome (Micron). Localization of 11 -HSD2 was carried out usingindirect immunoperoxidase labeling as described ( 19 ). Theprimary and secondary antibodies were the same as described above forimmunoblotting. For immunoperoxidase labeling, counterstaining was doneusing Mayer's hematoxylin. Microscopy was carried out with a LeicaDMRE light microscope.
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Comparative genomic analysis of 5'-flanking region of 11 -HSD2gene. The human 11 -HSD2 mRNA sequence was downloaded from GenBank viaEntrez ( http://www.ncbi.nlm.nih.gov/Entrez/index.html ). The mRNAsequence was used in a homology search of the human genome using thegenome browser available at the University of California, Santa Cruz(Jim Kent curator, http://genome.ucsc.edu/cgi-bin/hgBlat? command=start) toidentify the human gene. The browser was used to locate the 5'-flankingregion and a 7,000-bp length immediately upstream of the transcriptionstart site was chosen for further analysis. Regions that were conservedbetween human and mouse were identified by the browser. The conservedhuman and mouse sequences were further analyzed by a string searchusing TESS ( http://www.cbil.upenn.edu/tess/index.html ) to comparethe conserved sequences with elements of a transcription factor bindingmotif database (TRANSFAC version). The conserved sequences betweenhuman and mouse were entered into TESS, which returned a list ofpotential transcription factor binding sites. Only perfect matches with elements of the database were included, and sites not found in commonbetween the two species were eliminated.

RESULTS
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To generate hypotheses concerning the direct or indirect long-termaction of vasopressin in the renal collecting duct, we carried out cDNAarray experiments using mRNA isolated from Brattleboro rat innermedullary tissue samples after 72 h of dDAVP or vehicle treatment.Figure 1 summarizes the distribution ofdot-density ratio responses for an experiment that compared RNA pooledfrom three dDAVP-infused Brattleboro rats with RNA pooled from three vehicle-infused rats. Of the 1,176 genes on the array, 137 transcripts were increased by 2-fold or more in response to dDAVP infusion, and 10 transcripts were decreased to 0.5-fold or less (dashed lines, Fig. 1 ).Full results including TIFF images of the arrays are presented inaccordance with MIAME guidelines ( 4 ) in the SupplementalMaterials.

Fig. 1. Response to long-term desamino-Cys1, D -Arg8 vasopressin(dDAVP) infusion in Brattleboro rat inner medullas; distribution ofdot-density ratios for all 1,176 features on cDNA arrays. Verticaldashed lines indicate demarcation points for 1:2 and 2:1 densityratios, respectively. Note that the data are plotted as the base 10 logarithm of the absolute ratio.
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Validation of array results. Among the 1,176 genes on the arrays, several of them were selected forfurther analysis because of the potential physiological significance ofchanges (or lack of changes) that were detected. These targets werestudied further using both real-time RT-PCR to confirm the responses atan mRNA level and semiquantitative immunoblotting to test whether themRNA changes are associated with corresponding changes in proteinabundance. The results of real-time RT-PCR determinations of mRNAresponses to dDAVP are shown in Table 1.Real-time RT-PCR determinations were carried out in different total RNAsamples than used for the arrays. These samples were isolated frominner medullas of separate, identically treated Brattleboro rats (5 dDAVP-treated vs. 5 vehicle-infused Brattleboro rats). As can be seenin Table 1, the genes with the largest increases in mRNA in response todDAVP on the cDNA arrays (namely, WT1, -arrestin 2, neurofibromin,and casein kinase II ) were found to be associated with significantincreases in mRNA in response to dDAVP infusion by real-time RT-PCR.These are novel responses, which have potential significance regarding the mechanism of the cellular response to vasopressin in collecting duct cells (see DISCUSSION ). In addition, among the threeaquaporins expressed in the renal collecting duct, aquaporin-2, -3, and-4, there was reasonable agreement between cDNA array results and real-time RT-PCR results. Both aquaporin-3 and aquaporin-4 manifested substantial increases in mRNA in response to dDAVP, while, somewhat surprisingly, aquaporin-2 either did not change (cDNA array) or increased only modestly (real-time RT-PCR). Interestingly, GAPDH and -actin, both of which are considered housekeeping genes, manifestedincreases in mRNA in response to dDAVP when measured by real-timeRT-PCR.
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Table 1. Comparison of mRNA changes in inner medullas of Brattleboro ratsestimated using cDNA arrays vs. real-time RT-PCR  I% k) ~) W1 ]5 F& d$ p( a: \. G
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The lack of an increase in aquaporin-2 mRNA abundance was surprising inview of previous reports of upregulation of aquaporin-2 protein ininner medulla in response to dDAVP ( 8, 51 ). Therefore, wedecided to run Northern blots to assess aquaporin-2 mRNA abundance changes in renal inner medulla and cortex in response to long-term dDAVP infusion (Fig. 2 ). Consistent withfindings with the cDNA arrays and real-time RT-PCR, there was nosubstantial change in aquaporin-2 mRNA in the inner medullas ofBrattleboro rats in response to dDAVP infusion (normalized banddensities: vehicle infused, 100 ± 18; dDAVP infused, 85 ± 17, not significant). In contrast, a substantial increase inaquaporin-2 mRNA was detected in RNA samples isolated from the renalcortices of the same rats (normalized band densities: vehicle infused,100 ± 82; dDAVP infused, 1,072 ± 485, P on Northern blot analysisof whole kidney samples ( 12 ).
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Fig. 2. Northern blots for aquaporin-2 in inner medullas (IM) andcortices (CTX) of control (vehicle-infused) and dDAVP-infusedBrattleboro rats (3-day infusion). Five micrograms of total RNA/lanewere loaded for IM, and 10 µg/lane were loaded for cortex. Blots werehybridized with a digoxigenin-labeled aquaporin-2 cDNA probe.Aquaporin-2 transcript size, 1.8 kb.5 a5 J4 C$ f/ t$ V- k

