Combined Proteomics and Pathways Analysis of Collecting Duct Reveals a Protein Regulatory Network Activated in Vasopressin Escape
Ewout J. Hoorn,
Jason D. Hoffert and
Mark A. Knepper
Laboratory of Kidney and Electrolyte Metabolism; National Heart, Lung, and Blood Institute; National Institutes of Health, Bethesda, Maryland
Address correspondence to: Dr. Mark A. Knepper, National Institutes of Health, 10 Center Drive, Building 10, Room 6N260, Bethesda, MD 20892. Phone: 301-496-3064; Fax: 301-402-1443; E-mail: knep{at}helix.nih.gov
Received for publication March 26, 2005.
Accepted for publication June 25, 2005.
Low sensitivity is characteristic of many proteomics methods.Presented here is an approach that combines proteomics basedon difference gel electrophoresis (DIGE) with bioinformaticpathways analysis to identify both abundant and relatively nonabundantproteins in inner medullary collecting duct (IMCD) altered inabundance during escape from vasopressin-induced antidiuresis.Rats received the vasopressin analog dDAVP by osmotic minipumpplus either a daily water load (vasopressin escape) or onlyenough water to replace losses (control). Immunoblotting confirmedthe hallmark of vasopressin escape, a decrease in aquaporin-2,and demonstrated a decrease in the abundance of the urea transporterUT-A3. DIGE identified 22 mostly high-abundance proteins regulatedduring vasopressin escape. These proteins were analyzed usingpathways analysis software to reveal protein clusters incorporatingthe proteins identified by DIGE. A single dominant cluster emergedthat included many relatively low-abundance proteins (abundancestoo low for DIGE identification), including several transcriptionfactors. Immunoblotting confirmed a decrease in total and phosphorylatedc-myc, a decrease in c-fos, and increases in c-jun and p53.Furthermore, immunoblotting confirmed hypothesized changes inother proteins in the proposed network: Increases in c-src,receptor for activated C kinase 1, calreticulin, and caspase3 and decreases in steroid receptor co-activator 1, Grp78/BiP,and annexin A4. This combined approach proved capable of uncoveringregulatory proteins that are altered in response to a specificphysiologic perturbation without being detected directly byDIGE. The results demonstrate a dominant protein regulatorynetwork in IMCD cells that is altered in association with vasopressinescape, providing a new framework for further studies of signalingin IMCD.
Escape from the antidiuretic action of vasopressin ("vasopressinescape") is an important physiologic process that limits theseverity of the syndrome of inappropriate antidiuresis (SIADH)and other hyponatremic disorders (1,2). During vasopressin escape,humans and experimental animals undergo a brisk water diuresis,despite high circulating levels of vasopressin (2–4).Vasopressin escape is of considerable clinical importance withregard to SIADH and other vasopressin-dependent dilutional hyponatremicstates, because without escape, further water retention andhyponatremia could be fatal (4–6).
Studies in a rat model of vasopressin escape have demonstratedthat the central feature of the vasopressin-independent increasein water excretion is a marked suppression of the expressionof the water channel aquaporin-2 (AQP2) (2). The fall in AQP2protein abundance is due in part to decreased levels of AQP2mRNA in collecting duct (2). This response is associated witha decrease in the capacity of inner medullary collecting ducts(IMCD) to produce cAMP in response to vasopressin (7) in associationwith a fall in vasopressin-binding capacity of the V2R receptor(8). In contrast, there is upregulation of AQP-3 (2) and the-subunit of the epithelial Na channel (ENaC) (9), suggestingthat the process that is responsible for suppression of AQP2expression is selective. With regard to possible mediators ofescape, roles have been suggested for nitric oxide and prostaglandins(10) and aldosterone (11). However, little is known about whichintracellular signaling processes orchestrate the escape andhow the vasopressin escape phenomenon is triggered and maintained.
To address which proteins are co-regulated with AQP2 in IMCDduring vasopressin escape, we used a relatively new differentialproteomics method called difference gel electrophoresis (DIGE)coupled with matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) mass spectrometry to identify differentially expressedproteins. Recently, we demonstrated the feasibility of suchan approach using DIGE-based proteomics to identify vasopressin-responsiveproteins in the collecting duct of Brattleboro rats that weretreated with the selective V2R agonist dDAVP (12).
