Sodium loading changes urinary protein excretion: a proteomicanalysis - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:38 |只看该作者 |倒序浏览 |打印

作者：VisithThongboonkerd, Jon B.Klein, William M.Pierce, Anthony W.Jevans,  John M.Arthur,作者单位：1 Core Proteomics Laboratory, Kidney DiseaseProgram, Department of Medicine, and Departments of Biochemistry and Molecular Biology and Pharmacology and Toxicology, University of Louisville, Veterans Affairs Medical Center, and Pathology Department, Jewish Hospital, Louisville,Kentucky 40202; and Div ; M, u, }7 c9 ^+ V
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      【摘要】
   Plasma sodium concentration ismaintained even when sodium intake is altered. Sodium homeostasis mayinvolve changes in renal tubular protein expression that are reflectedin the urine. We used proteomic analysis to investigate changes inurinary protein excretion in response to acute sodium loading. Ratswere given deionized water followed by hypertonic (2.7%) saline for28 h each. Urinary protein expression was determined during thefinal 4 h of each treatment. Acute sodium loading increasedurinary sodium excretion (4.53 ± 1.74 vs. 1.70 ± 0.27 mmol/day, P = 0.029). Urinary proteins were separatedby two-dimensional PAGE and visualized by Sypro ruby staining.Differentially expressed proteins were identified by matrix-assistedlaser desorption ionization-time-of-flight mass spectrometry followedby peptide mass fingerprinting. The abundance of a total of 45 proteincomponents was changed after acute sodium loading. Neutralendopeptidase, solute carrier family 3, meprin 1, diphor-1,chaperone heat shock protein 72, vacuolar H -ATPase, ezrin,ezrin/radixin/moesin-binding protein, glutamine synthetase, guaninenucleotide-binding protein, Rho GDI-1, and chloride intracellularchannel protein 1 were decreased, whereas albumin and -2u globulinwere increased. Some of these proteins have previously been shown to beassociated with tubular transport. These data indicate that alterationsin the excretion of several urinary proteins occur during acute sodium loading. % [+ Y) I) J  `7 D+ k& v
      【关键词】 urine tubular transport
   INTRODUCTION
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RENAL SODIUM HANDLING IS IMPORTANT in maintaining intracellular and extracellular sodiumand water homeostasis. Renal sodium excretion is controlled by changesin body fluid volume and the stimulation of osmoreceptors ( 1, 5 ). The mechanisms that occur in the kidney can be divided intothree main categories: humoral signals (especially vasopressin and therenin-angiotensin system), renal nerve activity, and physical factors( 15 ). Passive and active sodium transporters along renaltubules, mostly at proximal tubules, are generally considered as thetargets of these mechanisms. However, a coherent understanding of thesecomplex pathways and the roles of regulatory targets other than these transporters is lacking.

