Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1603
Submitted 22 May 2003 ; accepted in final form 27 September 2003
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ABSTRACT |
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vasopressin; mass spectrometry; collecting duct
In addition to these short-term effects, AVP has long-term effects on the collecting duct to regulate the abundances of the water channels AQP2 (12) and AQP3 (16, 40) and of two of the subunits of the epithelial sodium channel ENaC (- and
-subunits; see Ref. 14). These long-term actions of AVP are of paramount importance to the regulation of salt and water excretion. However, the mechanisms by which the long-term effects of AVP occur remain to be elucidated.
Recently, several groups have begun to investigate AVP actions on the collecting duct using discovery techniques focusing on the transcriptosome, such as serial analysis of gene expression (SAGE; see Ref. 35), subtractive hybridization differential display (9), and cDNA arrays (3). However, recent work has made it clear that changes in the levels of specific proteins in cells are not necessarily predictable from changes in the levels of the corresponding mRNA transcripts (3, 24). Apparently, regulation of protein abundance by mechanisms such as translational regulation and regulation of protein degradation are more important than previously appreciated (28). Thus, to gain a complete view of the long-term response of the collecting duct to vasopressin, proteomic techniques are needed.
To identify proteins involved in the direct and indirect responses to vasopressin in the IMCD, we used a relatively new proteomic differential display method called "difference gel electrophoresis" or DIGE (25, 43) coupled with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. In a recent paper, we showed that DIGE can be a very effective technique for identification of proteins in native IMCD cells isolated from whole inner medulla suspensions (21). The experimental model used in the present study is the Brattleboro rat (44) infused with either the V2R-selective vasopressin analog DDAVP or vehicle for 3 days. The Brattleboro rat lacks endogenous vasopressin because of a mutation in the neurophysin-vasopressin gene (37). Proteins in IMCD cells isolated from inner medullas of DDAVP-infused vs. vehicle-infused Brattleboro rats were labeled with different fluorescent dyes, mixed, and run on the same two-dimensional (2-D) gel to identify differentially expressed or modified proteins. These proteins were identified by MALDI-TOF mass spectrometry. Identification of DDAVP-responsive proteins was selectively verified by immunoblotting.
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METHODS |
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Sample preparation. For 2-D gel electrophoresis and immunoblotting, IMCD suspensions were made as described by Chou et al. (6). Briefly, inner medullas were dissected, finely minced with a razor blade, and transferred to a 12 x 75-mm glass tube containing tubule suspension solution (in mM: 118 NaCl, 5 KCl, 4 Na2HPO4, 25 NaHCO3, 2 CaCl2, 1.2 MgSO4, 5.5 glucose, and 5 sodium acetate) supplemented with 2 mg/ml collagenase B (Boehringer Mannheim, Indianapolis, IN) and 540 U/ml hyaluronidase (Worthington Biochemical, Freehold, NJ). Suspensions were incubated at 37°C with 95% air-5% CO2 superfusion. Every 15 min, suspensions were aspirated with a large-bore Pasteur pipette until all large tissue clumps were digested (75-90 min). Next, suspensions were centrifuged at 50 g, after which the supernatant was discarded, and the pellet was resuspended in tubule suspension solution. The procedure was repeated two times, and, after the third centrifugation, the resuspended pellet was centrifuged at 1,000 g for 5 min. Previously, we have demonstrated that this procedure gives an 10-fold enrichment of a collecting duct marker AQP2 and that the resulting pellet is contaminated with only very small amounts of non-IMCD cells (21). For 2-D gel electrophoresis, the pellet from the final step was solubilized in 200 µl 2-D sample buffer (7 M urea, 2 M thiourea, 30 mM Tris·Cl, and 4% CHAPS, pH 8.5) per two kidneys. CHAPS is a zwitterionic detergent that can efficiently solubilize most cellular proteins with the exception of integral membrane proteins. (In general, strong ionic detergents like SDS are needed for complete solubilization of integral membrane proteins. However, ionic detergents are incompatible with the isoelectric focusing step of the 2-D separation and cannot be used for this procedure. Hence, we emphasize that the DIGE method is likely to be relatively inefficient at identifying integral membrane proteins regulated by vasopressin.)
For immunoblotting, IMCDs were prepared as above and homogenized using a tissue homogenizer (Omni 1000 fitted with a micro-sawtooth generator) in ice-cold isolation solution (pH 7.6) containing 250 mM sucrose, 10 mM triethanolamine (Calbiochem), 1 mg/ml leupeptin (Bachem), and 0.1 mg/ml phenylmethylsulfonyl fluoride (United States Biochemical), and total protein concentration (bicinchoninic acid kit; Pierce) was adjusted to equal concentrations with isolation solution. The samples were solubilized in 5x Laemmli sample buffer (1 vol/4 vol sample) followed by heating to 60°C for 15 min.
