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【摘要】 Proximal tubules were isolated from control and acidotic rats by collagenase digestion and Percoll density gradient centrifugation. Western blot analysis indicated that the tubules were 95% pure. The samples were analyzed by two-dimensional difference gel electrophoresis (DIGE) and DeCyder software was used to quantify the temporal changes in proteins that exhibit enhanced or reduced expression. The mass-to-charge ratios and the amino acid sequences of the recovered tryptic peptides were determined by MALDI-TOF/TOF mass spectrometry and the proteins were identified using Mascot software. This analysis confirmed the well-characterized adaptive responses in glutaminase (GA), glutamate dehydrogenase (GDH), and phospho enol pyruvate carboxykinase (PEPCK). This approach also identified 17 previously unrecognized proteins that are increased with ratios of 1.5 to 5.6 and 16 proteins that are decreased with ratios of 0.67 to 0.03 when tubules from 7-day acidotic vs. control rats were compared. Some of these changes were confirmed by Western blot analysis. Temporal studies identified proteins that were induced either with rapid kinetics similar to PEPCK or with more gradual profiles similar to GA and GDH. All of the mRNAs that encode the latter proteins contain an AU sequence that is homologous to the pH response element found in GA mRNA. Thus selective mRNA stabilization may be a predominant mechanism by which protein expression is increased in response to acidosis.
【关键词】 difference gel electrophoresis mass spectrometry phospho enol pyruvate carboxykinase glutaminase mRNA stabilization
METABOLIC ACIDOSIS IS A COMMON clinical condition that is caused by the overproduction of an acid or the reduced recovery of bicarbonate and is characterized by a decrease in blood pH and bicarbonate concentration ( 37 ). In response to acidosis, the kidneys exhibit a complex set of adaptive responses. Within the renal proximal tubule, this process is characterized by a rapid increase in the catabolism of plasma glutamine ( 40 ). Within 1 to 3 h, the arterial plasma glutamine concentration is increased twofold ( 19 ) and significant renal extraction becomes evident. Net extraction rapidly reaches 30% of the plasma glutamine, a level that exceeds the percentage filtered by the glomeruli. Thus both apical and basolateral transport of glutamine must contribute to uptake by the epithelial cells of the proximal tubule. In addition, mitochondrial transport and catabolism of glutamine are acutely activated ( 34 ). The resulting increases in renal synthesis of ammonium and bicarbonate ions partially restore acid-base balance ( 4 ). Further responses include a prompt acidification of the urine that results from an acute activation of NHE3, the apical Na + /H + exchanger ( 30 ). This process facilitates the rapid removal of cellular ammonium ions ( 39 ) and ensures that the bulk of the ammonium ions generated in the proximal tubule is excreted in the urine. Finally, a pH-induced activation of -ketoglutarate dehydrogenase reduces the intracellular concentrations of -ketoglutarate and glutamate ( 29 ). Thus the increased catabolism of glutamine initially results from a rapid activation of key transport processes, an increased availability of glutamine, and a decreased concentration of the products of the glutaminase (GA) and glutamate dehydrogenase (GDH) reactions.