A sodium-excess status induced by salt loading causes changes in therenal expression of several proteins, which can be divided into twomain groups; sodium excretion-controlling proteins ( 8, 9 )and regulated proteins that cause systemic effects ( 17, 19, 20, 29, 30 ). Previous studies have demonstrated that aquaporin-2,Na /H exchanger type 3 (NHE3),Na -K -2Cl cotransporter, andthiazide-sensitive Na -Cl cotransporter wereexcreted into the urine ( 7, 14, 21 ). Western blotting andRIA were used to identify expression of the proteins in those studies.However, these techniques are limited by the relatively small number ofproteins that can be studied for each experiment and the need forspecific antibodies to those proteins. In addition, antibody-basedstudies only identify proteins that are suspected to be there a priori.Other tubular proteins are also excreted into the urine, and sodiumloading may alter urinary excretion of unsuspected proteins that areinvolved in sodium homeostasis. The global study of a large complementof urinary proteins may contribute to understanding renal sodium handling.9 l; O9 l" Z5 {
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In 1975, O'Farrell ( 18 ) developed a technique for theresolution of proteins using two-dimensional PAGE (2D PAGE or 2-DE), and 1,100 proteins from Escherichia coli were visualized.Using this technique, a large number of proteins can be studiedsimultaneously without specific antibodies. The proteins are separatedby isoelectric point (pI) in the horizontal dimension and by molecularweight (MW) in the vertical dimension. The protein spots can bevisualized by several staining methods. Recently, up to 10,000 proteinspots have been visualized by high-resolution 2-DE ( 12 ).The analysis of separated proteins by mass spectrometry has permittedanalysis of proteins on a "genomic" scale ( 11 ). Theanalysis of proteins on a genomic scale has acquired the name"proteomics" ( 2 ). A common approach for proteomicanalysis uses resolution of proteins by high-resolution 2-DE, peptidemass fingerprinting, and bioinformatics to identify the proteins in ahigh-throughput fashion. Once the proteins are visualized, the proteinspots are excised and undergo in-gel tryptic digestion. Peptide massesare obtained by matrix-assisted laser desorptionionization-time-of-flight (MALDI-TOF) mass spectrometry (MS). Peptidemass fingerprinting is then performed to identify the protein, usingsearch engines to match the peptide masses to the theoretical masses inprotein databases. The National Center for Biotechnology Information(NCBI) is an annotated protein database containing more than 1.2 × 10 6 peptide sequences and 3.9 × 10 8 residues ( http://www.ncbi.nlm.nih.gov ).
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We used proteomic analysis to determine alterations in urinary proteinexcretion during acute sodium loading. A self-controlled study wasconducted in young male Sprague-Dawley rats fed with deionized (dI)water that was then replaced with 2.7% NaCl. Urinary sodium excretionwas significantly increased, and urinary excretion of several proteinswas altered after acute sodium loading.5 o  k7 L- z  S4 u) U, p  N
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MATERIALS AND METHODS6 n# v! K& I; H% F& O' R9 P

Urine collection. All studies using rats were approved by the University of LouisvilleInstitutional Animal Care and Use Committee. A self-controlled studywas conducted in four young male Sprague-Dawley rats (body wt 383 ± 16 g). The rats were transferred to metabolic cages and fedwith dI (18 M ) water and rat chow obtained from PMI Nutrition (Richmond, IN) for 24 h. Twenty-four-hour urine was collected forurinary Na concentration measurement. The rats were thentransferred to another cleaned metabolic cage with dI water without anyfood (to prevent contamination with proteins from food particles). Four-hour urine for protein analysis was collected with a protease inhibitor cocktail (0.1 mg/ml leupeptin, 0.1 mg/ml PMSF, and 1 mMsodium azide in 1 M Tris, pH 6.8). The rats were then immediately changed to 2.7% NaCl feeding, and the rat chow was returned. Urine wascollected over 24 h for urinary Na concentrationmeasurement. After 24 h, the rats were then transferred to cleanedmetabolic cages for 2.7% NaCl feeding but no food. Four-hour urine forprotein analysis was again collected with a protease inhibitorcocktail. Urine protein collections were done at the same time of dayto avoid diurnal variation.
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Urinary Na concentrationmeasurement. Urinary Na concentration measurement was performed with anindirect ion-specific electrode on a Beckman Coulter LX20.0 ^* S1 z6 n$ |# i" q! V4 [