The array results showed that there was an apparent increase in themRNA abundance for UT-A in response to dDAVP infusion (dDAVP-to-vehicleratio: 2.8). This gene codes for a major urea transporter UT-A1 ininner medullary collecting duct. This finding contrasts with theobserved lack of increase in UT-A1 protein in inner medulla in responseto dDAVP infusion ( 52 ). Real-time RT-PCR was impracticalfor UT-A1 because its sequence overlaps with other splice variants fromthe same gene ( 33 ). Hence, we carried out Northernblotting for UT-A isoforms, using the same samples used for real-timequantitative RT-PCR studies (Fig. 3 ). There was a striking increase in the abundance of UT-A2 mRNA(transcript size: 2.9 kb), whereas the abundance of UT-A1 mRNA(transcript size: 3.9 kb) changed comparatively little. Consequently,real-time RT-PCR was run for UT-A2, targeting the single exon unique to this isoform. The real-time RT-PCR analysis indicated that UT-A2 mRNAabundance was increased 56-fold in inner medulla by dDAVP infusion. Acheck of the sequence of the cDNA probe for UT-A on the array indicatedthat it overlaps sequence for both UT-A1 and UT-A2 (proprietaryinformation, Clontech). Thus, although UT-A1 is the dominant isoform inthe inner medulla, an extremely large increase in UT-A2 (a splicevariant expressed in thin descending limbs of Henle's loops) wasapparently chiefly responsible for the marked increase detected by cDNAarray analysis.
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Fig. 3. Northern blot for UT-A isoforms in inner medullas ofcontrol (vehicle-infused) and dDAVP-infused Brattleboro rats (3-dayinfusion). Ten micrograms/lane of total RNA were loaded. The blot washybridized with a 32 P-labeled full-length urea transporterA (UT-A) cDNA probe. Abundance of UT-A2 transcript was markedlyelevated in response to dDAVP infusion.