During chronic metabolic acidosis, the acute decreases in the renal concentrations of glutamate and -ketoglutarate are partially compensated and the arterial plasma glutamine concentration is decreased to 70% of normal ( 4 ). However, the kidney continues to extract more than one-third of the total plasma glutamine ( 36 ) in a single pass through this organ. Renal catabolism of glutamine is now sustained by increased expression of various transporters and key enzymes of glutamine metabolism ( 7 ). Following onset of acidosis, a rapid induction of phospho enol pyruvate carboxykinase (PEPCK) gene expression occurs within the S1 and S2 segments of the proximal tubule ( 11 ). A more gradual increase in the level of the mitochondrial GA also occurs solely within the proximal convoluted tubule ( 9, 43 ). The adaptations in GA and PEPCK levels result from increased rates of synthesis of the proteins ( 22, 41 ) that correlate with comparable increases in the levels of their respective mRNAs ( 6, 20 ). However, the increase in GA results from the selective stabilization of the GA mRNA ( 13, 26, 27 ), whereas the increase in PEPCK activity results primarily from enhanced transcription of the PEPCK gene ( 15 ). The activities of the mitochondrial glutamine transporter ( 34 ) and GDH ( 42 ), the apical NHE3 ( 32 ), the basolateral SN1 glutamine transporter ( 25 ), and NBC1, the basolateral Na + -3HCO 3 - cotransporter ( 32 ), are also increased in the proximal tubule during chronic acidosis. The combined adaptations facilitate the increased reabsorption of bicarbonate ions, the increased synthesis of ammonium and bicarbonate ions, and their vectoral transport across the apical and basolateral membranes, respectively. The onset of acidosis also causes an increased expression of the apical Na + /dicarboxylate cotransporter, NaDC-1, the cytoplasmic citrate lyase, and the mitochondrial aconitase ( 1 ). The latter adaptations contribute to the increased reabsorption and metabolism of citrate that support gluconeogenesis and produce HCO 3 -. Finally, the renal proximal tubule also undergoes an extensive hypertrophy during chronic metabolic acidosis ( 28 ).
Our current understanding of the renal adaptations to metabolic acidosis has been derived from the work of numerous investigators who have characterized one or more of the individual responses. However, it is highly probable that the cumulative studies in this field have uncovered only a small fraction of the adaptive responses that occur in the proximal tubule and are essential to maintenance of acid-base balance. Proteomics and bioinformatics offer an experimental approach to accurately quantify the altered expression of multiple proteins and to identify commonalities that provide insight into the signal transduction pathways and molecular mechanisms that mediate this essential adaptive response. The current study reports the application of difference gel electrophoresis (DIGE) to further characterize the temporal changes in multiple proteins that occur within the rat renal proximal tubule during the development of chronic metabolic acidosis.
MATERIALS AND METHODS
Materials. Male Sprague-Dawley rats (150-200 g) were obtained from the National Cancer Institute-Frederick Cancer Research Facility or purchased from Charles River. The N -hydroxysuccinimide ester derivatives of Cy2, Cy3, and Cy5 dyes and all other reagents for two-dimensional (2-D) gel electrophoresis were obtained from GE Healthcare. Sypro Ruby stain was purchased from Molecular Probes. Primary antibodies were obtained from Calbiochem, Cayman Chemicals, Lab Vision, Mito Sciences, R&D Systems, Rockland, and Upstate. The antibodies specific for arginine-glycine amidinotransferase and dimethylglycine dehydrogenase were obtained from Dr. J. Van Pilsum (University of Minnesota) and Dr. R. Brandsch (University of Freiburg), respectively. The rabbit anti-rat renal glutaminase antibody was prepared as described previously ( 8 ). Antibody specific for the GAC isoform of glutaminase was prepared vs. a unique peptide sequence (LKETVWKKVSPESN) that is contained in the COOH terminus of the GAC isoform (Macromolecular Resources, Ft. Collins, CO). The secondary antibodies, goat anti-rabbit IgG IRDye800, and goat-anti mouse IgG AlexaFluor680, were obtained from Invitrogen and Rockland, respectively. The Odyssey blocking buffer and the black washing boxes were purchased from Li-Cor Biosciences. All other biochemicals were purchased from Sigma.