Sample preparation. The samples were passed through 0.34-mm Whatman chromatography paperand then centrifuged at 1,000 g for 5 min. The supernatants were saved and centrifuged at 200,000 g for 120 min. Thepellets were resuspended in 100 µl of 250 mM sucrose in 10 mMtriethanolamine. The concentration of proteins was measured byspectrophotometry using a protein microassay (Bio-Rad Laboratories,Hercules, CA) based on Bradford's method ( 6 ).$ B; M. d. t  C0 D5 w& f
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First dimension of 2-DE. A tube gel mobile ampholyte running system (Genomic Solutions, AnnArbor, MI) was used for first-dimensional isoelectric focusing. Thecathode buffer was 100 mM sodium hydroxide, and 10 mM phosphoric acidwas used as an anode buffer. Precast carrier ampholyte tube gels [pH3-10, 1 mm × 18 cm (Genomic Solutions)] were prefocused with a maximum of 1,500 V and 110 µA/tube. A total of 50 µg from each sample was loaded into each tube and was focused for 17 h, 30 min to reach 18,000 volt hours.( h/ P) @& {+ H$ m! L9 C9 D
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Second dimension of 2-DE. The gels were extruded from the tubes after completion of focusing andwere incubated in premixed Tris/acetate equilibration buffer with0.01% bromophenol blue and 50 mM DTT for 2 min. The tube gels werethen loaded onto precast 8-18% gradient, 22 × 22-cm slabgels (Genomic Solutions). Lower running buffer contained 25 mM Trisbase, 192 mM glycine, and 0.1% SDS. Upper running buffer was a 2×solution of the lower buffer. The system was run with a maximum of 500 V and 20,000 mW/gel.( d3 ?! E' n' Y) d3 ?6 S
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Sypro ruby staining and visualization. The gel slabs were fixed in 10% methanol and 7% acetic acid for30 min. The fixative solution was removed, and 500 ml of Sypro ruby gelstain (Bio-Rad Laboratories) were added to each gel and incubated on acontinuous rocker at room temperature for 18 h. A high-resolution,12-bit camera with a UV light-box system (Genomic Solutions) was usedto visualize the protein spots with an optimal exposure time point of3 s. The images were digitally inverted before analysis with 2D software.
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Matching and analysis of protein spots. Investigator HT analyzer (Genomic Solutions) software was used formatching and analysis of spot expression on the gels. A representativegel was constructed as a reference for each group. An average mode ofbackground subtraction was used for normalization of intensity volumeon each spot and for compatibility of the intensity among gels. Thedata were reported as "normalized intensity," which were correctedby total intensity of all spots from all the gels, instead of the rawintensity values being used. This normalized value provides aratiometric comparison of protein abundance. The representative gel wasthen used for determination of existence and difference of proteinexpression between groups.4 N9 w# j, j: n) o. B
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In-gel tryptic digestion, MALDI-TOF MS, and peptide massfingerprinting. In-gel tryptic digestion and sample preparation for MALDI-TOF MS wereperformed as described previously by our laboratory ( 3, 25 ). Peptide mass fingerprinting was used for protein identification from tryptic fragment sizes by using the Mascot searchengine ( http://www.matrixscience.com ). The search was based on theentire NCBI protein database on the assumption that peptides aremonoisotopic, oxidized at methionine residues, and carbamidomethylated at cysteine residues. Up to one missed trypsin cleavage was allowed, although most matches did not contain any missed cleavages. Mass tolerance of 150 parts/million (ppm) was the window of error allowed for matching the peptide mass values. Probability-based MWsearch scores were estimated by a comparison of search results against an estimated random match population and were reported as 10 · log 10 ( P ), where P 71 were consideredsignificant ( P lessthan the significant level were reported as unidentified.; d; \- K- x, U8 P/ k% i

Prediction of posttranslational modifications. Potential posttranslational modifications (PTMs) were predicted usingthe FindMod search engine ( http://ca.expasy.org/tools/ findmod/).Because the presence of a PTM causes a peptide mass shift, thepotential PTMs can be predicted by matching the mass difference (massdifference = theoretical mass observed mass) to the massesof known PTMs. To date, there are at least 30 known PTMs provided inthe database. A window of error ( mass) of 150 ppm was allowed.

Western blotting. Urinary proteins were processed as for 2-DE analysis. SDS sample buffer(Tris · HCl, glycerol, SDS, DTT, and bromophenolblue) was added 1:1 to the protein solution. The mixture was heated at100°C for 5 min. The protein concentration of each sample was measured by the spectrophotometric method using the HP 8453 UV-visible system (Hewlett-Packard, Palo Alto, CA) and Bio-Rad Protein Assay (Bio-Rad Laboratories), and 20 µg of total proteins were equally loaded onto each lane on 10% SDS-PAGE gels. Proteins on the gel weretransferred to a nitrocellulose membrane by electroblotting. Themembrane was incubated with mouse monoclonal anti-ezrin (Sigma, St.Louis, MO) 1:1,000 in 5% milk/Tween 20 Tris-base sodium (TTBS) at4°C overnight. Immunoreactive proteins were detected by radiography using IgG conjugated with horseradish peroxidase. The membrane was thenstripped in 0.2 N NaOH for 5 min and reblotted with goat anti-mousealbumin (Bethyl Laboratories, Montgomery, TX) 1:1,000 in 5% milk/TTBS,mouse monoclonal anti-actin (A4700, Sigma) 1:200 in 5%milk/TTBS, andmouse monoclonal anti-calbindin-D 28K (Sigma), respectively.The intensity analyses of immunoreactive bands were performed using aPDSI Densitometer (Amersham Biosciences, Piscataway, NJ).
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Statistical analyses. The Mann-Whitney test (version 10.0, SPSS) was used for acomparison of the differences between two groups. Exact and Monte Carloresampling methods were used to reassign the data for multiple analysesof a single data set. Therefore, Exact and Monte Carlo P values were calculated on the basis of the permutated data, adjustedfor multiple inferences, and corrected for tied values. Only Exact P values testwere considered statistically significant. This significance level isbased on the reassignment of a test statistic, which is more accuratethan using asymptotic significance values when the sample size is small( 4 ). To avoid changes by chance or normal variability,only changes greater than twofold (0.5-fold less or 2-fold greater thanthe control) were considered significant. The data are reported asmeans ± SE.