Correlation between mRNA changes and protein changes for selectedgenes. To correlate changes in mRNA expression detected on the cDNA arrayswith changes in protein expression, we carried out quantitative immunoblotting for 28 of the proteins corresponding to genes on thearray. For this, we utilized kidneys from a different set ofdDAVP-infused Brattleboro rats than those used for the arrays (Fig. 4, Table 2 ). As shown in Fig. 4, severaltranscripts exhibiting very large increases in mRNA levels in responseto dDAVP infusion (based on cDNA array analysis) were alsoassociated with significant increases in the corresponding proteinabundances. This group included neurofibromin, casein kinase II, and11 -HSD2. WT1 (24-fold increase in mRNA) was not detectable in theinner medulla by immunoblotting despite positive controls showingstrong labeling of heterologously expressed WT1 protein (Sean Lee,National Institute of Diabetes and Digestive and Kidney Diseases,National Institutes of Health, Bethesda, MD, personal communication).Two proteins previously recognized to be upregulated by vasopressin, -ENaC ( 11 ) and aquaporin-3 ( 13, 51 ), alsoshowed substantial increases at the protein and mRNA levels in thepresent study (Fig. 4 ). In addition, c-Fos showed a relatively modestincrease in mRNA abundance by cDNA array analysis (1.7-fold) butexhibited a large increase in protein abundance (4.9-fold). Conversely,two proteins (the 1 -subunit of the Na-K-ATPase and thetype 2 Na-K-2Cl cotransporter) showed corresponding decreases in mRNAand protein (Fig. 4 ). [Analysis of serum samples from these ratsrevealed a significant increase in serum concentrations of aldosteronein dDAVP-infused (1.9 ± 0.5 nM, n = 6) comparedwith vehicle-infused Brattleboro rats (0.5 ± 0.1 nM, n = 6), ruling out a decrease in circulatingaldosterone level as a cause of the decrease in Na-K-ATPase 1 -subunit protein expression.]

Fig. 4. Comparison of mRNA abundance changes with proteinabundance changes in response to long-term dDAVP infusion. Data arepresented as the base 10 logarithm of the ratio of value for dDAVPinfusion to vehicle infusion. AQP1, AQP2, and AQP3, aquaporin-1, -2, and -3, respectively; 11 -HSD2, 11 -hydroxysteroid dehydrogenasetype 2; eNOS, endothelial isoform of nitric oxide synthase; nNOS,neuronal isoform of nitric oxide synthase; ET-BR, endothelin Breceptor; Syt-5, synaptotagmin type 5 (also called synaptotagmin IX);CB-28, calbindin 28; CD5, lymphocyte differentiation antigen-CD5;ENaC, epithelial sodium channel; VAMP2, vesicle-associated membraneprotein type 2; UT-A1, UT-A isoform 1; NKCC2, type 2 Na-K-2Clcotransporter.: z, ~, D! Q" q- W! n
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Table 2. Immunoblotting results from long-term dDAVP infusionexperiment

For the 17 genes in Table 2 for which there were a greater than1.5-fold increase in transcript abundance on the array, 7 were notassociated with significant increases in protein abundance (endothelinB receptor, CD5, syntaxin 2, renin, UT-A1, c-Jun, and calbindin 28).Conversely, several genes for which there was no substantial change intranscript abundance on the array (0.67- to 1.5-fold changes in dotdensity) showed significant changes in protein abundance with threeincreasing (eNOS, -ENaC, and aquaporin-2) and one decreasing(syntaxin-3).