Labeling of proteins with fluorescent dyes. In preparation for 2-D gel electrophoresis, IMCD samples from control and DDAVP-treated rats (solubilized in 2-D sample buffer) were labeled using specially designed fluorescent cyanine dyes. According to the manufacturer's guidelines, samples were labeled using Cy2 (control) or Cy3 (DDAVP-treated) N-hydroxysuccinamide (NHS) ester DIGE dyes (product numbers RPK0272, RPK0273; Amersham Biosciences) freshly dissolved in anhydrous dimethylformamide by mixing 50 µg protein with 1 µl CyDye (400 pmol/µl) and incubating this mixture on ice in the dark for 30 min. The reaction was terminated by the addition of 10 nmol lysine and subsequent incubation on ice in the dark for an additional 10 min.
2-D electrophoresis. For the first dimension, 50 µg of each of the two protein samples labeled with different fluorescent Cy dyes were mixed with 50-900 µg unlabeled protein (a 1:1 mixture of control and DDAVP-treated rat IMCD lysate). Rehydration buffer [7 M urea, 2 M thiourea, and 4% CHAPS, 2% dithiothreitol (DTT), and 2% (pH 3-10) Pharmalyte] was added to give a total volume of 450 µl. This was loaded on a 24-cm Immobiline DryStrip (pH 3-10 linear) by active rehydration for 10 h (Ettan IPGphor; Amersham Biosciences). Isoelectric focusing was accomplished by subsequently applying 500 V for 500 Vh, 1,000 V for 1,000 Vh, and 8,000 V for 62,533 Vh. After isoelectric focusing, strips were equilibrated in SDS equilibration buffer (50 mM Tris·HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 0.025% bromphenol blue) containing 0.5% DTT for 10 min at room temperature followed by an incubation in SDS equilibration buffer containing 4.5% iodoacetemide for 10 min at room temperature. After equilibration, strips were loaded on a 12% SDS-PAGE gel and run at 5 W/gel on an Ettan DALT-12 separation unit (Amersham Biosciences) until the blue front reached the bottom of the gel.
Image acquisition and analysis. Images were scanned on a Typhoon 9400 variable mode imager (Amersham Biosciences) at an excitation wavelength of 520/40 (maxima/bandwidth) for Cy2- or 580/30 for Cy3-labeled samples. The laser power was chosen so that no saturated signal was obtained. The resolution was 100 µm. Images were then processed with DeCyder Differential In Gel Analysis V4.0 software (Amersham Biosciences) to identify spot fluorescence intensities that were increased or decreased with DDAVP treatment.
After protein spots were designated to be picked, gels were fixed in 10% methanol-7% acetic acid for 1 h and incubated in SYPRO Ruby protein gel stain (Molecular Probes) for at least 2 h, after which they were washed in 10% methanol-7% acetic for 1 h. Next, the gels were scanned at an excitation wavelength of 610/30 to visualize all (including nonlabeled) proteins present in the gel. CyDye- and SYPRO-derived images were matched using DeCyder Biological Variation Analysis V4.00.07 software (Amersham Biosciences), from which a picklist was generated.
Spot picking and protein processing. The Ettan Spot Handling Workstation was used to subsequently cut out the selected protein spots from the gel, perform an in-gel tryptic digestion, extract the peptides from the gel, and transfer the digested proteins on a MALDI-TOF/Pro (Amersham Biosciences) sample slide. Peptides were analyzed using an Ettan MALDI-TOF/Pro mass spectrometer (Amersham Biosciences), and proteins were identified from the acquired spectra using a ProFound database (48).