Isolation of proximal tubules. Rats were made acidotic by stomach loading with 20 mmol NH 4 Cl and then providing 0.28 M NH 4 Cl as their sole source of drinking water for 2 h and for 1, 3, and 7 days. The protocols used in this study were approved by the university Animal Use and Care Committee (protocol 93-250A-12). Proximal tubules were isolated using a modification of a protocol developed to isolate mouse proximal tubules ( 10 ). Briefly, the rat was decapitated, the kidneys were removed, and the cortex was dissected and then sliced with a razor blade into 1- to 2-mm chunks. Approximately 0.8 g of minced cortex was incubated in a 37°C shaker at 140 rpm in 13 ml of PBS-glucose (138 mM NaCl, 5 mM KCl, 2 mM NaH 2 PO 4, 1 mM MgCl 2, and 5 mM glucose) supplemented with 4 mM glycine, 1 mM heptonoate, 2 mg/ml collagenase, 1 mg/ml BSA, and 0.1 mg/ml DNase I. After 15 min, the tissue was dispersed by drawing up and down 10 times with a 25-ml pipette. The released tubules were decanted and the process was repeated two more times using a 10-ml pipette to disperse the tissue. The combined tubules were centrifuged at 100 g for 1 min, washed once, and resuspended with 40 ml of 42.5% Percoll in PBS-glucose. The sample was centrifuged for 35 min at 35,000 g at 4°C in an SS34 rotor. After centrifugation, the band of tubules that formed about three/fourths of the way down the tube was removed, diluted twofold with PBS-glucose, and pelleted by centrifugation at 100 g for 1 min. The resulting proximal tubules were divided into two samples that were either homogenized in PBS-glucose containing 0.1 mg/ml PMSF and 1 µg/ml leupeptin or solubilized in lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS {3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate} and 30 mM Tris, pH 8.8. Homogenates of kidney cortex were also prepared. The samples were divided into 0.2-ml aliquots and stored at -80°C. Protein concentrations were determined using the Bradford assay ( 3 ) with bovine serum albumin as the standard. The protocol produced 5-10 mg of proximal tubule protein from the kidneys of a single rat.
DIGE analysis. DIGE was performed as described previously ( 18 ). Briefly, for single gel analyses, samples containing 50 µg of a proximal tubule lysate obtained from control or acidotic rats were labeled with Cy3 or Cy5 dye, respectively. The samples analyzed for increased protein expression were supplemented with 300 µg of unlabeled protein from an acidotic proximal tubule lysate, whereas the samples analyzed for decreased proteins were supplemented with 300 µg of a control proximal tubule lysate. Temporal analyses were performed by merging the images from four separate 2-D gels. Each gel included a common 50-µg control containing 6.25 µg of each of duplicate control, 1-day acidotic, 3-day acidotic, and 7-day acidotic tubules that were labeled with Cy2 dye. The four control samples were mixed individually with two 50-µg samples that were obtained from duplicate control, 1-day acidotic, 3-day acidotic, or 7-day acidotic tubules that had been labeled separately with Cy3 or Cy5 dye. For either type of analysis, the combined samples were diluted to 450 µl with rehydration buffer and isoelectric focusing (IPGphor apparatus, GE Healthcare) was performed using 24-cm, pH 3-10-nl strips. The initial isoelectric focusing step requires the use of a nonionic detergent. As a result, DIGE analysis is usually not effective for characterization of membrane proteins. After focusing, the strips were rinsed, reduced with DTT, and treated with iodoacetamide. The samples were then subjected to SDS-PAGE using 10 or 12% polyacrylamide gel. The gels were imaged using a Typhoon scanner (GE Healthcare) to individually quantify the fluorescence emitted from each of the Cy dyes. The gels were subsequently fixed in 30% ethanol/7.5% acetic acid, stained with Sypro Ruby, and reimaged to detect total protein. The images were merged and analyzed using Decyder software to identify protein spots that exhibited greater than 1.5-fold increases or 0.67-fold decreases in abundance. Approximately 50-70 spots were picked from each gel using an automated Spot Handling Workstation (Ettan, GE Healthcare) that also performed the trypsin digestion, extraction, and spotting of peptides on a MALDI plate. Further analysis was performed with an ABI 4700 MALDI/TOF/TOF mass spectrometer and proteins were identified using Mascot software to match the spectra to a rat database. The corresponding mRNA sequences were downloaded from GenBank and searched for pH response elements ( 13, 35 ) and AU-rich elements ( 2 ) using Omiga software.