11 -HSD2. One of the upregulated transcripts was 11 -HSD2, whose dot density oncDNA arrays was increased by 3.1-fold (Table 1 ) and whose proteinabundance was increased by 4.0-fold (Table 2 ) in response to long-termdDAVP infusion. Because of its potential physiological importance inthe regulation of glucocorticoid action in the collecting duct( 16 ), we chose to study 11 -HSD2 regulation byvasopressin in greater detail. Additional Brattleboro rats were studiedto localize the increase in 11 -HSD2 protein abundance in response todDAVP (Fig. 5 ). As shown in Fig. 5 A, immunoblot analysis of inner medullary protein samplesfrom Brattleboro rats infused with dDAVP for 3 days again showed anincrease in 11 -HSD2 protein abundance compared with vehicle-infusedrats. Densitometry analysis of the immunoblots revealed that thenormalized band density was significantly increased to 169 ± 20 compared with 100 ± 25 in control inner medulla, P were seen in renalcortical samples (not shown; dDAVP infused, 242 ± 9 vs.vehicle-infused, 100 ± 12, P 5 B shows immunoperoxidase labeling of 11 -HSD2 in renalinner medullas of vehicle- and dDAVP-infused Brattleboro rats. Labelingconditions and exposure settings on the microscope were identical forboth images. The 11 -HSD2 labeling was present in the principal cells of the collecting ducts (arrows, Fig. 5 B ), whereasintercalated cells were not labeled (arrowheads). dDAVP treatmentmarkedly increased 11 -HSD2 immunostaining in principal cells(arrows, Fig. 5 B -B) compared with vehicle-infusedBrattleboro rats (arrow, Fig. 5 B -A). Similar observationswere made in two additional pairs of rats. Thus we conclude thatlong-term dDAVP infusion increases the abundance of 11 -HSD2 proteinin the inner medullary collecting ducts of Brattleboro rats.
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Fig. 5. Effect of dDAVP on 11 -HSD2 protein abundance in innermedullas of Brattleboro rats. A : immunoblot of homogenatesof inner medullary samples from Brattleboro rats treated with vehicleor dDAVP for 7 days. Blot was loaded with 10 µg of total protein/laneand probed with sheep polyclonal antibody to 11 -HSD2. Band densityfor 11 -HSD2 was significantly increased by dDAVP infusion in theinner medulla (* P B :immunoperoxidase labeling of 11 -HSD2 in renal inner medullas ofvehicle ( A )- and dDAVP-infused ( B ) Brattlebororats probed with the same antibody as in Fig. 2 A. Labelingconditions and exposure settings on the microscope were identical forboth images. Inner medullary collecting ducts (arrows) and intercalatedcells (arrowheads) are shown.
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To assess putative binding sites for transcription factors in the5'-flanking region of the 11 -HSD2 gene, we carried out comparative genomic analysis using the newly available genomic sequencedata for humans and mice as described in METHODS. Figure 6 reports the putative transcriptionfactor binding motifs present in the 5'-flanking regions that wereconserved between the human and mouse 11 -HSD2 genes.Notably, there was no conserved cAMP responsive element binding protein(CREB) site. (A CREB site was present in the human sequence, but thecorresponding mouse sequence had a 1-bp difference.) The analysis alsoconfirms previous identification of a conserved Sp1 site in both mouseand human 11 -HSD2, positioned between B438 and B1088( 32 ). However, our analysis also identified threeadditional putative Sp1 sites upstream of this region, which wereconserved between mice and humans. Sp1 cis -regulatoryelements have been associated with cAMP-mediated transcriptionalregulation ( 43 ). Finally, Fig. 6 also shows the presenceof a conserved putative AP1 binding element that binds c-Fos/c-Junheterodimers. Such sites have also been shown to be involved incAMP-mediated regulation of transcription ( 50 ). The c-fos gene is known to be transcriptionally regulated bycAMP, although CREB-dependent and CREB-independent means( 3 ), and an AP1 site in the 5'-flanking region of theaquaporin-2 gene has previously been demonstrated to play a centralrole in the upregulation of its transcription by vasopressin inLLC-PK 1 cells ( 57 ).
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Fig. 6. Conserved transcription factor binding motifs in the 5'-flankingregions of 11 -HSD2. Nucleotide positions are shown in gray, and thestart of the open reading frame for the 11 -HSD2 protein wheremarked. See http://www.cbil.upenn.edu/tess/index.html fora full listing of transcription factor abbreviations.0 h* J% d7 p) c

AP1 mediated transcriptional regulation typically occurs via twomechanisms: 1 ) regulation of c-Fos protein abundance and 2 ) phosphorylation of the c-Jun protein. Addressing thesetwo mechanisms, Fig. 7 shows immunoblotsof inner medullary homogenates from Brattleboro rats. dDAVP infusionindeed increased c-Fos abundance to 489 ± 108%( P 7 A ). Application of a phosphorylation-specific (Ser73) c-Junantibody using the same protein samples (Fig. 7 B ) revealedan increase in phospho-c-Jun abundance relative to vehicle-infusedBrattleboro rats (normalized band densities: 722 ± 225 vs.100 ± 28% in control rats, P