Semiquantitative immunoblotting. Inner medullas were homogenized and prepared for immunoblotting. Equal loading was confirmed by staining identically loaded gels with Coomassie blue, as described previously (26). Most primary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA): annexin II (sc-1924), heat shock protein (HSP)-70 (sc-1060), nitric oxide synthase (NOS) type 2 (sc-8310), GRP78/BiP (sc-1050), cathepsin D (sc-6486), and adenylyl cyclase VI (sc-590). Additional polyclonal antibodies were supplied by independent investigators: rabbit anti-carbonic anhydrase II [Dr. William Sly (23)], rabbit anti-glutaminase [Dr. Norman Curthoys (10)], and goat anti-aldehyde reductase I [Dr. Peter Kador (41)]. Antibodies were diluted in a diluent containing 50 mM NaPO4, 150 mM NaCl, 0.05% Tween 20, and 0.1% BSA. The peroxidase-conjugated secondary antibodies were diluted at 1:5,000 in blot wash buffer (50 mM NaPO4, 150 mM NaCl, and 0.05% Tween 20) containing 5% nonfat dry milk. Band visualization was achieved using an enhanced chemiluminescence substrate (LumiGLO for Western blotting no. VC110; Kirkegaard and Perry) before exposure to X-ray film (Kodak 165-1579). The band densities were quantitated by laser densitometry (model PDS1-P90; Molecular Dynamics). To facilitate comparisons, the densitometry values were normalized to control, defining the mean for the control group as 100%.
Immunocytochemistry. Rat kidneys were perfusion fixed with a paraformaldehyde-based fixative, and 2-µm paraffin sections were prepared as described previously (31). Antibodies were among those used for immunoblotting (see RESULTS). Sections were labeled using the immunoperoxidase method described by Hager et al. (20).
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RESULTS |
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DDAVP-induced changes in the Brattleboro rat IMCD proteome. Brattleboro rats were given DDAVP (5 ng/h in isotonic saline, n = 3) or vehicle (n = 3) via osmotic minipumps for 72 h, after which the IMCD cells were isolated by collagenase/hyaluronidase dissociation and centrifugation (see METHODS). Whole homogenates were solubilized in 2-D sample buffer, and a single sample for each treatment was prepared by pooling equal amounts of protein from each of the three rats undergoing that treatment. The pooled samples were derivatized with NHS-Cy2 (vehicle) or NHS-Cy3 (DDAVP treated). Figure 2 shows a superimposition of the Cy2 (control) and Cy3 (DDAVP-treated) images from the same 2-D gel. In this image, spots corresponding to proteins expressed at nearly equal levels in the two samples appear yellow, those upregulated in response to DDAVP infusion appear green, and those downregulated in response to DDAVP appear red. Flanking the 2-D gel image are three-dimensional pixel density plots for eight selected proteins that were identified: adenylyl cyclase VI, GRP78 (BiP), HSP-70, cathepsin D, NOS2, aldehyde reductase I, proteasome subunit , and annexin II. Protein spots that were decreased or increased >1.5-fold were selected for MALDI-TOF identification. As seen in Table 1, a total of 113 protein spots was identified by MALDI-TOF mass spectrometry in seven different gels loaded with varying protein amounts (150-1,000 µg). Also listed in Table 1 are the theoretical molecular weights, theoretical isoelectric point values, and the expectation values of the identifications. The expectation value is an indicator of the degree of certainty of an identification, with low values indicating the greatest certainty (18). (For example, an expectation value of 0.01 indicates a 99% chance of a correct identification, or a 1% chance of an incorrect identification.) The molecular weights and isoelectric point values for all of these proteins matched those derived from the spot position on the gel, providing an initial verification of the identifications (16 protein identifications did not match expected molecular weight and isoelectric point values and were therefore not reported). The identified proteins included proteins that were downregulated, proteins that were upregulated, and proteins that shifted positions in the gel in response to DDAVP. Several proteins of regulatory significance in the IMCD were identified to be up- or downregulated in response to DDAVP (see DISCUSSION).
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Of particular interest is the group of proteins that appear to be shifted in the gel in response to DDAVP infusion (Table 1), namely, HSP-27, cytokeratin 8, and -actin. Each protein was identified in two discrete protein spots, one of which increased and one of which decreased in intensity in response to DDAVP. Figure 3 shows magnified Cy2 and Cy3 gel images for two of these proteins. For HSP-27 (Fig. 3, left) and cytokeratin 8 (Fig. 3, right) and
-actin (data not shown), the two spots were at virtually the same molecular weight but at different isoelectric points, suggestive of posttranslational modifications consisting of addition or removal of a small charged moiety, for example, a phosphate group. The specific nature of these modifications, however, cannot be ascertained from the present data.
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Confirmation of IMCD expression of identified proteins by immunocytochemistry. Immunocytochemistry was performed for some of the proteins identified by MALDI-TOF peptide-mass fingerprinting to confirm their presence in IMCD cells. Results of this analysis, shown in Fig. 4, indicate that, for all six proteins analyzed, localization in the IMCD could be confirmed, although expression was not absolutely restricted to the IMCD for any protein.