To address further the signaling pathways that are activatedduring vasopressin escape, we analyzed the DIGE results usingbioinformatic pathways analysis (13,14). The pathways analysissoftware generates hypothetical protein networks, based on largedatabases of protein interactions culled from the biologic literature,including physical binding reactions, cis-trans interactionsin transcriptional regulation, and enzyme–substrate relationships.Such networks can be used to predict signaling pathways thatare activated during vasopressin escape. The rationale for usingthe pathways analysis approach was not only to facilitate theinterpretation of the relationships between the identified proteinsbut also to identify relatively low-abundance proteins (abundancestoo low for DIGE identification) that may be involved in vasopressinescape (15). The hypothetical changes in these low-abundanceproteins then can be tested by immunoblotting. This integratedapproach identified a single dominant network of proteins thatincludes several proteins that may play key regulatory rolesin vasopressin escape.
Animals and Sample Preparation
Osmotic minipumps (model 2001; Alzet, Palo Alto, CA) that deliver5 ng/h dDAVP (Peninsula Laboratories, San Carlos, CA; ACUC Protocol2-KE-3) were implanted subcutaneously in Male Sprague-Dawleyrats (Taconic Farms, Germantown, NY). After 3 d, rats were dividedinto two groups: "escape" and "control." Escape rats were givenexcess daily water (0.4 ml/g body wt) via a gelled-agar diet(71% water, 28% finely ground rat food, 1% agar; BACTO-AGAR;Difco Laboratories, Detroit, MI). This diet forced the ratsto take the water load to consume the food ration. Control ratswere given the same amount of food but with only enough water(0.075 ml/g body wt) to compensate for insensible losses plus0.015 ml/g body wt per d urine. Rats did not receive ad libitumwater. This represents a small modification from our previousstudies in which control rats received no water mixed with thefood but were allowed to receive ad libitum water (2,7,9). Therats were maintained in metabolic cages in a temperature- andhumidity-controlled room with a 12:12-h light-dark cycle, andurine was collected daily for measurement of volume and osmolality.Because the onset of escape is known to occur between 1 and2 d after the start of water loading (2), three time pointswere analyzed (four control and four escape for each time point):After 1 and 2 d of water loading (early stages of escape) andafter 4 d of water loading (late stages of escape). Thus, atotal of 24 rats were separated into 12 control rats and 12escape rats, and four versus four rats were selected arbitrarilyfor IMCD analysis at each of the three time points. The day1 and day 4 time points were selected for DIGE analysis, whereasall three time points were analyzed by immunoblotting. IMCDsuspensions were prepared using the method of Stokes et al.(16) with some modifications (17) (Supplemental Materials availableonline).
Semiquantitative Immunoblotting
Immunoblotting was carried out as described previously (18)(see Supplemental Materials). The IMCD pellet was solubilizedin 5x Laemmli sample buffer (1 vol per 4 vol of sample) followedby heating to 60°C for 15 min before electrophoresis. Equalloading was confirmed by staining gels loaded for all threetime points (24 samples) with Coomassie blue (18). This loadinggel was scanned with a linear fluorescence scanner (Odyssey;Li-Cor Biosciences, Lincoln, NE) at an excitation wavelengthof 700 nm (Supplemental Figure 1 available online).
DIGE
DIGE analysis was carried out (for day 1 and day 4 time points,each four versus four samples) as described previously (12,19)(see Supplemental Materials). Briefly, before two-dimensional(2-D) gel electrophoresis, IMCD proteins were solubilized in2-D sample buffer (7 M urea, 2 M thiourea, 30 mM Tris Cl, and4% CHAPS [pH 8.5]). The samples were labeled on lysine sidechains with Cy3- (control), Cy5- (escape), or Cy2- (mixtureof control + escape samples, internal standard) fluorophoresusing N-hydroxysuccinimide chemistry (Amersham). Isoelectricfocusing was performed using an IPGphor apparatus (Amersham,Piscataway, NJ). Isoelectric focusing strips were loaded ontoEttan DALT-6 electrophoresis unit (Amersham) and further separatedon a 10% SDS-PAGE gel (5 W/gel).