Fig. 7. Effect of long-term dDAVP infusion on c-Fos, c-Jun, andphosphorylated (phospho-)Jun protein abundance in Brattleboro rat innermedullas. Immunoblots were performed using inner medullary samples fromvehicle- and dDAVP-infused Brattleboro rats. Blots were loaded with 15 µg of total protein/lane and probed with polyclonal antibodies,anti-c-Fos, anti-c-Jun, and anti-phospho-Jun (Ser73). Band density forc-Fos and phospho-Jun were significantly increased by dDAVP infusion( P
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Response to 4-h dDAVP infusion. Figure 8 summarizes the overall patternof mRNA abundance changes in response to 4-h treatment with dDAVPcompared with 72-h treatment, as determined by cDNA microarrayanalysis. Predictably, the range of response ratios is not as greatafter 4-h compared with 72-h dDAVP infusion. Furthermore, in a largenumber of cases, responding genes at 72 h were different fromresponding genes at 4 h. Among the 18 transcripts that wereincreased in Tables 1 or 2 1.5) after 4 h of dDAVP infusion(casein kinase II, syntaxin-2, -ENaC, UT-A, and aquaporin-3),while 9 were unchanged after 4-h dDAVP treatment (neurofibromin, CD5,11- -HSD2, renin, synaptotagmin 5, c-Jun, calbindin 28, -ENaC, andc-Fos) and 4 were decreased (dDAVP:vehicle -arrestin 2, and aquaporin-4). Fullresults of the 4-h dDAVP/Brattleboro rat array experiments, includingTIFF images of the arrays, are presented in accordance with MIAMEguidelines ( 4 ) in the Supplemental Materials.

Fig. 8. Comparison of mRNA abundance changes with 4-h vs. 3-daydDAVP infusion vs. 3-day dDAVP infusion for 1,176 transcripts on cDNAarray. Data are presented as the base 10 logarithm of the ratio ofvalue for dDAVP infusion to vehicle infusion for both time points.$ S& w* b, Y& B" J# _

DISCUSSION
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Previous studies have established that vasopressin regulates geneexpression in the kidney, at least with regard to the genes that codefor aquaporin-2 and -3 ( 12, 51 ), the three ENaC subunits( 11, 34 ), the urea transporter UT-A2 ( 55 ),and the Na-K-2Cl cotransporter NKCC2 ( 22 ). In this paper,we used cDNA arrays to broaden these observations, identifying several additional genes for which expression is altered in the renal innermedulla in response to administration of the V 2 receptor-selective vasopressin analog dDAVP. The studies were done inthe Brattleboro rat, which lacks circulating vasopressin due to amutation in the vasopressin-neurophysin gene ( 46 ) and thusprovides a vasopressin-free host in which to test responses to dDAVPinfusion. Because these studies were done in vivo, responses to dDAVPinfusion in this study could be either direct, i.e., responses toincreased phosphorylation of transcription factors byvasopressin-activated kinases (protein kinase A or calmodulin-dependentkinases) in collecting duct cells, or indirect, i.e., due to a morecomplex response that is triggered by increased V 2 -receptoroccupation but is not an immediate consequence of vasopressinreceptor-mediated signaling. The latter would include severalcategories of responses including 1 ) activation ofadditional signaling cascades in collecting duct cells such as the MAPkinase pathway; 2 ) induction of hierarchical transcriptionfactors downstream of transcription factors immediately activated byvasopressin-induced signaling; 3 ) secondary changes incirculating hormone levels; or 4 ) responses to an alteredinner medullary interstitial environment. This paper does not attemptto discriminate these different types of responses. In the following,we discuss, first, the regulatory targets identified and then addresssome general issues raised by the results.

-Arrestin 2. -Arrestin 2 is a member of a family of proteins involved in Gprotein-coupled receptor desensitization ( 39 ). Theseproteins bind to the phosphorylated COOH termini of G protein-coupledreceptors and mediate receptor desensitization in part by inducingreceptor endocytosis. In the present studies, identification of -arrestin 2 upregulation in response to dDAVP infusion points to apotential role for the induction of this protein in thevasopressin-escape phenomenon ( 12 ), which has been foundto be due to V 2 receptor downregulation ( 10, 53 ) and is crucial in limiting the degree of extracellular fluiddilution in the syndrome of inappropriate antidiuresis. Recently, the -arrestins have also been found to play a role as ligand-activatedscaffold proteins for two MAP kinase pathways, the ERK cascade( 7, 27 ) and the c-Jun NH 2 -terminal kinase 3 (JNK3) cascade ( 31 ). It is therefore conceivable that induction of -arrestin 2 by vasopressin is involved in the observed activation of MAP kinase cascades in the renal inner medulla during antidiuresis ( 56 ) and may provide part of the explanationfor the dDAVP-induced increase in c-Jun phosphorylation demonstrated inthe present study (Fig. 7 ).# c) D3 K. `6 F& L: }4 w2 I( `' d