Western blot analyses. Samples containing 9.2 µg of a rat renal proximal tubule homogenate were run on 7.5 to 15% SDS-PAGE gels in a Bio-Rad Mini-Protean 3 electrophoresis unit. Proteins were transferred to an Immobilon-FL PVDF membrane (Millipore) in a Bio-Rad Mini Trans-Blot cell. The gel probed for succinate dehydrogenase was soaked in a 10 mM CAPS, 10% methanol, pH 11 solution for 30 min before transfer in this buffer. All other gels were transferred using a 25 mM Tris, 192 mM glycine, 20% (vol/vol) methanol, pH 8.3 transfer buffer. Following transfer, the membranes were incubated overnight at 4°C in 25 ml of either 20 mM Tris, 150 mM NaCl, pH 7.5 (TBS) supplemented with 5% (wt/vol) nonfat dry milk (for rabbit primary antibodies) or Odyssey blocking buffer (for mouse primary antibodies). Blots were then equilibrated to room temperature, rinsed twice with 15 ml TBS, and then incubated with 5 ml of primary antibodies for 1-2 h with gentle rocking. The primary antibodies were diluted as recommended by the supplier in either TBS containing 1% nonfat dry milk powder and 0.1% Tween (rabbit antibodies) or in Odyssey blocking buffer with 0.1% Tween (mouse antibodies). The blots were rinsed twice in TBS-0.1% Tween (vol/vol) and then washed four times for 5-10 min each. Goat anti-rabbit IgG IRDye800 secondary antibodies were diluted 1:10,000 in TBS containing 1% milk, 0.1% Tween, and 0.05% SDS and goat-anti mouse IgG AlexaFluor680 secondary antibodies were diluted 1:10,000 in Odyssey blocking buffer with 0.1% Tween and 0.05% SDS. Blots were incubated in 5 ml of the appropriate secondary antibody for 1-2 h and then rinsed and washed as above, but in black washing boxes to minimize exposure to light. The final wash was carried out in TBS lacking Tween. The blots were stored at room temperature in the dark, in the final wash solution, until scanned on a Li-Cor Odyssey Infrared Imager.
RESULTS
Proteomic analysis. Before initiation of a proteomic analysis of the differentially expressed proteins, it was necessary to develop a protocol to isolate rat renal proximal tubules. This was accomplished by modifying an existing protocol to isolate mouse renal proximal tubules ( 10 ). The final protocol involved incubation of minced rat renal cortex with 2 mg/ml of collagenase to digest the extracellular matrix, followed by Percoll density gradient centrifugation. The resulting proximal tubules migrate well into the gradient, while glomeruli and other tubular segments collect at the top of the Percoll. The purity of the isolated proximal tubules was confirmed by Western blot analyses using antibodies to proteins that are expressed only within a single cortical segment of the nephron ( Fig. 1 ). This analysis indicated that the purified tubules retained PEPCK, a specific marker for the proximal tubule, but essentially lacked the Na + -K + -2Cl - cotransporter (NKCC2), a marker for thick ascending limbs; the thiazide-sensitive NaCl transporter (TSC), a marker for distal tubules; and aquaporin-2 (AQP-2), a marker for cortical collecting ducts. From this analysis and a visual inspection under a light microscope, the isolated proximal tubules appear to be 95% pure. Proximal tubules were prepared from control rats ( n = 3) and from rats that were made acidotic for 2 h and for 1, 3, and 7 days ( n = 2). The resulting samples were analyzed by DIGE.
Fig. 1. Western blot analysis of isolated proximal tubules. Duplicate samples of rat renal cortex or isolated proximal tubules were separated by SDS-PAGE and probed with antibodies to phosph enol pyruvate carboxykinase (PEPCK), the Na + -K + -2Cl - cotransporter (NKCC2), the thiazide-sensitive NaCl transporter (TSC), and aquaporin-2 (AQP-2) proteins that are expressed specifically in the proximal tubule (PT), thick ascending limb (TAL), distal tubule (DT), and collecting duct (CD), respectively.