Fluorescence analytical gel images were obtained (Typhoon scanner;100 µm resolution; Amersham) using the following emissionfilters: Cy2 (520 BP 40), Cy3 (580 BP 30), and Cy5 (670 BP 30).Spot matching, quantification, and statistical analyses wereperformed using DeCyder software (Version 5.0; Amersham). Thecorresponding Cy3 (control) and Cy5 (escape) images were normalizedto the pooled internal standard (Cy2) for that gel using a Least-Means-Squared-Gradient-Descentalgorithm. One gel was chosen as the "master," and all remaininganalytical gels were matched and normalized to the Cy2 masterspot map. The resulting protein abundance ratios, now representedas standardized log abundance values, were compared using anunpaired t test. The inverse log of these values is presentedin Table 1 as protein abundance ratio. A protein abundance ratio>1 corresponds to an increase in escape compared with controlsamples, whereas a ratio <1 corresponds to a decrease inescape (significance criterion, P 0.05). For picking, gelswere fixed in 30% ethanol/7.5% acetic acid for 2 h followedby SYPRO Ruby (610 BP 30) staining overnight for total proteinvisualization.
Table 1. IMCD proteins regulated at early (day 1) or later (day 4) stages of vasopressin escapea
A robotic workstation (Ettan; Amersham) was used to excise proteinspots, perform in-gel tryptic digestion, extract peptides fromthe gel, and transfer the extracts onto a MALDI substrate. Spectrawere acquired with an ABI 4700 MALDI-TOF/TOF mass spectrometer,and proteins were identified by database matching using Mascot.
Bioinformatic Pathways Analysis
Regulated proteins identified by DIGE were analyzed furtherby bioinformatic pathways analysis (Ingenuity Pathway Analysis[IPA]; Ingenuity Systems, Mountain View, CA; www.ingenuity.com).IPA constructs hypothetical protein interaction clusters onthe basis of a regularly updated "Ingenuity Pathways KnowledgeBase." The Ingenuity Pathways Knowledge Base is a very largecurated database that consists of millions of individual relationshipsbetween proteins, culled from the biologic literature. Theserelationships involve direct protein interactions, includingphysical binding interactions, enzyme substrate relationships,and cis-trans relationships in transcriptional control. Thenetworks are displayed graphically as nodes (individual proteins)and edges (the biologic relationships between the nodes).
In practice, a data set that contains the GenBank identifiersof differentially expressed proteins identified in the DIGEexperiment is uploaded into IPA. IPA then builds hypotheticalnetworks from these proteins, and other non–DIGE-identifiedproteins from the database that are needed fill out a proteincluster. Network generation is optimized for inclusion of asmany proteins from the inputted expression profile as possibleand aims for highly connected networks.
IPA computes a score for each possible network according tothe fit of that network to the inputted proteins. The scoreis calculated as the negative base-10 logarithm of the P valuethat indicates the likelihood of the inputted proteins in agiven network being found together as a result of random chance.Therefore, scores of 2 or higher have at least a 99% confidenceof not being generated by random chance alone. For previousstudies using IPA, see Siripurapu et al. (13) and Raponi etal. (14).
Verifying Vasopressin Escape
In this model of vasopressin escape, both control and experimentalrats received a continuous dDAVP infusion starting on day –3,but only the experimental rats received a daily water load,mixed with the food, starting on day 0. As previously noted(2,7,9), rats began to "escape" from dDAVP-induced antidiuresison the second day, i.e., 24 to 48 h after initiation of waterloading, as evidenced by a marked increase in urine excretionrate (Figure 1A). Urine osmolality (Figure 1B) was reduced significantlyin escape on the second day of water loading. Plasma sodiumlevels (Figure 1C) showed an acute decrease between days 1 and2 (from 137 ± 2 to 105 ± 3 mmol/L) and, subsequently,a partial recovery on day 4 (115 ± 3 mmol/L) in responseto vasopressin escape. Plasma urea levels were significantlylower in escape animals both at early (day 1: 7.0 ± 0.7versus 5.0 ± 0.4 mmol/L) and late (day 4: 7.0 ±0.5 versus 5.0 ± 0.4 mmol/L) stages of vasopressin escape(Figure 1D).