Neurofibromin. The renal inner medullary expression of neurofibromin was markedlyincreased at both the mRNA (Table 1 ) and protein (Table 2 ) levels inresponse to long-term dDAVP infusion. Neurofibromin was originallyidentified as the protein product of the disease gene responsible forthe autosomal dominant genetic disease neurofibomatosis type 1 ( 18 ). It is a member of the GTPase-activating protein (GAP) family and has been implicated as a key factor in limiting of thegrowth-promoting action of the small GTP-binding protein Ras. Recently,however, an additional function has been identified for the protein,direct activation of CNS-specific adenylyl cyclase isoforms ( 17, 54 ). It is unknown whether neurofibromin interacts in a similarmanner with renal isoforms of adenylyl cyclase. If it does,upregulation of neurofibromin expression in the kidney could contributeto long-term regulation of water, urea, and sodium ion transport in therenal tubule.
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Casein kinase II. Casein kinase II expression was strongly increased in the renalinner medulla in response to dDAVP infusion at both mRNA and proteinlevels (Tables 1 and 2 ). A detectable increase in casein kinase II mRNA after a 4-h dDAVP infusion suggests rapid, direct induction.Casein kinase II is a regulatory subunit of the protein kinase CK2,a ubiquitous serine/threonine kinase, which is composed of tworegulatory -subunits and two catalytic -subunits. Many proteinsubstrates for CK2 have been identified, including growth factorreceptors, transcription factors, cytoskeletal proteins, cell cycleregulatory proteins, and vesicle trafficking proteins. The latterincludes syntaxin-4, which is believed to play a role invasopressin-dependent aquaporin-2 trafficking to the plasma membrane( 41 ). Recent findings point to a role for CK2 in thecellular response to various forms of cellular stress ( 1 ),raising the possibility that its induction by dDAVP may play a role inprotecting inner medullary cells against osmotic stress.
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11 -HSD2. 11 -HSD2 was upregulated in response to long-term dDAVPinfusion at both mRNA and protein levels (Figs. 4 and 5 ). This enzyme is believed to play a central role in the regulation of ion transport in the renal collecting duct through its ability to break down glucocorticoids (cortisol in humans; corticosterone in rodents) toinactive forms ( 16 ). In the absence of 11 -HSD2 incollecting duct cells, glucocorticoids at circulating concentrationswould be expected to bind to and fully activate the mineralocorticoid receptor (MR), impairing the ability of regulated changes incirculating aldosterone levels to alter gene expression. Theconventional view of the role of 11 -HSD2 is that it isconstitutively expressed at such high levels that only themineralocorticoid aldosterone can reach the MR. However, previousstudies identified 11 -HSD2 as a regulatory target for the short-termactions of vasopressin by a nontranscriptional mechanism( 2 ), suggesting that regulation of 11 -HSD2 activity mayplay a physiological role, perhaps by controlling glucocorticoid accessto the MR and glucocorticoid receptor in the collecting duct. Thepresent finding of upregulation of 11 -HSD2 gene expression inresponse to long-term vasopressin treatment extends this view and couldhave implications for the regulation of transporter proteins in thecollecting duct that are recognized targets for glucocorticoid ormineralocorticoid regulation, including the UT-A1 urea transporter( 38 ), the Na-K-ATPase ( 15 ), and ENaC( 48 )., ?  x8 x% S! K; H) E* T; s# N) V2 J
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The interpretation of the effects of dDAVP infusion oncorticosteroid-regulated transporters is complicated by the fact that dDAVP infusion increased the circulating level of aldosterone in thisstudy by almost fourfold. This response might be expected to oppose theeffect of increased 11 -HSD2 levels. Indeed, in the present study -ENaC, whose abundance is strongly upregulated by aldosterone( 30 ), was substantially increased at both the mRNA andprotein levels in response to dDAVP infusion (Fig. 4, Table 2 ). Theincrease in plasma aldosterone concentration may be due in part to theability of dDAVP to bind and activate the V 1b receptor incorticotroph cells of the anterior pituitary ( 44 ), therebyincreasing ACTH secretion, which is considered a minor factor in theregulation of adrenal aldosterone secretion.: i: `$ S/ q/ z( g+ G& R; l5 [
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WT1. Among all cDNAs on the array, WT1 manifested the largest increasein dot density in response to long-term infusion of dDAVP (Table 1 ). Alarge increase in mRNA abundance was confirmed by real-timequantitative RT-PCR. WT1 is a zinc-finger transcription factorexpressed chiefly in kidney, gonads, uterus, and spleen, whichfunctions as a tumor suppressor ( 26 ). In addition, WT1 appears to be involved in posttranscriptional processing of mRNA ( 45 ). Mutations in WT1 are associated with a highincidence of Wilms' tumor, a renal neoplasm arising from renal tissueof metanephric origin. In the mature kidney, WT1 is generally believed to be expressed only in the glomerular podocyte ( 45 ),although the possibility of its expression in the renal inner medullaof the adult kidney has not been investigated in detail. Available antibodies to WT1 could not convincingly demonstrate WT1 protein in the rat inner medulla (Table 2 ), although absolute expression levelsof a functional transcription factor could conceivably be quite low.Consequently, further studies will be needed to localize WT1 expressionin the renal inner medulla and to determine its role invasopressin-mediated transcriptional regulation.