Initially, equivalent aliquots of proximal tubule lysates from a control and a 7-day acidotic animal were labeled with Cy3 and Cy5 fluorescent dyes, respectively, mixed and then resolved on a 2-D gel. The gel was imaged with a Typhoon instrument to discriminate light emitted by the two fluorescent dyes. DeCyder software was used to ratio the light intensities emitted by nearly 2,000 different proteins that are resolved on the gel ( Fig. 2 ). The software also identifies and quantifies the proteins that are enhanced (pseudo-colored as red) or reduced (pseudo-colored as green) in the sample obtained from the acidotic animal. A robotic workstation was then used to pick 120 spots containing proteins that are differentially expressed, to perform trypsin digestion, and to spot the resulting peptides on a MALDI plate. The mass-to-charge ratios and the amino acid sequences of the recovered peptides were subsequently determined by MALDI-TOF/TOF mass spectrometry. Mascot software was used to compare the resulting data to a rat protein database and to identify the specific proteins.
Fig. 2. Difference gel electrophoresis of proximal tubules isolated from control and 7-day acidotic rats. Separate samples of proximal tubules isolated from a control (Cy3) and a 7-day acidotic (Cy5) rat were labeled and separated by 2-dimensional (2-D) gel electrophoresis. Images were analyzed using DeCyder software. Yellow spots represent proteins that are unchanged, whereas red or green spots represent proteins that are increased or decreased, respectively. The image is representative of that obtained from 4 separate 2-D gels.
The DIGE analysis identified PEPCK and GA as the two proteins that exhibit the greatest fold-induction in response to acidosis ( Fig. 3 ). The major bright red spot in this region of the 2-D gel corresponds to a protein that is increased sevenfold in proximal tubules isolated from 7-day acidotic rats relative to its level in control tubules. The protein contained in this spot was identified by MALDI-TOF/TOF analysis to be PEPCK. Similarly, the two red spots, that are increased 8.7- and 8.4-fold, were identified as the 68- and 66-kDa subunits of the mitochondrial GA, respectively. The two major orange spots in this field were identified as isoforms of mitochondrial GDH that are induced 2.4- and 3.0-fold. Additional proteins identified in this field include: isoforms of succinate dehydrogenase (increased 1.8- and 2.7-fold) and a selenium-binding protein (increased 2.2- and 2.3-fold), the GAC isoform of glutaminase (increased 1.8-fold), multiple species of arginine-glycine amidinotransferase (decreased 0.48-, 0.26-, and 0.17-fold), and enolase (decreased 0.38-fold). The pattern of spots identified as arginine-glycine amidinotransferase is characteristic of a protein that is phosphorylated at multiple sites.
Fig. 3. Identification of proteins that may be increased or decreased after 7 days of acidosis. The labeled spots from the 2-D gel were identified by MALDI-TOF/TOF analysis as PEPCK, the 68- and 66-kDa subunits of GA, GDH, and the GAC isoform of glutaminase, succinate dehydrogenase (SDH), a selenium binding protein (SE-BP), arginine-glycine amidinotransferase (AGAT), and enolase. The numbers in parentheses indicate the ratio of protein levels in 7-day acidotic vs. control proximal tubules. The reported data are representative of that obtained from duplicate 2-D gels.
In total, the DIGE analysis identified 21 proteins, including 17 previously unrecognized proteins, that increased between 1.5- and 5.6-fold ( Table 1 ) and 16 additional proteins that decreased between 0.67- and 0.03-fold ( Table 2 ) within the renal proximal tubule during chronic metabolic acidosis. Additional enzymes that exhibited a significant increase and catalyze key reactions in renal amino acid and glutathione metabolism include: glycine decarboxylase (1.7-fold), pyruvate dehydrogenase kinase (1.7-fold), dimethylglycine dehydrogenase (1.9-fold), 5-oxoprolinase (2.1-fold), aconitase (2.1-fold), the P i (2.2-fold), and Mu (5.6-fold) isoforms of glutathione S -transferase, and phenylalanine 4-hydroxylase (3.5-fold). The enzymes that exhibited a significant decrease included: argininosuccinate synthetase (0.67-fold), pyruvate carboxylase (0.63-fold), cathepsin B (0.28), and multiple isoforms of arginine-glycine amindinotransferase (0.48- to 0.17-fold). Finally, the levels of two plasma fatty acid binding proteins that are extracted by the proximal tubule, plasma retinol binding protein (0.33-fold) and multiple isoforms of 2 -microglobulin (0.16- to 0.03-fold), exhibited pronounced decreases ( Fig. 4 ). In addition, calmodulin, an important Ca 2+ -binding regulatory protein, was induced 5.6-fold within 2 h after onset of acidosis and returned to control levels by 7 days of acidosis.