Figure 1. Urine excretion rate, urine osmolality, plasma sodium concentration, and plasma urea concentration during vasopressin escape. (A) Urine excretion rate over the course of the experiment with water loading commencing on day 0. Urine excretion rate was increased significantly from day 2 in the escape group relative to control (four versus four rats per time point). Negative time points represent the equilibration period. (B) Urine osmolality over the course of the experiment. Osmolality was decreased significantly from day 1 to the end of the experiment in the escape group relative to control (four versus four rats per time point). (C) Plasma sodium concentrations at days 1, 2, and 4. Plasma sodium was significantly lower in the escape group relative to control on all 3 d (four versus four rats per time point). (D) Plasma urea concentrations at days 1 and 4. Plasma urea was significantly lower in the escape group relative to control on both days (four versus four rats per time point). *P < 0.05 for all.
Changes in Abundances of Transport Proteins in IMCD Figure 2 shows immunoblots for AQP2, -ENaC, and collecting ducturea transporters in IMCD on day 4 of vasopressin escape anddensitometry values for all three time points. Downregulationof AQP2 and upregulation of -ENaC is consistent with previousstudies (7,9). A novel finding was that UT-A3 showed a 50% decreasein band density on day 2 of vasopressin escape and was decreasedfurther on day 4. Conversely, UT-A1 did not show a significantdecrease.
Figure 2. Immunoblots showing changes in abundances of aquaporin 2 (AQP2), subunit of epithelial Na channel (-ENaC), and collecting duct urea transporters in inner medullary collecting ducts (IMCD) from rats that underwent vasopressin escape versus control rats. Immunoblots are of IMCD cells purified from rat renal medullas at late stage of vasopressin escape (day 4 time point). Each lane is loaded with a sample from a different rat (n = 4 rats per treatment). Thirty micrograms of total protein was loaded in each lane, and the resulting immunoblots were probed with anti-AQP2 antibody (L127), anti–-ENaC (Q3560-2), anti–UT-A3 (Q2695-2), or anti–UT-A1 (L403). To the right of each immunoblot is a bar graph showing densitometry values for all three time points studied. *P < 0.05.
Changes in Rat IMCD Proteome in Vasopressin Escape Figure 3 shows an example of a DIGE gel with superimpositionof Cy3 (control, red) and Cy5 (escape, green) images of thegel. Spots corresponding to proteins expressed at nearly equallevels in the two samples appear yellow, those upregulated inresponse to vasopressin escape appear green, and those downregulatedin response to vasopressin escape appear red. Flanking the 2-Dgel image are 3-D pixel density plots for nine selected proteinsidentified by MALDI-TOF/TOF mass spectrometry, including heat-shockprotein 70 (HSP70), ATP synthase, calreticulin, prohibitin,mitochondrial aconitase, Sec23B, annexin A2, malate dehydrogenase,and protein disulfide isomerase (PDI). PDI had an apparent shiftin isoelectric point, suggestive of a posttranslational modification.Only protein spots with statistically significant abundanceratios (P 0.05 for four pairs of samples) were selected forMALDI-TOF/TOF identification. Moreover, only those identificationswith expectation values (i.e., an indicator of the degree ofcertainty of an identification) larger than 95% were accepted.A total of 22 protein spots were identified by MALDI-TOF/TOFmass spectrometry (Table 1). More proteins with altered abundancelevels were identified at day 1 than at day 4. Those identifiedincluded proteins that were downregulated and upregulated andone protein that shifted position in the gel (Table 1; Figure 3)in response to vasopressin escape. Also listed in Table 1are the theoretical molecular weights and theoretical isoelectricpoints. The molecular weights and isoelectric points for allof these proteins matched those derived from the spot positionon the gel, providing additional verification of the identifications.