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Verification of protein abundance changes by semiquantitative immunoblotting. For selected proteins, we used semiquantitative immunoblotting to verify the responses seen with the DIGE method. For this, separate IMCD protein samples were prepared from inner medullas of six vehicle-infused and six DDAVP-infused rats. Urine osmolality increased from 210 ± 12 to 1,248 ± 286 after 3 days of DDAVP administration. A preliminary immunoblot for AQP2 confirmed the efficacy of the DDAVP infusion by showing the expected increase in AQP2 abundance [mean band density increased to 174 ± 14% (SE) of control, not shown; see Ref. 12]. The quantification of the immunoblots for selected proteins identified in this study is summarized in Table 2. The changes in protein abundances for these proteins are plotted against Cy2-to-Cy3 ratios from DIGE analysis in Fig. 5. Although DIGE and immunoblotting gave percent changes that differed somewhat in some cases, in general, immunoblotting confirmed the qualitative responses detected with the DIGE technique.
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DISCUSSION |
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In the present study, using DIGE coupled to MALDI-TOF identification of proteins, we have identified several IMCD proteins regulated in response to DDAVP infusion in Brattleboro rats. Previously, complementary experiments by Brooks and colleagues (3) were carried out to identify the effects of DDAVP (using the same animal-treatment protocol) on mRNA abundances in the renal inner medulla using cDNA arrays. The experimental model was designed to determine the overall physiological response to V2R occupation in the IMCD, including both direct effects of vasopressin and indirect effects that may result from changes in tubule fluid flow rate or interstitial composition. Both cDNA array studies and proteomics studies are "discovery" approaches whose outputs are hypotheses that can be tested with more detailed hypothesis-driven experimentation. The present experiments identified three categories of responses to DDAVP infusion, namely, decreases in protein abundance, increases in protein abundance, and shifts in the position of protein spots on the 2-D gels (Table 1). The latter response is likely to be the result of posttranslational modifications that alter the isoelectric point of proteins, e.g., phosphorylation.
In general, protein identifications with MALDI-TOF can be preliminarily validated by comparison of the theoretical physical properties (i.e., molecular weights and isoelectric points) of the identified proteins with corresponding values measured from the gel images. This internal check of the validity of the identification was carried out for all identifications reported herein. Nevertheless, it is theoretically possible that the mass spectrometer can identify one protein in a spot that actually was derived from an overlap of two or more proteins and consequently could yield a false identification of a regulated protein. For this reason, we carried out immunoblotting for 9 of the 43 proteins listed in Table 1, comparing samples from six DDAVP-treated Brattleboro rats vs. six vehicle-treated Brattleboro rats to determine how well quantification by the DIGE method corresponded to quantification by densitometry of immunoblots. Of the nine proteins examined by immunoblotting, six were increased in response to DDAVP infusion: NOS-2, GRP78, HSP-70, annexin II, glutaminase, and cathepsin D. The remaining three were decreased in response to DDAVP: aldehyde reductase I, adenylyl cyclase VI, and carbonic anhydrase II. In general, the immunoblots confirmed the changes seen by DIGE, although the magnitude of the changes differed in some cases (compare Fig. 5 and Table 1). Thus we conclude that the DIGE method can give valid identification of differentially expressed proteins in IMCD cells.
IMCD proteins regulated in response to DDAVP infusion are listed in Table 1. There were a substantial number of proteins in this list that are known to play roles in cell signaling in various cell types. These included proteins with decreased abundance in response to DDAVP infusion (NADPH oxidase -subunit, GTP-binding protein Gh, annexin V, arginase II, adenylyl cyclase V/VI, and leukotriene A-4 hydrolase) and proteins with increased abundance in response to DDAVP [annexin II, G protein-coupled receptor kinase (GPCR) 4B, and NOS2 (iNOS)].
These results point to the hypothesis that vasopressin may trigger coordinate control of several regulators of nitric oxide production in the IMCD cell. Specifically, an enzyme that produces nitric oxide (NOS2) was upregulated by vasopressin, whereas an enzyme that indirectly consumes nitric oxide through generation of oxygen free radicals (NADPH oxidase) was downregulated by vasopressin. Furthermore, although NOS2 was upregulated by vasopressin, another enzyme that competes for its substrate (arginine) but does not produce nitric oxide, namely, arginase II ("nonhepatic arginase"; see Ref. 29), was downregulated in response to DDAVP infusion. These observations fit with previous observations showing that vasopressin increases nitric oxide production in the IMCD as a result of vasopressin-induced calcium moblization (30) and that long-term increases in circulating vasopressin secondary to water restriction of rats is associated with increased NOS2 mRNA and protein in the IMCD (38). NOS activity is higher in the IMCD than in all other renal tubule segments (47), and nitric oxide production at this site has been implicated as an important factor that homeostatically counters the antinatriuretic effects of vasopressin, thereby preventing the development of hypertension when circulating levels of vasopressin are high (39). Nitric oxide, acting through increases in cellular cGMP, has been implicated both as a positive (2) and a negative (19) regulator of water permeability in the collecting duct.