Table 1. Proteins that are potentially increased in proximal tubules of 7-day acidotic rats
Table 2. Proteins that are potentially decreased in proximal tubules of 7-day acidotic rats
Fig. 4. DIGE analysis of temporal changes in calmodulin and 2 -micro- globulin expression. Proximal tubules from control rats and from rats made acidotic for 2 h ( A ), 1 day ( B ), or 7 days ( C ) were separated on 2-D gels and imaged. The numbers in parentheses indicate fold-increases (red) or decreases (green) in expression of calmodulin (CaM) and 2 -microglobulin ( 2 -µG). The reported data are representative of that obtained from duplicate 2-D gels.
Duplicate samples of proximal tubules isolated from control rats and from rats that were made acidotic for 1, 3, and 7 days were analyzed to determine the temporal pattern of the adaptive responses ( Fig. 5 A ). This analysis confirmed that PEPCK is maximally induced within 1 day, whereas GA and GDH are gradually increased over 7 days ( 7 ). By contrast, the multiple iosforms of arginine-glycine amidinotransferase exhibit a gradual decrease and approach a new steady state after 3 days of acidosis ( Fig. 5 B ). In total, the adaptive increases for 10 of the identified proteins were determined ( Table 3 ). The induced proteins were grouped into two distinct kinetic profiles. Only PEPCK and anchorin CII were fully induced within 1 day of acidosis. The two genes were not sufficient to identify common promoter elements that may mediate a pH-responsive increase in transcription. The second group exhibited a more gradual induction with kinetics similar to GA. All eight of the corresponding mRNAs and the PEPCK mRNA contain, within their 3'-untranslated regions, one or more sequences in which seven of eight nucleotides are identical to either of the two 8-nucleotide AU sequences that function to stabilize the GA mRNA during acidosis ( 26, 27 ). Two of the four sequences within GDH mRNA that satisfy this criteria were previously shown to function as a pH response element ( 35 ). In addition, 4 of 11 mRNAs that encode induced proteins, for which kinetic profiles were not determined, also contained a putative pH response element. Thus 12 of 21 proteins that are increased during acidosis are encoded by mRNAs that contain sequences that may function as pH-responsive stabilizing elements.
Fig. 5. Time course of the changes in various proteins following onset of acidosis. Duplicate samples of proximal tubules isolated from control rats and from rats that were made acidotic for 1, 3, and 7 days were separated using four 2-D gels and the resulting images were merged and analyzed using DeCyder software to quantify the temporal changes in PEPCK, GA, and GDH ( A ) and arginine-glycine amidinotransferase (AGAT; B ). The data points represent means ± range of duplicate samples.
Table 3. Induction kinetics of proteins that are increased in proximal tubules after 1, 3, and 7 days of acidosis
Western blot analysis. Multiple samples of proximal tubules were isolated from control and 7-day acidotic rats and analyzed by semiquantitative Western blot analysis ( Fig. 6 ). The analyses confirmed the well-characterized increases in GA, PEPCK, and GDH and verified the previously uncharacterized increases in dimethylglycine dehydrogenase and the GAC isoform of glutaminase and decreases in the plasma retinol binding protein and arginine-glycine amidinotransferase. Analysis of duplicate tubule preparations confirmed that calmodulin is increased 2.6-fold within 2 h after onset of acidosis and then decreases gradually (data not shown). However, the Western analysis failed to confirm the putative increase in succinate dehydrogenase or the decreases in the multiple isoforms of the plasma 2 -microglobulin ( Fig. 6 ).