RESULTS1 c( A1 N! ~' k; S2 _

The animals were fed with dI water for 28 h and then with2.7% NaCl for 28 h. Urinary Na concentration wasmeasured over the first 24 h of each phase. All of the animalswere in a sodium-excess status as determined by an increase in urinaryNa concentration and 24-h urinary Na (Table 1 ). After the first 24 h of eachphase, the animals were transferred to another clean metabolic cagewithout rat chow to avoid contamination of dietary proteins from foodparticles. Preliminary data showed that contamination of proteinsfrom food particles could interfere with analysis of urinaryproteins because proteins in food particles incubated in water wereseen on gels (data not shown). We collected the urinary samples inclean metabolic cages without the presence of food to eliminate thisproblem. Urinary proteins were resolved by 2-DE as outlined in MATERIALS AND METHODS.

Table 1. Urinary parameters+ F' j$ \# c2 `8 ^

Up to 277 protein spots were visualized by Sypro ruby staining on eachgel (Fig. 1 ). HT analyzer 2D software wasemployed to measure and compare spot intensity, which represents theamount of protein per spot. Differentially expressed protein spots were excised and underwent in-gel tryptic digestion. Figure 2 A demonstrates mass spectraof peptide masses obtained by MALDI-TOF MS from spot 1 inFig. 1. Peptide mass fingerprinting was then performed to identify theproteins by using the Mascot search engine, as demonstrated in Fig. 2 B. The peptide masses shown in Fig. 2, A and B, were significantly matched with the theoretical masses ofthe protein NEP 24.11 ( P
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Fig. 1. Proteome map for significant changes in rat urinary proteins afteracute NaCl loading. Urinary proteins during deionized (dI) waterfeeding or control ( A ) and 2.7% NaCl feeding ( B )were resolved by 2-dimensional PAGE (2-DE) based on differentialisoelectric point (pI) for the 1st dimension ( x -axis) anddifferential molecular weight for the 2nd dimension( y -axis). The protein spots were visualized by Sypro rubystaining, and normalized intensity was compared by HT analyzer 2Dsoftware. The quantities of yellow-highlighted proteins weredecreased, whereas those of blue-highlighted proteins were increasedafter acute NaCl loading. Differentially expressed protein spotsunderwent in-gel tryptic digestion and matrix-assisted laser desorptionionization-time-of-flight mass spectrometry (MALDI-TOF MS)followed by peptide mass fingerprinting. Nos. at labeling spotscorrespond to spot numbers in Table 2.

Fig. 2. Protein identification by MALDI-TOF MS and peptide massfingerprinting. A : typical mass spectra of NEP 24.11 (enkephalinase or neprilysin) obtained from spot 1 byMALDI-TOF MS. B 71 was considered statistically significant for matching( P m / z ) 843.58, 1045.60, 2212.07, and 2284.17] and spectra with m / z were excluded from the analysis. The observed masses (18 of 24 total)were significantly matched to the theoretical masses of NEP 24.11 (gi|6981210) with9 q& D6 N; o7 B! f
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Urinary excretion of 45 protein components was significantly changedafter acute sodium loading, as summarized in Table 2. Only significant changes, 0.5-foldless or 2-fold greater than control, were included. Forty-oneprotein components were significantly decreased, whereas only fourcomponents were increased in intensity after acute sodium loading.Peptide mass fingerprinting did not identify 5 of the 45 differentiallyexpressed spots. All of the identified spots had probability-basedprotein MW search scores 71 ( P cleavage sites by trypsin, and awindow of error was much less than 150 ppm. The expected pI and MW ofthe identified proteins corresponded with their positions in the 2Dgels. GenInfo identification numbers in the NCBI database are alsoprovided in Table 2. Of the identified spots, several spots were in aseries of the same protein with similar MWs but different pIs,suggesting PTMs. We predicted several PTMs in these proteins bybioinformatic analyses using the FindMod search tool (Table 3 ).