Examination of the data in Table 1 also leads to hypotheses relevant to the mechanism of the vasopressin-escape phenomenon in the collecting duct. Vasopressin escape is associated with downregulation of AQP2 (15) resulting from decreased cAMP production in response to vasopressin (13). The results in these papers point to two potential mediators of this response, namely, adenylyl cyclase VI (the predominant form of adenylyl cyclase in the collecting duct; see Ref. 5), which was downregulated, and GPCR kinase 4B, which was upregulated. Adenylyl cyclase VI catalyzes cAMP production. GPCR kinase 4B stimulates receptor internalization by phosphorylating their COOH-terminal tails, providing a target for the binding of -arrestins, which in turn stimulate clathrin-mediated endocytosis (33). Previous studies using the same animal model have demonstrated upregulation of
-arrestin 2 in the IMCD in response to DDAVP infusion (3). It is conceivable that coordinate downregulation of the V2R (mediated by increased GPCR kinase 4B and
-arrestin 2) couples with the demonstrated reduction in adenylyl cyclase VI to account for the phenomenon of vasopressin escape.
An additional protein upregulated in response to DDAVP infusion was annexin II (Table 1 and Figs. 2 and 3). Annexin II is a calcium-binding protein that is expressed in the IMCD and in other structures of the inner medulla (21). Various roles for the annexins have been proposed, including involvement in vesicle fusion (17). Annexin II is potentially one of the downstream targets for intracellular calcium in the vasopressin-induced trafficking of AQP2 (7). A recent study also demonstrated association of annexin II in a protein complex with the epithelial calcium channel ECaC and the usual binding partner of annexin II, S100A10 (45). ECaC is believed to be the major apical entry pathway for calcium in the calcium reabsorptive process in the renal connecting tubule and collecting duct. Vasopressin is known to decrease calcium absorption in the cortical collecting duct by a PGE2-dependent mechanism (22), but regulation of calcium transport in the IMCD has not been investigated to our knowledge. In addition to annexin II, the DIGE analysis also demonstrated regulation of another annexin in response to DDAVP infusion, namely, annexin V, which, decreased (Table 1).
Also upregulated in IMCD cells in response to long-term DDAVP infusion was the molecular chaperone HSP-70. This protein has previously been demonstrated to be upregulated in cultured MDCK cells in response to increased tonicity (8), and we speculate that the increase in HSP-70 expression in the present study is a response to altered inner medullary tonicity rather than to DDAVP itself.
Another protein upregulated in response to DDAVP infusion was GRP78 (or BiP), an endoplasmic reticulum-resident molecular chaperone belonging to the 70-kDa HSP family. Upregulated expression of GRP78 in response to DDAVP was associated with changes in the abundances of other proteins involved in endoplasmic reticulum function, including protein disulfide isomerases A6 (increased) and A3 (decreased). Considering the fact that vasopressin also altered the abundance of several proteins involved in protein degradation, like proteasome 28 subunit- (decreased), the renin-like protease cathepsin D (increased), and calpain 1 (decreased), it seems likely that many changes in the IMCD proteome in this experiment may have been mediated in part by posttranscriptional processes.
Although the use of the DIGE approach with MALDI-TOF identification of proteins has provided important new data concerning direct and indirect actions of vasopressin in the IMCD, certain limitations should be emphasized. First of all, as described in METHODS, integral membrane proteins generally do not enter the 2-D gel because detergents that are compatible with isoelectric focusing often do not solubilize them. In this study, only one integral membrane protein was identified (adenylyl cyclase type VI). Second, proteins at the extremes of molecular weight or isoelectric point often are not identified. Third, there is a bias toward abundant proteins. Because these problems are inherent in proteomics techniques based on 2-D electrophoresis, it is likely that some of them will be solved through use of alternative separation methods (32a). We emphasize that no single method is likely to be capable of identifying all of the key regulatory pathways involved in the response of the collecting duct to vasopressin. Hence, it will continue to be necessary to employ a broad spectrum of techniques, including gene expression studies that identify regulated transcripts, as well as classic hypothesis-driven physiology and biochemistry.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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