Figure 3. Two-dimensional (2-D) gel showing changes in the IMCD proteome in vasopressin escape. Superimposed images from samples labeled with Cy3 (control, red pseudocolor), and Cy5 (escape, green pseudocolor) and 3-D representation of spot intensities. Spots that appear red or green represent proteins that are respectively down- or upregulated in vasopressin escape, whereas proteins that are equally abundant in both samples appear yellow. Full range of horizontal axis is from 3 pH units (left) to 10 pH units (right). Full range of vertical axis is 15 kD (bottom) to 120 kD (top). pI, isoelectric point; MW, molecular weight.
Pathways Analysis of Vasopressin Escape Figure 4 shows the largest protein cluster that was generatedby the pathways analysis of the proteins listed in Table 1.This "vasopressin escape cluster" consists of a network of 33proteins, including eight of the 22 proteins that were identifiedby DIGE-based proteomics and 25 additional proteins that wererecognized as being related because of their reported interactionswith the proteins identified by DIGE. The nodes represent individualproteins listed by gene name (see Table 2 for glossary), whereasthe edges represent the interactions, which include direct physicalbinding, substrate–enzyme interactions, and/or cis-/transrelationship in transcriptional regulation. In Figure 4, nodesare displayed using different gray levels that represent howthe protein was identified and studied. Proteins that were identifiedby DIGE only are represented as light gray nodes, whereas proteinsthat were identified by DIGE and studied further by immunoblottingare represented as black nodes. Proteins that were identifiedby IPA only are represented as white nodes, whereas proteinsthat were identified by IPA and studied further by immunoblottingare represented as dark gray nodes. Finally, the index proteinAQP2 is shown. Because we were chiefly interested in identifyingcandidate proteins for follow-up by immunoblotting, we did notdiscriminate between the early and late time points for thepathways analysis. Annotation of the interactions in Figure4 is provided as Supplemental Materials.
Figure 4. Protein regulatory network associated with vasopressin escape. Protein regulatory network was generated by bioinformatic pathways analysis through the use of the Ingenuity Pathways Analysis (IPA) software. Proteins that are listed in Table 1 were analyzed. Individual proteins are displayed as nodes, using different shades of gray to represent how the protein was identified and studied. In addition, different shapes are used to represent the functional class of the gene product (see figure insert). The edges describe the nature of the relationship between the nodes: An edge with arrowhead means that protein A acts on protein B, whereas an edge without an arrowhead represents binding only between two proteins. P indicates phosphorylation as a special case of the former. The gene names associated with the proteins are shown; see Table 2 for glossary. See Supplemental Materials for detailed descriptions of individual protein–protein interactions, including literature references. The overall score for the depicted network was 34, indicating that the probability of matching the indicated proteins by a purely random event was 10–34.
Table 2. Glossary: Protein regulatory network associated with vasopressin escape
Confirmation by Semiquantitative Immunoblotting
Four of the 22 proteins that were identified by DIGE were selectedfor semiquantitative immunoblotting on the basis of the availabilityof high-quality antibodies. These were PDI, calreticulin, -tubulin,and HSP70 (Figure 5). Although DIGE and immunoblotting gavepercentage changes that differed somewhat for these four proteins,in general, immunoblotting confirmed the qualitative responsesdetected with the DIGE technique, as found previously (12,19).Of the 25 proteins identified by pathways analysis as potentiallyinvolved in vasopressin escape (Figure 4), 10 were selectedfor semiquantitative immunoblotting on the basis of the centralpositions in the protein cluster and of antibody availability.One of these is c-myc, a transcription factor that is regulatedin part by phosphorylation. At early stages of escape, totalc-myc abundance was decreased, whereas at later stages, theabundance of the phosphorylated form of c-myc was decreased(Figure 6). The abundances of four additional proteins thatwere identified by pathways analysis were decreased (annexinA4, GRP78/BiP, steroid receptor co-activator 1 [SRC-1], andc-fos; Figure 7A), whereas the abundances of five other proteinswere increased (caspase 3, p53, receptor for activated C kinase1 [RACK1], c-src, and c-jun; Figure 7B). The abundances of theproteins shown in Figures 5, 6, and 7 were changed only at thereported time point and were unchanged at the other time points(data not shown). To address whether the protein abundance changesare specific for the IMCD or occur in other tissues, we immunoblottedrenal cortex and brain samples from the same experiments andprobed with selected antibodies (Figure 8). Among the respondingproteins in IMCD, only c-src showed a similar response in renalcortex, and only HSP70 showed a similar response in brain.