Table 2. Significant changes in urinary protein excretion after NaCl loading% Y$ _7 I9 R4 d2 d+ L2 W

Table 3. Potential PTMs7 X) k1 p, q7 z3 s

Although we have shown in previous studies that the data obtainedfrom proteomic analysis are consistent with other standard conventionalmethods ( 3, 24 ), we also confirmed the proteomic data byWestern blot analyses in the present study. Western blotting for ezrinclearly showed that excretion of ezrin was significantly decreasedafter acute sodium loading, consistent with the proteomic data (Fig. 3 A ). Conversely, the level ofalbumin was increased by acute sodium loading (Fig. 3 B ). Wehypothesized that using strict analytic criteria in the present studylikely caused us to underestimate the number of proteins that werechanged. To address this hypothesis, we examined the effect of acutesodium loading on excretion of two other abundant proteins in thekidneys, actin and calbindin ( 3 ). Urinary excretion ofactin was decreased, but excretion of calbindin was increased afteracute sodium loading (Fig. 3, C and D ).9 o( G1 r$ o  @  K, J

Fig. 3. Western blot analyses. Western blot analyses showdecreased excretion of ezrin ( A ) and actin ( C )and increased excretion of albumin ( B ) and calbindin( D ). A total of 20 µg of protein was equally loaded ineach lane ( n = 3 animals).) e9 U6 u% S# T: ]7 X9 k4 d
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We used proteomic analysis to study changes in the relativeabundance of a large number of urinary proteins simultaneously. Using2D analysis software, protein expression could be compared based on theintensity of staining, which represented the amount of protein perspot. Acute sodium loading caused changes in urinary excretion of 45 protein components. Some of the altered proteins have never beenstudied in relationship to salt loading, and their roles in sodiumregulation have not been established. Several proteins, such as NEP24.11 (enkephalinase or neprilysin), solute carrier family 3, meprin1, and ezrin (villin-2), are typical membrane proteins. Someproteins, such as vacuolar H -ATPase, dnaK-type chaperone(heat shock protein 72), albumin precursor, and chloride intracellularchannel protein 1, were identified in rat kidney cortex and medulla inour previous study ( 3 ). Cells, debris, and particles werecompletely removed from the samples, as determined by a hemacytometriccounting chamber. Thus most of these proteins probably originate in therenal tubules and may play important roles in sodium regulation.5 I. y% Y9 ?" V' g( ?& [