c-Fos and c-Jun. c-Fos and c-Jun are immediate early genes,whose products together constitute the transcription factor AP1. OncDNA arrays, dot densities for both c-Jun and c-Fos mRNA were observed to increase with long-term dDAVPinfusion (Fig. 4 ). Immunoblotting demonstrated that c-Fos proteinabundance was markedly increased in response to dDAVP administration,consistent with the findings of Yasui et al. ( 57 ) inLLC-PK 1 cells. However, there was no demonstrable increasein the abundance of c-Jun protein. Nevertheless, there was a markedincrease in the abundance of phosphorylated c-Jun, possibly resultingfrom activation of MAP kinases in response to dDAVP. Thus, althoughdirect studies of transcriptional regulation are beyond the scopeof this study, we postulate a critical role of the AP1 binding motif inmediating the long-term responses to vasopressin in the renal innermedulla. One gene that may be transcriptionally upregulated by AP1 maybe 11 -HSD2, which has a conserved AP1 binding site in its5'-flanking region and is markedly upregulated by vasopressin (see above).

Synaptotagmin 5. The synaptotagmins are postulated to play calcium-sensing roles in theregulation of exocytosis ( 49 ). In the present study, wefound that synaptotagmin 5 ( 20 ) (also termed synaptotagmin IX) was upregulated in the inner medulla in response to long-term dDAVPinfusion at both mRNA and protein levels (Fig. 4 ). Synaptotagmin 5 hasrecently been demonstrated to be a binding partner for the -subunitof the protein serine/threonine kinase CK2 ( 9 ), another dDAVP-responsive protein (see above).* B7 A6 P! P- t( v- v! u
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Aquaporin-2 and -3. Aquaporin-3, a basolateral water channel in collecting ductprincipal cells, was found to be upregulated at both the mRNA (Table 1 )and protein (Table 2 ) levels, consistent with prior results ( 12, 13, 51 ). The inner medullary protein abundance of aquaporin-2,the apical water channel in collecting duct principal cells, was alsoincreased (Table 2 ), consistent with previous findings ( 8, 51 ). In the context of these previous findings, we weresurprised to find that there was little or no increase inaquaporin-2 mRNA abundance in the inner medulla in response to dDAVPinfusion as demonstrated on cDNA arrays (Fig. 4 ), by Northern blotting(Fig. 2 ), and by real-time RT-PCR (Table 1 ). This result suggests thatposttranscriptional mechanisms may be involved in the regulation ofaquaporin-2 protein abundance in the renal inner medulla. In contrast,aquaporin-2 mRNA abundance was strongly increased in the cortex inresponse to dDAVP infusion (Fig. 2 ), consistent with previous findings( 10 ).
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Urea transporters. The UT-A gene codes for several urea transporterproteins expressed in inner medulla that arise from alternativesplicing ( 33 ). UT-A1, the predominant form in the innermedullary collecting duct ( 37, 47 ), has been shown to bedownregulated in the inner medulla in response to dDAVP infusion( 52 ). Hence, we were somewhat surprised to find that themRNA dot density for UT-A was increased 2.8-fold on the cDNA array(Table 1 ), whereas immunoblotting confirmed a lack of increase in UT-A1protein in the inner medulla in response to dDAVP infusion (Fig. 4 ).Further analysis by real-time quantitative RT-PCR revealed a56-fold increase in the abundance of a second splicing variant,UT-A2, which is expressed chiefly in the thin descending limbs of Henlein the outer medulla ( 37, 47 ) but is relativelynonabundant in the inner medulla ( 55 ). Interrogation ofthe commercial supplier of the array revealed that the sequence of theUT-A cDNA on the array overlaps both UT-A1 and UT-A2. Hence, it appearsthat a very large change in a relatively nonabundant splicing variantgave a result on cDNA array analysis that was not representative ofchanges in the most abundant splice variant.  E$ a; Z2 P9 F% W2 z, Z. {/ ?