Figure 5. Immunoblots confirming selected protein abundance changes identified by DIGE-based proteomics. IMCD suspensions, prepared at the day 1 and day 4 time points. Each lane is loaded with a sample from a different rat (n = 4 rats/treatment). Thirty micrograms of total protein were loaded in each lane, and the resulting immunoblots were probed with (for day 1) anti–protein disulfide isomerase (PDI), anti-calreticulin, anti–-tubulin, and (for day 4) anti–heat shock protein 70 (HSP70). The apparent change in mobility of -tubulin may represent an unidentified posttranslational modification. *P < 0.05.
Figure 6. Immunoblots of IMCD proteins using antibodies to total and phosphorylated c-myc at early and late stages of vasopressin escape. Each lane is loaded with a sample from a different rat (n = 4 rats/treatment). Thirty micrograms of total protein were loaded in each lane, and the resulting immunoblots were probed with anti–c-myc and anti-phosphorylated c-myc. *P < 0.05.
Figure 7. Immunoblots for selected proteins that were identified by pathways analysis. IMCD proteins that were identified by pathways analysis were immunoblotted (30 µg of total protein per lane). Immunoblots show the regulation of proteins at late stages of vasopressin escape (day 4 time point); all proteins were unchanged at early stages of vasopressin escape (day 1; data not shown). Each lane is loaded with a sample from a different rat (n = 4 rats/treatment). Immunoblots were probed with antibodies to proteins indicated at left. *P < 0.05.
Figure 8. Immunoblots for selected proteins in renal cortex and brain tissue during vasopressin escape (day 4). Each lane is loaded with a sample from a different rat (n = 4 rats/treatment), using renal cortex (A) and whole brain (B) homogenates. Thirty micrograms of total protein were loaded in each lane, and the resulting immunoblots were probed with antibodies to proteins indicated at left. *P < 0.05.
Proteomics analysis is seeing increasing use as a means of identifyingnew mechanistic hypotheses in physiology (20). An importantdisadvantage of all 2-D gel-based proteomics approaches is thatlow-abundance proteins, including many cell-signaling proteins,are unlikely to be identified (15,21). Hence, alternative approachesare needed to discover transcription factors and other regulatoryproteins that are involved in the responses to physiologic perturbations.In this study, we used a combination of DIGE-based proteomicsand pathways analysis to identify a protein regulatory networkwhose state is altered in association with the vasopressin escapeprocess and the associated downregulation of AQP2 expression.Proteins that were identified by DIGE were used as input datafor bioinformatic pathways analysis, which pointed to severaladditional proteins that hypothetically could be involved inthe escape process. These additional proteins corresponded individuallyto "hypotheses" that could be tested by immunoblotting askingwhether each protein was up- or downregulated as part of theescape process. Indeed, 10 of 10 proteins that were tested inthis way displayed the hypothesized regulation (Figures 6 and7). The outcome of this integrated approach was a cluster ofproteins, discussed below, that could participate in the onsetand maintenance of escape or alternatively in secondary responsesafter vasopressin escape. Most of the demonstrated protein abundancechanges occurred only in IMCD, not in renal cortex or brain,indicative of a selective response in the IMCD.
UT-A3 but Not UT-A1 Is Downregulated during Vasopressin Escape
As a preliminary step in these experiments, we carried out immunoblottingto verify the downregulation of AQP2 seen in previous studies(2,9). This study confirmed the decrease in AQP2 expressionand showed that the -subunit of ENaC is upregulated in IMCDin the same manner as in more proximal parts of the collectingduct (9). In addition, we extended our studies to collectingduct urea transporter abundances and demonstrated that UT-A3but not UT-A1 is markedly decreased in abundance during vasopressinescape (Figure 2). The decrease occurred in parallel with thereduction in AQP2. Because both UT-A1 (unchanged) and UT-A3(downregulated) share the same transcription start site andupstream regulatory regions (22), it is unlikely that transcriptionalregulation is the basis of the decrease in UT-A3 expression.Instead, recognizing that the 3' end of UT-A1 and UT-A3 transcriptsdiffer (22), it seems possible that the differential regulationof these two proteins is based on the different 3' ends of themRNA. Both mRNA stability regulation and translational regulationare based on specialized processes involving the 3' end of mRNAmolecules (23), raising the hypothesis that either of thesemechanisms may be involved in UT-A3 regulation.