Fig. 6. Western blot analysis of proteins identified by difference gel electrophoresis (DIGE). A : antibodies were obtained for 9 of the proteins identified by DIGE to be significantly increased or decreased following 7 days of acidosis. Western blot analysis was performed to compare the relative levels of the proteins in separate preparation of proximal tubules isolated from 5 control and from 5 7-day acidotic rats. The resulting bands were imaged and quantified using an Odyssey Infrared Imager. B : fold-changes calculated by Western blot analysis were plotted vs. the fold differences measured as the mean of the duplicate 7-day acidotic samples used in the temporal analysis ( Table 3 ). The line represents equivalent fold-differences measured by the 2 techniques.
DISCUSSION
Previous experiments determined that the increase in rat renal PEPCK mRNA is initiated within 1 h following onset of acidosis and reaches a maximum within 7 h at a level that is sixfold greater than normal ( 20 ). The sixfold induced level of renal PEPCK mRNA is sustained in rats that are made chronically acidotic for periods up to 7 days ( 21 ). In contrast, the rat renal GA activity increases gradually within the proximal tubule (2-fold within 24 h) and reaches a plateau after 7 days of acidosis that is eightfold greater than normal ( 9, 43 ). The adaptive increases in rat renal GDH mRNA levels ( 24 ) and activity ( 42 ) also occur gradually within the proximal tubule and reach a maximum threefold induction only after 7 days of chronic metabolic acidosis. Thus the combined DIGE analyses produced data that verified both the fold induction and the kinetic profiles of the three proteins that were previously characterized by more extensive biochemical analysis. This finding verifies the ability to isolate highly purified proximal tubules and adds credibility to the proteomic protocol.
Nearly all of the proteins identified in the temporal studies ( Table 3 ) were increased gradually with kinetics similar to GA and GDH. All eight of these proteins are encoded by mRNAs that contain an AU sequence that has a high degree of identity 85%) to the pH-responsive element that binds -crystallin ( 38 ) and that contributes to the selective stabilization of the GA ( 26, 27 ) and GDH ( 35 ) mRNAs during acidosis. The PEPCK mRNA also contains a similar AU sequence within the loop portion of a highly conserved stem-loop structure that contributes to the rapid turnover of the PEPCK mRNA ( 12 ). More recent studies have established that this sequence also binds -crystallin and that a pH-responsive stabilization may contribute to the maintenance of the fully induced level of PEPCK during chronic acidosis (unpublished data of Hajarnis S, Taylor L, and Curthoys NP). By contrast, of the 21 mRNAs that encode proteins that are apparently increased ( Table 1 ), only the glutathione S -transferase-P i mRNA contains the consensus sequence (WWWUAUUUAUWW, where W is an A or U) for the classical AU-rich element that mediates the rapid decay of mRNAs that encode various cytokines, transcription factors, and immediate-early response proteins ( 2 ). Furthermore, only 3 of the 16 mRNAs that encode proteins that are apparently decreased contain a putative pH response element. Therefore, selective mRNA stabilization may be the predominant mechanism by which protein expression is increased in response to acidosis.
An important observation from the initial experiments is the rapid increase in calmodulin protein ( Fig. 4 ). This protein binds Ca 2+ ions with high affinity and mediates Ca 2+ -signaling through its ability to interact with and activate multiple downstream signaling molecules ( 5, 17, 23 ). Antibodies specific for the rat calmodulin protein confirmed the rapid but transient increase of calmodulin in the proximal tubule samples used in the DIGE experiments. Further verification of this observation would provide the basis to determine whether onset of acidosis triggers a rapid influx of Ca 2+ ions into the renal proximal tubule and the activation of various downstream signaling molecules. The influx of Ca 2+ ions into mitochondria activates multiple dehydrogenase reactions of the tricarboxylic acid cycle, including -ketoglutarate dehydrogenase ( 14 ). Such a mechanism could contribute to the acute activation of the mitochondrial catabolism of glutamine that occurs during onset of acidosis and that clearly precedes the significant increases in GA and GDH levels ( 44 ).