General observations and conclusions. cDNA array analysis has revealed several genes that areupregulated in the inner medulla of the Brattleboro rat in response todDAVP infusion. Further studies will be needed to investigate furtherthe role of these genes and their protein products in the overallresponse to vasopressin. Clearly, the data presented here provide onlyan initial glimpse of the response to vasopressin, with a detailed viewonly of the long-term response, which may consist of both direct andindirect effects of vasopressin on gene expression. A generalcomparison of the response to vasopressin at a 4-h vs. a 72-h timepoint (Fig. 8 ) reveals a much different pattern of mRNA abundancechanges at the pre-steady-state time point. Detailed time coursestudies will be required to work out the sequence of events involved inthe vasopressin response and to determine which genes are upregulatedin direct response to vasopressin-mediated signaling vs. secondaryresponses, which might be related to vasopressin-mediated changes inlocal osmolality, local calcium ion concentrations, luminal pH, luminalflow rate, and other factors altered by vasopressin.
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An important component of the present study was a detailed comparisonof mRNA abundance and protein abundance responses to dDAVP infusion,with an assessment of 28 different protein products by quantitativeimmunoblotting (Table 2, Fig. 4 ). For this element of the study,we used a large number of polyclonal antibodies developed in thislaboratory for targeted proteomic studies ( 24 ) as well asa selection of antibodies from commercial sources for which thespecificities were clearly documented. Although for many of the genesexamined in this manner there was a clear correlation between changesin mRNA and those in protein, it is important to emphasize that severalgenes exhibited mRNA responses that were qualitatively different fromprotein responses. Some genes showed increases in protein with nochange in mRNA levels, whereas some showed changes in mRNA abundancewithout coordinate changes in protein abundance. Similar observationshave been made in studies in yeast using large-scale proteomics andgene expression arrays ( 21 ). Clearly, there arephysiologically important mechanisms by which levels of specificproteins can change without changes in mRNA levels, includingtranslational regulation and regulation of protein half-life( 23 ). Hence, although cDNA arrays provide an importantmeans of generating new hypotheses about physiological regulation at amolecular level, a complete evaluation of such mechanisms requiresprotein measurements.

A previous study reported genes whose transcript abundances wereupregulated or downregulated in response to exposure of cultured mpkCCD c14 to 10 8 M arginine vasopressin for4 h as determined by SAGE ( 42 ). There was no overlapbetween the responding genes reported in that study to those up- ordownregulated in the present study at either the 4- or 24-h time point(see list of responding genes in Supplemental Materials). However, itis well recognized that there are many differences in gene expressionand regulation between the cortical collecting duct and the innermedullary collecting duct. One difference was illustrated in thepresent study: aquaporin-2 mRNA levels are increased by vasopressin inthe renal cortex but not the renal medulla (Fig. 2 )./ C5 [' h2 Z3 Z  q0 m
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The strength of the cDNA array approach is that a large number oftranscripts can be assessed simultaneously. However, costs limit thenumber of observations per transcript to relatively low numbers,yielding severe statistical limitations with regard to probabilities ofboth false positives and false negatives. Furthermore, even if largenumbers of array experiments can be completed, ambiguities can remain,as evidenced by the finding that the "urea transporter" spot on thearray actually recognizes two different splicing variants of the UT-Agene with much different physiological roles (see above). Completeevaluation of complex physiological responses therefore requiresapplication of other methodologies, such as Northern blotting andreal-time RT-PCR, for quantitative assessment of abundance changes inspecific transcripts. As noted above, assessment of protein abundancechanges can, in principle, substitute for confirmation at an mRNA levelif the question being addressed pertains to the physiological function of proteins.! u, L/ b+ N! P( ?0 B1 `! s8 u( |
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ACKNOWLEDGEMENTS0 p% t6 A* S8 g
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The authors thank Dr. Sean Lee (National Institute of Diabetes andDigestive and Kidney Diseases, Bethesda, MD) for advice regarding WT1 antibodies.
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