The observed downregulation of UT-A3 could contribute to theincrease in water excretion seen during the escape process bya mechanism similar to that demonstrated in knockout mice thatlack both UT-A1 and UT-A3 (24). The knockout mice developeda urinary concentrating defect because urea failed to accumulatein the inner medulla, resulting in a urea-dependent osmoticdiuresis. Vasopressin escape is associated with modest extracellularfluid volume (ECF) expansion and hypertension (10). This ECFvolume expansion could play a role in the decreased expressionof collecting duct urea transporters, as suggested in a previousstudy that implicated aldosterone/salt-induced extracellularvolume expansion in regulation of collecting duct urea transporterexpression (25). In our study, serum urea levels were markedlydecreased as seen previously (11), a presumed consequence ofUT-A3 downregulation.
Abundances of Several Transcription Factors Are Altered during Vasopressin Escape Process
Amid the protein regulatory network identified in this studyare several transcription factors, whose cellular abundancesare presumably too low to be detected via the DIGE technique.Several of these were demonstrated to undergo changes in abundanceby immunoblotting. The earliest transcription factor to exhibitabundance changes was c-myc, whose abundance was found to bedecreased just before the increase in water excretion (day 1).c-Myc is a basic helix-loop-helix leucine-zipper protein thatbinds as a heterodimer to so-called E-box cis-elements. Thepresence of three such E-box elements in the 5'-flanking regionof the AQP2 gene (26) raises the possibility that c-myc abundancechanges could contribute to the fall in AQP2 expression duringthe escape response. Although c-myc is best known as a tumorpromotor oncogene, it also has physiologic functions in allcells that seem to be related to the state of differentiationand proliferation through general regulation of transcriptionand translation (27). Its role in translational regulation wasrevealed in DNA array studies, which showed that changes inc-myc levels are associated with parallel changes in the expressionof a host of ribosomal proteins (28). The demonstrated fallin c-myc expression therefore may be expected to be associatedwith a decrease in total cellular transcription and translation,at least at early stages of escape.
At later stages of vasopressin escape, we found a decrease inphosphorylated c-myc. The antibody recognizes c-myc phosphorylatedat threonine-58 and serine-62. These modifications result ina decrease in half-life of the c-myc protein, thereby increasingthe abundance of total c-myc. The phosphorylation is mediatedlargely by two kinases: c-jun N-terminal kinase (29) and glycogensynthase kinase-3 (30). The decrease in phosphorylation mayplay a role in the restoration of total c-myc toward controllevels on day 4 of the vasopressin escape protocol.
In addition to c-myc, several other transcription factors wereidentified whose abundances were altered at later stages ofvasopressin escape, viz. c-fos (decreased), c-jun (increased),and p53 (increased). In addition, the abundance of a transcriptionalco-factor, the SRC-1, was decreased.
c-Fos and c-jun together form the transcription factor called"activated protein 1" (AP-1), for which there is an enhancersite in the 5'-flanking region of the human AQP2 gene (31–33).Previously, we demonstrated that c-fos and c-jun are upregulatedin the rat renal inner medulla in response to long-term vasopressininfusion (34). Previous studies have demonstrated that AP-1and a cAMP response element are necessary for maximal transcriptionalactivation of the AQP2 gene in response to increased intracellularcAMP (31). Conceivably, the demonstrated decrease in c-fos expressioncontributes to the fall in AQP2 expression in vasopressin escape.c-Fos is itself regulated in part via a cAMP binding elementin its 5'-flanking region (33). SRC-1 is a transcriptional co-regulatorthat interacts with AP-1 and other transcription factors tomediate transcriptional regulation (35,36).
Abundances of Several Other Regulatory Proteins Are Altered during Vasopressin Escape
Aside from transcription factors, the protein regulatory networkthat was identified by pathways analysis included several otherregulatory proteins that potentially could play a role in vasopressinescape. One of these was c-src, a nonreceptor tyrosine kinasewhose abundance was increased three-fold during late stagesof vasopressin escape (Figure 7). c-Src is a critical proteinin the coupling between G-protein–coupled receptors andmitogen-activated protein kinase pathways (37). c-Src as wellas some of its substrates binds to -arrestin 2 (38), a key proteinin the internalization of the V2R. Note that c-src was alsoincreased in renal cortical samples (Figure 8), suggesting thatc-src upregulation may have been due to a systemic factor. Similarly,HSP70 was decreased in abundance not only in IMCD but also inbrain, perhaps related to the fall in systemic tonicity (39).Another protein whose abundance was upregulated was RACK1. Itsfunction seems to be broader than its name suggests, becauseit constitutes a component of the ribosome and thus may playa role in translational regulation (40). RACK1 is also antiapoptoticand a binding partner for c-src (41). Finally, many of the identifiedregulated proteins also play a role in the endoplasmic reticulum(ER) stress response, including calreticulin, GRP78/BiP, PDI,and caspase 3. ER stress results from situations in which theprotein folding capacity of the ER is exceeded such as generalizedacceleration of translation (42).
In conclusion, combined proteomics and pathways analysis servedto identify a protein network that is associated with vasopressinescape and contains both high- and low-abundance proteins. Thenetwork included several transcription factors that may be involvedin vasopressin escape as well as other relatively low-abundanceregulatory proteins. These findings provide a new frameworkfor the study of AQP2 regulation in the collecting duct, whichis critical to the understanding of SIADH and other forms ofhyponatremia.
Acknowledgments
This work was supported by the intramural budget of the NationalHeart, Lung, and Blood Institute (Z01-HL-01282-KE to M.A.K).E.J.H. was supported by the Dutch Kidney Foundation.
We thank Dr. M. Michalak (University of Alberta, Canada) forkindly providing a calreticulin antibody, Angel Aponte for experthelp with 2-D electrophoresis, Ellis Johns for assistance withimmunoblotting, David Caden for expert help with blood chemistry,and Dr. R. Zietse for helpful discussions.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Received for publication March 26, 2005.
Accepted for publication June 25, 2005.
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Top
Abstract
Introduction
-subunit of the epithelial Na channel (ENaC) (9), suggesting that the process that is responsible for suppression of AQP2 expression is selective. With regard to possible mediators of escape, roles have been suggested for nitric oxide and prostaglandins (10) and aldosterone (11). However, little is known about which intracellular signaling processes orchestrate the escape and how the vasopressin escape phenomenon is triggered and maintained. 
0.05). For picking, gels were fixed in 30% ethanol/7.5% acetic acid for 2 h followed by SYPRO Ruby (610 BP 30) staining overnight for total protein visualization. 
-tubulin, and HSP70 (Figure 5). Although DIGE and immunoblotting gave percentage changes that differed somewhat for these four proteins, in general, immunoblotting confirmed the qualitative responses detected with the DIGE technique, as found previously (12,19). Of the 25 proteins identified by pathways analysis as potentially involved in vasopressin escape (Figure 4), 10 were selected for semiquantitative immunoblotting on the basis of the central positions in the protein cluster and of antibody availability. One of these is c-myc, a transcription factor that is regulated in part by phosphorylation. At early stages of escape, total c-myc abundance was decreased, whereas at later stages, the abundance of the phosphorylated form of c-myc was decreased (Figure 6). The abundances of four additional proteins that were identified by pathways analysis were decreased (annexin A4, GRP78/BiP, steroid receptor co-activator 1 [SRC-1], and c-fos; Figure 7A), whereas the abundances of five other proteins were increased (caspase 3, p53, receptor for activated C kinase 1 [RACK1], c-src, and c-jun; Figure 7B). The abundances of the proteins shown in Figures 5, 6, and 7 were changed only at the reported time point and were unchanged at the other time points (data not shown). To address whether the protein abundance changes are specific for the IMCD or occur in other tissues, we immunoblotted renal cortex and brain samples from the same experiments and probed with selected antibodies (Figure 8). Among the responding proteins in IMCD, only c-src showed a similar response in renal cortex, and only HSP70 showed a similar response in brain. 