High-throughput proteomic technologies allow a large number of proteinsto be studied simultaneously. Proteomic analysis may lead to theidentification of both expected changes and unexpected changes in anexperimental condition. One of the strengths of proteomic analysis isthat identification of coordinated changes in protein expression maylead to more focused hypotheses in physiology and pathophysiology. Thisis illustrated by our previous work, in which we used proteomicanalysis to generate a new hypothesis to explain hypertension inducedby intermittent hypoxia in an animal model ( 24 ). Ourproteomic analysis indicated that several proteins in the renalkallikrein pathway play a role in episodic hypoxia (EH)-inducedhypertension. This hypothesis was then strongly supported bydemonstrating that transgenic hKLK1 rats, which overexpress human renalkallikrein, are resistant to EH-induced hypertension. Transgenic hKLK1animals were protected from EH-induced hypertension ( 24 ).A similar approach can be applied to proteomic data such as thoseproduced in the present study.$ \9 Y" Q2 Q, V' W( A# a! @9 I
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In the present study, we performed expression proteomics to demonstratechanges in protein excretion after acute sodium loading. Severalhypotheses and new insights regarding renal sodium handling can begenerated from these proteomic data. An example is a potential role ofezrin in renal sodium regulation. Ezrin (villin-2) is a member of theezrin/radixin/moesin family of actin-binding proteins, which functionas membrane-cytoskeletal cross-linkers ( 13 ). Ezrincolocalizes and closely associates with NHE3 andNa /H exchanger regulatory factor (NHERF)( 27 ). Ezrin binds with actin and NHERF and forms amultiprotein complex with NHE3. Formation of this complex facilitatesNHE3 phosphorylation and inhibits N /H exchange, resulting in inhibition of NaCl and NaHCO 3 reabsorption in the proximal tubules ( 22 ). Change of ezrinexpression obtained from proteomic analysis in the present study wasnot spurious or by chance. Western blotting for ezrin clearly confirmeda decrease in urinary ezrin excretion after acute sodium loading.Functional proteomics and other physiological studies are needed todetermine roles of the altered proteins in renal sodium regulation.
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Actin is a cytoskeletal protein that plays an important role in cellsignaling and cytoskeletal assembly. The role of actin in epithelialand renal tubular sodium transport has previously been established( 10, 16, 23 ). Alterations in renal expression of actin byhypertension ( 24 ) and by other experimental conditions (Thongboonkerd V and Klein JB, unpublished observations) were also shown in our previous studies. Additionally, actin binds to ezrinas a part of membrane-cytoskeletal cross-linkers ( 13 ). Therefore, a change in actin excretion in the present study was notunexpected. To test that the decreased excretion of ezrin and actin(Fig. 3, A and C ) was not the result of a smalleramount of protein loaded in the NaCl lanes of PAGE, we performedWestern blot analyses for albumin and calbindin, the two proteins that are not involved in ezrin/radixin/moesin-actin assembly. Excretion ofalbumin and calbindin was increased after acute sodium loading (Fig. 3, B and D ). Indeed, the equal amount of proteinloaded in each PAGE lane was controlled by spectrophotometry beforeimmunoblotting procedures were begun.4 ]4 M4 h/ ~0 V) h) C8 @
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Protein modification is one of the regulators of protein function.Proteomic approaches provide information about PTMs that is notobtained by many other methods. We used the FindMod tool to predictpotential PTMs of the identified proteins. PTMs cause changes inprotein pI, leading to the presentation of a row of multiple proteinspots of the same protein. This phenomenon is observed not onlywith regard to urine ( 26 ) but also serum and other bodyfluids ( 28 ).* {4 _/ ?, A- N4 Q
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We also have some concerns regarding an interpretation of theresults in the present study. First, several possible physiological explanations exist for differential urinary excretion of proteins. Aprotein that is necessary could have increased expression, leading toincreased appearance in the urine. Alternatively, a necessary proteincould be retained in the cell, leading to decreased appearance in theurine. Second, we could not identify some known sodium transporters inthe present study. Protein identification using 2-DE and MALDI-TOF islimited by the sensitivity of these techniques. Either using greateramounts of proteins loaded onto 2-DE, utilizing additionalprefractionated steps, or applying a more sensitive technique, such asliquid chromatography followed by tandem MS (LC-MS/MS), may be thesolution. Another concern is the strict criteria we used todetermine significant changes to avoid false-positive results. Thequantities of most of the proteins were decreased by sodium loading,whereas those of only a few proteins were increased. Thequantities of several proteins tended to increase, but the increase wasnot statistically significant. Use of these strict criterialikely caused us to underestimate the number of proteins that werechanged because we tested for actin and calbindin by immunoblotting(Fig. 3, C and D ). Our findings in the present study represent only the "tip of the iceberg" for the entire number of changes in urinary protein excretion caused by acute sodium loading.! w- a5 _8 K- c* F) K% B0 x" q+ O8 F
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In summary, we used proteomic analysis to determine global alterationsin urinary protein excretion during acute sodium loading. Severalproteins that play important roles in the transport of sodium and othersolutes, cellular pH regulation, and other cellular functions wereinvolved in this response. Several hypotheses can be generated fromthese data. Further functional studies are needed to determine thecoordination of these regulated proteins and their complex mechanismsin renal sodium handling.

ACKNOWLEDGEMENTS

This work was supported by the Carl W. Gottschalk Research ScholarAward from the American Society of Nephrology (to J. M. Arthur),National Institutes of Health Grants 21-DK-629686-01 andR01-HL-66358-01, and the Department of Veterans Affairs,Louisville, KY (to J. B. Klein). V. Thongboonkerd is a recipientof an International Fellowship Training Award from the InternationalSociety of Nephrology and from the National Kidney Foundation of Thailand.3 f5 w7 U/ I, k
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