The MALDI/TOF/TOF analysis identified the five low molecular weight proteins that appear to rapidly decrease following onset of acidosis ( Figs. 2 and 4 ) as multiple isoforms of 2 -microglobulin. This protein belongs to a family of proteins that bind free fatty acids, their CoA derivatives, and various sterols with high affinity ( 16 ). Expression of various fatty acid binding proteins correlates with the ability of a cell to catabolize fatty acids ( 45 ). However, 2 -microglobulin is expressed primarily in liver and is secreted into the plasma. In rodents, it is more highly expressed in male animals than females ( 33 ). The low molecular weight 2 -microglobulin is readily filtered by the glomeruli and constitutes a major urinary protein (MUP-1) that is used by male rodents to mark their territory. Some of this protein is reabsorbed by the proximal tubule where it is largely found in endocytic vesicles. In contrast to the rapid and pronounced decrease in 2 -microglobulin observed by DIGE analysis, Western blot analysis clearly established that the level of the 2 -microglobulin is unchanged during acidosis ( Fig. 6 ). The Western blot analysis also indicated that succinate dehydrogenase was not increased during acidosis. The two spots that were identified as succinate dehydrogenase had excellent peptide coverage (22 and 28 peptides) and Mascot scores (175 and 241), respectively. However, in both cases, the MALDI/TOF/TOF analysis also identified 18 peptides from PEPCK and assigned Mascot scores of 72 and 60, respectively, for this secondary identification. Close examination of the imaged gel ( Fig. 3 ) indicates a slight lateral streaking that emanates from the large bright red spot that is PEPCK. Thus the failure to completely focus this very abundant protein probably caused the apparent increase in the adjacent spots that are succinate dehydrogenase. These examples illustrate the importance of confirming the results of the DIGE analysis by using a traditional biochemical approach such as Western blotting.
Other potentially important data include the observed increase in pyruvate dehydrogenase kinase and decrease in pyruvate carboxylase that could reduce oxidation of pyruvate and contribute to the cataplerotic activity of PEPCK. Similarly, observed increases in glycine decarboxylase and dimethylglycine dehydrogenase and the decrease in arginine-glycine amidinotransferase proteins could shunt renal glycine utilization from the synthesis of creatine into the net production of ammonium ions. Multiple protein spots that either increase ( Table 1 ) or decrease ( Table 2 ) in response to acidosis were identified as a mitochondrial aldehyde dehydrogenase. However, Western blot analysis indicated that the level of this protein was not altered (data not shown). Thus the extent of phosphorylation or covalent modification of aldehyde dehydrogenase may be altered during acidosis. Significant increases in enzymes of glutathione (P i and Mu isoforms of glutathione S -tranferases) and phenylalanine (phenylalanine 4-hydroxylase) metabolism were also observed. Finally, cathepsin B is a lysosomal cysteine protease that participates in protein turnover. The observed decrease in this protein may contribute to the decreased rate of protein turnover that, in part, causes the acidosis-associated hypertrophy of the proximal tubule ( 31 ). This finding illustrates the fact that it is not possible to discriminate whether the changes observed in the reported proteomic analysis are direct effects of metabolic acidosis or if they are part of the hypertrophic response that is triggered by the acidosis. Thus the reported analysis has identified a number of potential changes in protein levels that could contribute significantly to the adaptive response to metabolic acidosis and/or renal hypertrophy. However, additional experiments will be necessary to confirm and extend the various hypotheses derived from the proteomic analysis.
GRANTS
This research was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-37124 and DK-43704 awarded to N. P. Curthoys and by the Intramural Budget of the National Heart, Lung, and Blood Institute (National Institutes of Health) Project No. ZO1-HL-01282-KE to M. A. Knepper.
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作者单位：1 Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado; and 2 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland