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DOI 10.1002/pmic.200700503

RESEARCH ARTICLE

Proteomic identification of tyrosine nitration targets in kidney of spontaneously hypertensive rats Raymond Tyther1, Ahmad Ahmeda2, Edward Johns2 and David Sheehan1 1 2

Proteomics Research Group, Department of Biochemistry, University College Cork, Ireland Department of Physiology, University College Cork, Ireland

Nitrosative and oxidative stress are implicated in the development of hypertension. Events in the renal medulla may play a key role in the development and progression of hypertension. This may arise through disruption of nitric oxide signalling in the medulla and be accompanied by enhanced nitrosative and oxidative stress as indicated by the presence of proteins containing 3nitrotyrosine. Here we demonstrate enhanced protein nitration in the medulla of spontaneously hypertensive rats. We have identified several nitrated proteins with both varied subcellular location and functional roles. These proteins are involved in nitric oxide signalling, antioxidant defense and energy metabolism. Moreover, increased nitration was observed in conjunction with enhanced oxidative damage as evidenced by the presence of protein carbonyl oxidative stress biomarkers. Our results suggest that kidney medulla is subject to enhanced nitrosative and oxidative stress, and that resulting protein modifications may contribute to the progression of hypertension.

Received: May 26, 2007 Revised: August 17, 2007 Accepted: September 3, 2007

Keywords: Carbonylation / Hypertension / Kidney / 3-Nitrotyrosine / Rat

1

Introduction

Renal dysfunction is crucial to onset of hypertension [1]. The spontaneously hypertensive rat (SHR) [2] and other models have revealed ROS as contributors to kidney pathophysiology [3, 4]. Nitric oxide (NO) signalling, essential for vascular and renal function [5], appears to be disrupted by ROS in some examples of kidney dysfunction [5, 6]. ROS such as superoxide (O22) and hydrogen peroxide (H2O2), arise from a Correspondence: Dr. David Sheehan, Proteomics Research Group, Department of Biochemistry, University College Cork, Lee Maltings, Prospect Row, Mardyke, Cork, Ireland E-mail: [email protected] Fax: 1353-21-4274034 Abbreviations: ADMA, asymmetric dimethylarginine; AKR, aldoketo-reductases; CA II, carbonic anhydrase II; DDAH1, dimethylarginine dimethylaminohydrolase 1; DNPH, 2,4-dinitrophenylhydrazine; HAOX, hydroxyacid oxidase; NOS, nitric oxide synthase; 3NT, 3-nitrotyrosine; OAT, ornithine aminotransferase; RNS, reactive nitrogen species; SHR, spontaneously hypertensive rat

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variety of sources including NAD(P)H-oxidase [7], leakage from mitochondrial electron transport [8] and nitric oxide synthase (NOS) activity [9]. Oxidative stress can generate nitrosative stress by reaction of ROS with NO producing reactive nitrogen species (RNS): (ONOO2); nitrosoperoxycarbonate (ONO2CO22); nitrogen dioxide radical (NO2); N2O3. Mechanisms of RNS formation are diverse and are influenced by local factors such as the presence of CO2 and heme proteins [10]. The proportion of nitration due to ONOO2 and its protonated form, peroxynitrous acid (ONOOH), is not yet known because nitration also occurs through oxidation of nitrite to nitrogen dioxide by myeloperoxidase and other enzymes [11– 13] and nonenzymatic acidification of nitrite [14]. Modification of tyrosine to 3-nitrotyrosine (3NT) is a key nitration biomarker associated with Alzheimer’s disease and diabetes [15–17]. Some nitrated proteins show increased turnover [18], but others accumulate [19]. Interestingly, some antioxidant enzymes including manganese-superoxide dismutase (Mn-SOD) [20] and catalase [21] are themselves nitration targets, perhaps further compromising ROS/RNS protection. www.proteomics-journal.com

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Renal medulla and cortex fulfil distinct roles. In the cortex, which is well perfused and oxygenated, fluids/electrolytes are filtered from proteins at the glomerulus, glucose, water and electrolytes are reabsorbed along the nephron and regulatory hormones are produced. The principal function of the medulla is to concentrate urine. In view of the dynamics of 3NT formation, the kidney presents an interesting model system because of the dichotomy between a well-perfused and oxygenated cortex, and a medulla on the threshold of anoxia [22]. Medulla cells are adapted to function in this hypoxic milieu, but O22 may perturb this situation causing dysfunction [23]. These contrasts extend to NO signalling/ metabolism: NO and nitrite/nitrate levels of medulla exceed those of cortex [24] as does NOS expression [25]. Kidney nitrosative stress is also associated with hypertension and induction of hypertension via angiotensin II is accompanied by an increase in 3NT [26]. The cortex and medulla proteomes in normotensive rats are quite similar [27], but activities/abundance of key antioxidant enzymes like SOD, catalase and glutathione peroxidase differ between the two kidney components in normotensive rats [28] and SHR [29]. Oxidative/nitrosative stress may be differentially regulated in the medulla and cortex [30]. Development of hypertension in SHR is accompanied by proteomic changes in myocardial tissue [31], but it is not known if kidney protein expression profiles are similarly altered. In this study, protein nitration in kidneys from hypertensive SHR and age-matched normotensive Wistars was studied. The relative amount of protein nitration in cortex and medulla was also investigated and we have identified several protein targets of tyrosine nitration; potential biomarkers for nitrosative stress. Characterisation of the ‘nitroproteome’ of SHR kidney may provide insights into the pathogenesis and progression of hypertension.

2

Materials and methods

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homogenate was centrifuged at 20 0006g at 47C for 20 min, and the supernatant collected (cytosolic fraction). The pellet was washed three times in PBS, resuspended in homogenisation buffer containing 1% C7BzO (Sigma, Germany) and incubated on ice for 20 min with frequent vortexing. It was then centrifuged for 10 min at 14 0006g and the supernatant was collected (membrane fraction). Protein concentrations were determined using the BioRad protein assay (BioRad, Germany) and fractions stored at –707C until use. 2.2 Protein preparation and electrophoresis methods Protein fractions (100 mg) were rehydrated in buffer containing 5 M urea, 2 M thiourea, 2% CHAPS, 4% carrier ampholyte (Pharmalyte 3–10, Amersham-Pharmacia Biotech, UK), 1% DeStreak reagent (Amersham-Pharmacia Biotech) and a trace amount of bromophenol blue. Final volumes of 125 mL were loaded on 7 cm pH 3–10 nonlinear IPG strips (BioRad, CA, USA) and rehydrated overnight for at least 15 h. IPG strips were focused on a Protean IEF Cell (BioRad) with linear voltage increases: 250 V for 15 min; 4000 V for 2 h; then up to 20 000 V?h. Following IEF, strips were equilibrated (20 min) in equilibration buffer (6 M urea, 0.375 M Tris, pH 8.8, 2% SDS, 20% glycerol) containing 2% DTT, and then for 20 min in equilibration buffer containing 2.5% iodoacetamide. Equilibrated strips were electrophoresed on 12% SDS-PAGE gels at a constant voltage (150 V) at 47C using an Atto AE-6450 mini PAGE system (Atto, Japan). For analytical gels, loading was determined by analyzing homogenate by 1-DE and staining using colloidal CBB G250, and IEF and 2-DE were carried out as described previously [32]. For 1-DE, protein fractions were diluted with sample buffer, and electrophoresis was performed [33] using 12% polyacrylamide gels on an Atto AE-6450 mini PAGE system (Atto).

2.1 Animals and tissue preparation

2.3 Detection of protein carbonyls

Rats were obtained from Harlan (UK) and maintained in the Biological Services Unit (University College Cork) for at least 1 wk prior to use. Animals received regular laboratory diet and tap water ad libitum. All procedures were performed in accordance with National guidelines and the European Community Directive 86/609/EC and approved by the Animal Experimentation Ethical Committee of University College Cork. Male Wistar and SHR weighing 250– 300 g were anaesthetised with 0.75–1.0 mL of a chloralose/ urethane mixture (16.5/250 mg/mL, respectively). Kidneys were exposed via retroperitoneal incision, quickly removed and placed on ice. Cortex was dissected from medulla, and tissues were weighed, diluted to 25% with homogenisation buffer (250 mM HEPES, pH 7.7/1 mM EDTA/0.1 mM neocuproine), and homogenised with a Polytron PCU2 Tissue Homogeniser (Kinematica, Switzerland). The

Protein carbonyls were detected using the 2,4-dinitrophenylhydrazine (DNPH) derivatisation method [34]. Briefly, 20 mg protein was derivatised with 10 mM DNPH in 10% TFA for 20 min with regular vortexing, and the reaction was stopped by incubation with neutralisation buffer (2 M Tris-base/30% glycerol) for 15 min. Samples were combined with Laemmli buffer and 1-DE was performed as described above. Following 1-DE, proteins were transferred (100 mA per blot, 55 min) to Protran NC (0.2 mM) membranes (Whatman Germany) using an AE-6677 HorizBlot (Atto) and equivalent protein loading was confirmed by staining with Ponceau S (0.2%) in 5% acetic acid. Membranes were blocked for 1 h at room temperature with 1% BSA in PBS containing 0.05% Tween (PBST). Membranes were incubated overnight at 47C with anti-DNP antibody (Dako Ref. V0401) at a 1/5000

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dilution in PBST/1% BSA. Membranes were washed 2615 min with PBST before incubation for 1 h at room temperature with goat antirabbit HRP antibody (Dako, Denmark) at 1/3000 dilution. Membranes were washed for 2615 min in PBST once more before carbonylated proteins were detected on X-OMAT film (Sigma) using the SuperSignal West Pico Chemiluminescence kit (Pierce). Blot images were acquired using an image scanner (GS-800 calibrated densitometer, BioRad) and protein carbonylation was quantified by densitometric analysis using Quantity One 4.5.2 Analysis software (BioRad, CA, USA). 2.4 Detection of nitrated proteins For 1-DE, 100 mg protein was loaded per well and electrophoresis and protein transfer performed as described above. Following transfer, NC membranes were blocked for 1 h in 5% milk/TBS/0.05% Tween (TBST- 20 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.5). Membranes were incubated overnight at 47C with antinitrotyrosine, clone 1A6 antibody (Upstate, Lake Placid, NY, USA) at a 1/1000 dilution in 5% milk/TBST. Membranes were washed for 3615 min with TBST, prior to incubation for 1 h at room temperature with rabbit antimouse HRP-conjugated antibody (DAKO). Following 3615 min washes with TBST; immunodetection was performed using the SuperSignal West Femto Chemiluminescence kit (Pierce). Analyses involving 2-DE gel blots were carried out as above, except equivalent protein loading was confirmed using BLOT-FastStain™ (Geno Technology, MO, USA). To specifically reduce 3-NT to aminotyrosine for control experiments [35], membranes were treated with sodium dithionite (Na2S2O4) as previously described [20]. 2.5 Preparation of nitrated BSA To generate a positive control for Western blot analysis, BSA (Sigma) was treated with 500 mM peroxynitrite (Cayman Chemical, USA) at a final concentration of 1 mg/mL in 200 mM sodium bicarbonate (Sigma) at pH 7.8 [36]. Nitration of BSA was confirmed spectrophotometrically by detection of the yellow chromophore at 430 nm [37]. 2.6 Image analysis and statistics Differentially nitrated protein spots were determined by comparing 2-DE gel blots representing Wistar medulla cytosolic fraction versus SHR medulla cytosolic fractions. SHR and Wistar samples were each represented by duplicate gels originating from four individual animals. Each blot and stained membrane was scanned (BioRad GS-700 densitometer) and imported into BioRad PDQuest 2D analysis software. Replicate gels were combined into groups, normalised to the total density of detected spots, and 3NT immunoreactive spots were matched across the set of 16 blots. Resulting spot intensities were an average across each replicate group, and SDs were calculated based on spot intensities © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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of individual gels. Blots were analysed for different spot intensity (quantitative differences) and identification of spots present in only one of the two sample groups (qualitative differences). Differences in spot intensity were determined using a Mann-Whitney test within PDQuest with significance level set to either 95% (p,0.05) or 99% (p,0.01). Resulting band sets were visually inspected to verify band quality and the integrity of the statistical significance. For 1-DE blot analyses, the total intensity of the 3NT-positive bands in each lane has been divided by the intensity of four prominent bands (common to all lanes) from the corresponding lane in the Ponceau S image. This normalised quantity was used for statistical analysis so that the results would not be skewed by any slight differences in protein loading. All data are mean 6 SD, and significance (p,0.05) was investigated using unpaired, two-sample unequal variance Student’s ttest. 2.7 Spot excision, tryptic digestion and LC-MS/MS Following 1-DE or 2-DE, proteins were visualised using colloidal CBB G-250 and spots of interest excised from the gel. Proteins were in-gel digested with trypsin (sequencing grade, modified; Promega, UK,) using an Investigator ProGest robotic workstation (Genomic Solutions, Huntingdon, UK). Briefly, proteins were reduced with DTT (607C, 20 min), S-alkylated with iodoacetamide (257C, 10 min) then digested with trypsin (377C, 8 h). The resulting tryptic peptide extract was dried by rotary evaporation (SC110 Speedvac; Savant Instruments, NY, USA) and dissolved in 0.1% formic acid for LC-MS/MS analysis. Peptide solutions were analysed using an HCTultra PTM Discovery System (Bruker Daltonics, UK) coupled to an UltiMate 3000 LC System (Dionex, UK). Peptides were separated on a monolithic capillary column (200 mm id65 cm; Dionex part no. 161409). Eluent A was 3% ACN in water containing 0.05% formic acid, eluent B – 80% ACN in water containing 0.04% formic acid with a gradient of 3–45% B in 12 min at a flow rate of 2.5 mL/min. Peptide fragment mass spectra were acquired in data-dependent AutoMS (2) mode with a scan range of 300–1500 m/z, three averages, and up to three precursor ions selected from the MS scan 100–2200 m/z). Precursors were actively excluded within a 1.0 min window, and all singly charged ions were excluded. Peptide peaks were detected and deconvoluted automatically using Data Analysis software (Bruker). Mass lists in the form of MASCOT generic files were created automatically and used as the input for MASCOT MS/MS ions searches of the NCBI database using the Matrix Science web server (www.matrixscience.com). Default search parameters used were: enzyme = trypsin, max. Missed cleavages = 1; fixed modifications = carbamidomethyl (C); variable modifications = oxidation (M); peptide tolerance 6 1.5 Da; MS/ MS tolerance 6 0.5 Da; peptide charge = 21 and 31; instrument = ESI-TRAP. www.proteomics-journal.com

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Results

3.1 Protein nitration is enhanced in SHR compared to normotensive kidney medulla Cytosolic medulla fractions from SHR and Wistar cortex and medulla were compared by Western blot with anti-3NT (Fig. 1). Nitrated proteins were detected in both tissues, with prominent bands visible at ,40 kDa, ,25 kDa in Wistar and SHR medulla (Fig. 1A) and at ,38 KD only in SHR medulla cytosol (Fig. 1A). Densitometry revealed a statistically significant difference in 3NT immunoreactivity between medulla from the normotensive and hypertensive strains (Fig. 1C), principally due to the presence of the extra band, but similar analysis of Wistar and SHR medulla membrane, and cortical membrane and cytosolic fractions exhibited no differential immunoreactivity (data not shown). Our data suggest enhanced nitrosative stress within the SHR medulla cytosol compared to normotensive kidney. Although some studies have focused on the dynamics of protein nitration in mitochondria [17, 38, 39], this may not feature as significantly in the medulla due to the relatively low abundance of mitochondria in this tissue [40].

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spots unique to the SHR group were matched by PDQUEST and assessed for qualitative (spots unique to SHR) and quantitative (p,0.01–0.05, greater intensity in SHR vs. Wistar) differences. Examples of differences in 3NT immunoreactivity between SHR and Wistar samples are illustrated by zoom boxes in Fig. 2E. Sodium dithionite reduction eliminated anti-3NT immunoreactivity in both BSA nitrated in vitro (Fig. 3A) and in blots generated from SHR medulla cytosolic protein (Fig. 3B), which indicates that 3NT residues were successfully converted to aminotyrosines, and that any nonspecific binding of anti-3NT antibody is unlikely. In addition, LC-MS/ MS analysis of nitrated BSA revealed specific nitration of the protein at only one of six possible tyrosines. A y1 series ion was found at LGEY*GFQNALIVR (Tyr 424) with the correct mass of 208 (Fig. 3C). Analysis revealed 23 spots of interest; 12 spots were unique to SHR medulla, and 11 spots exhibited greater intensity in SHR than in Wistar. These were matched and excised from a corresponding Coomassie-stained polyacrylamide gel, followed by in-gel tryptic digestion and identification by LC-MS/MS. Database searching with the peptide masses gave protein identifications for 23 of the spots (Table 1).

3.2 Proteomic analysis reveals differential anti-3NT immunopositive cytosolic proteins in SHR and Wistar medulla

3.3 SHR medulla cytosol proteins exhibit more carbonylation than Wistar

Proteins from both SHR and Wistar medulla cytosolic fractions, separated by 2-DE and probed with anti-3NT, revealed enhanced nitration in the SHR medulla compared to normotensive Wistar controls (Fig. 2). Immunopositive spots common to both SHR (Fig. 2A) and Wistar (Fig. 2B), and

Protein carbonyl formation is another important marker of oxidative stress, and results from the action of ROS on such amino acids as lysine, arginine, threonine, and proline. Protein carbonyls readily react with the hydrazine moiety of DNPH, facilitating detection with anti-DNP antibodies [34].

Figure 1. Representative examples of protein tyrosine nitration (3-nitrotyrosine immunoreactivity) from SHR and Wistar rat kidney medulla. Tyrosine nitration was compared in both SHR and Wistar medulla cytosolic (A), and ponceau S staining of blots after transfer revealed equivalent loading of total protein (B). Bands obtained from three experiments were analysed by densitometry. Histogram (C) represents mean OD of total bands in lane from three independent experiments. Data are mean 6 SD. n = 3. *p,0.05.

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Figure 2. Representative 2-D immunoblots of nitrated proteins from medulla cytosolic fraction of (A) SHR and (B) Wistar (100 mg per gel) and corresponding nitrocelluose membranes (SHR medulla, C; Wistar Medulla, D) stained with BLOT-FastStain. Zoom boxes (E) illustrate examples of differences in spot immunoreactivity between SHR and Wistar blots. All qualitatively or quantitatively different immunoreactive spots are indicated by arrows. Numbers correspond to those in Table 1.

A greater level of carbonylation was observed in the SHR medulla cytosolic extract when compared with Wistar (Fig. 4) indicating that proteins in hypertensive animals are subject to enhanced oxidative stress. Immunoblotting of DNPHtreated SHR medulla samples revealed significantly greater carbonylation in the 3NT-postive proteins- Eno 1 protein, dihydrolipoamide dehydrogenase, catalase, carbonic anhydrase II (CA II), and predicted: similar to aldehyde dehydrogenase family 7 member A1 than in Wistar medulla (data not shown). In this case it would seem nitration can occur in concert with carbonylation, but the latter may not necessarily be a prerequisite for the former. This is an important con© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

sideration when protein loss/gain of function is attributed solely to nitration but where the protein may be subject to other types of oxidative modification.

4

Discussion

The medulla of SHR kidneys is the principal site of nitrosative stress, and a nitroproteome of 23 protein spots exhibiting differential immunoreactivity, representing at least 19 proteins, has been identified. Nitrated proteins were most prevalent in medulla cytosol, consistent with the observation www.proteomics-journal.com

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Figure 3. Assessment of 3NT antibody specificity. Nitrated and untreated BSA (2 mg) were immunoblotted for 3NT, with and without treatment with 100 mM sodium dithionite, which reduces nitrotyrosine to aminotyrosine (A). 2-D immunoblots of SHR medulla cytosolic protein fraction (100 mg) treated with 100 mM sodium dithionite prior to incubation with 3NT antibody (B). MS/MS spectrum of the 3NT-containing peptide L421-R433 from nitrated BSA, showing nitration at position Y424 (C).

that NO metabolism/signalling is more significant in medulla [24, 25] and suggesting that SHR medulla may be subject to enhanced nitrosative stress. Comparable immunohistochemical studies of angiotensin II-infused rats also found prominent anti-3NT staining in the renal inner medulla [20]. Greater protein carbonylation was detected in SHR medulla than in that of normotensive Wistars, but we only observed enhanced carbonylation in five of the proteins immunopositive for 3NT. This suggests that nitration and carbonylation are not necessarily mutually inclusive, and similar studies have arrived at the same conclusion [41, 42]. The amino acids subject to carbonylation are far more abundant in proteins than tyrosines, but protein structure, subcellular location and what type of RNS/ROS the local cellular environment gives rise to, may all be factors in what residues are modified. CA II is the predominant CA isoform found in kidney, where it facilitates H1 secretion by catalysing formation of HCO32 from OH2 in the presence of CO2. CA isoforms have previously been identified as nitration targets in inflammatory disease models [43], asthma [21] and as a sensitive marker of oxidative stress [42]. In kidney, CA II is involved in maintaining acid–base and fluid balance, but it is unclear what consequences its nitration may have. Acidosis may favour generation of RNS by acidification of nitrite and nitrate. This route to 3NT formation has primarily been considered in relation to intragastric compartments with low pH, but may contribute in other acidic compartments such as lysosomes, or in regions where local pH may be low (e.g. adjacent to H1 pumps). Conversely, inhibition of CA II may mitigate against 3NT formation if lowering cytosolic pH reduces the stability of ONOO2, or reduces the CO2 concentration, which appears to be a key requirement for 3NT formation [10]. Catalase is a key peroxisomal antioxidant enzyme catalyzing decomposition of H2O2 to water and oxygen. It has © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

been identified as a nitration target in nitrosative stress [21], and, when coadministered in the kidney medulla with the SOD mimetic Tempol, it can prevent blood pressure increases in SHR [44], suggesting a role in managing hypertension. Attempts have been made to exploit the therapeutic potential of catalase in lung transplantation [45], and a similar strategy may prove beneficial in hypertension. A second peroxisomal protein identified in this study was hydroxyacid oxidase 3 (HAOX3), a member of the HAOX family, which catalyse oxidation of a-hydroxy acids, a-amino acids and glycolate with production of H2O2. HAOX3 was originally reported as a pancreatic HAOX [46], but was misidentified as murine a-hydroxyacid oxidase 2 (HAOX2). This suggests that we have identified the rat kidney isoform HAOX2. Due to co-compartmentalisation with catalase, H2O2 generated by HAOXs should be rapidly decomposed but, in rat liver, HAOX1 is down-regulated during oxidative stress [47]. Nitration of HAOX2 may cause a similar downregulation of activity in kidney, and could be an adaptive response to diminish ROS generation. Furthermore, differential gene expression analysis identified the HAOX2 gene as a qualitative trait locus for blood pressure in hypertensive Dahl-salt-sensitive rats, suggesting HAOX2 may restrict vasodilation though NOS inhibition [48]. We identified various aldo-keto-reductases (AKR). A growing body of evidence implicates these proteins in both protection against, and propagation of, oxidative and nitrosative stress. The AKR superfamily comprises approximately 60 proteins, all monomeric NADPH-dependent oxidoreductases, with broad tissue distribution and substrate specificity for aldehydes and ketones. AKR metabolise a range of toxic aromatic and aliphatic carbonyl compounds including lipid peroxidation byproducts [49]. The ability to protect against diseases like vasculitis [50] has earned AKR a nominal cytoprotective role but, under certain conditions, they www.proteomics-journal.com

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Table 1. Differentially nitrated proteins in SHR medulla

Spot Protein no.

1 2 3 4 8 5

Accession no.

3NT qualitative difference

3NT quantitative difference

MW (kDa)/pI predicted– observed

Sequence MASCOT Peptides coverage score matched (%)

Functional grouping

p,0.01 p,0.05 p,0.05

p,0.05

29/6.9–29/7.13 71/5.4–69.19/5.3 70/6.1–70.7/5.6 51/7.2–49.3/6 51/7.2–49.3/5.48 38/6.2–38/6.16

53 44 71 74 60 53

472 1383 2419 1849 1429 812

18 42 67 58 37 22

CO2 metabolism Chaperone/stress response Transport/homoeostasis Gluconeogeneis/glycolysis Gluconeogeneis/glycolysis Gluconeogeneis/glycolysis

p,0.01 p,0.05 p,0.05

38/6.2–38/6.04 37/6.2–38.4/5.9 37/6.8–38.6/6.7

51 62 59

806 801 1048

24 31 37

39/6.8–48.7/6.68

49

705

28

37/6.8–45/6.5

36

407

11

32/5.8–37/5.6 49/6.3–48.9/5.96 56/6.7–63/5.91

54 48 39

888 1092 988

23 25 26

38/6.2–67.2/7.2

51

806

24

57/5.9–65.7/5.55

41

791

21

42/5.3–38.3/5.41 32 60/7.07–67.6/7.01 37 70/6.1/–68.3/6.05 18

419 542 414

15 16 8

CA II Heat shock protein 8 Albumin Eno 1 protein Eno 1 protein Glycerol-3-phosphate dehydrogenase

AAH65577 NP_077327 AAH85359 AAH81847 AAH81847 NP_071551

13 6 7

Malate dehydrogenase AKR family 1, member A1

NP_150238 NP_112262

20

Afar protein

AAH78872

23

AKR family 1, member A1

NP_112262

9 10 11

DDAH1 Ornithine aminotransferase Mitochondrial aldehyde dehydrogenase precursor Hypothetical protein LOC361730 PDI A3 precursor

NP_071633 NP_071966 AAS75814

P11598

3

Actin–beta Catalase Phosphoenolpyruvate carboxykinase 1 Dihydrolipoamide dehydrogenase Predicted: similar to aldehyde dehydrogenase family 7 member A1 HAOX3

ATRTC NP_036652 NP_942075

3 3 3

NP_955417

3

55/7.96–66.3/6.35 32

440

12

XP_214535

3

59/7.9–63/6.5

37

697

16

p,0.01

40/7.5–44.3/6.44

48

792

25

p,0.05

57/8.7–61.2/7.07

48

1015

29

12 14 15 16 17 18 19

21 22

3 3

3 p,0.01 3 3 3

NP_001034120

p,0.05

NP_114471

Predicted: similar to 3 XP_001053666 oxoacid CoA transferase 1 isoform 2

p,0.05

Gluconeogeneis/glycolysis Oxidoreductase/antioxidant defence Oxidoreductase/antioxidant defence Oxidoreductase/antioxidant defence NOS regulation Urea cycle Aldehyde metabolism

Unknown: homologous to dihydroxyacetone kinase Chaperone/disulphide bridge formation Structural Antioxidant defence Gluconeogeneis/glycolysis Pyruvate dehydrogenase complex component Aldehyde dehydrogenase superfamily a-Hydroxy acid oxidation/ H2O2 generation Ketone body metabolism

Figure 4. Representative examples of carbonylated proteins in both SHR and Wistar medulla cytosol (A). Ponceau S staining of blots after transfer revealed equivalent loading of total protein. Bands obtained from at least four experiments were measured densitometrically. Histogram (B) represents mean OD of total bands per lane from four experiments. Data are mean 6 SD. n = 3. *p,0.05.

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may contribute to pathology [51]. Having discovered that AKR are susceptible to nitration, it was proposed that nitration of a conserved Tyr-55 residue in the catalytic site may enhance rather than inhibit activity [38]. Ornithine aminotransferase (OAT) was also identified as a member of the nitroproteome, but is generally associated with mitochondria. This could have arisen through incomplete separation of the membrane/mitochondrial and cytosolic fractions, but there is an unusually high proportion of cytosolic OAT in regions of the medulla [52] and mitochondria are much less abundant in the inner medulla than in other kidney regions [40]. OAT is a component of the urea cycle catalyzing conversion of L-ornithine and a-ketoglutarate to glutamate and glutamate semialdehyde. The metabolic fate of ornithine in kidney is linked to both the urea cycle and glutamate synthesis. This, in turn, implicates ornithine in glutathione biosynthesis and in the citric acid cycle. It is not known if ornithine utilisation is perturbed in SHR kidney, but it is interesting to consider possible consequences of OAT modification. Enhanced OAT activity might deplete the arginine pool utilised by NOS, thus impairing NO signalling, whereas decreased OAT activity could impair antioxidant defence through diminished glutathione synthesis. Nitration of dimethylarginine dimethylaminohydrolase 1, (DDAH1) may have more direct consequences for NO bioavailability. DDAH1 was originally thought to be mainly responsible for preventing bioaccumulation of the endogenous arginine analogues, asymmetric dimethylarginine (ADMA) and N-monomethylarginine, byproducts of protein degradation. However, ADMA and N-monomethylarginine are inhibitors of NOS [53]. DDAH1 is therefore understood to regulate NOS activity through controlling the concentration of these inhibitors. ADMA is eliminated by renal excretion and the action of DDAH1. Inhibition of DDAH1 could cause ADMA accumulation, inhibiting NOS and promoting vasoconstriction over vasodilation. There is evidence that DDAH1 itself is regulated through S-nitrosylation, so alterations in NO levels could have important activity implications [54]. Protein disulphide isomerase (PDI) and heat shock proteins 70 isoforms (hsp70) have been identified in nitrosative stress [21, 35], suggesting that ER-associated proteins are also RNS targets. Under oxidative and nitrosative stress, both increased degradation and accumulation of proteins have been observed. The latter outcome may entail ER-associated stress induced by peroxynitrite leading to accumulation of misfolded proteins [55], a risk factor in kidney disease. Another nitration target, albumin, which is involved in osmotic pressure maintenance, has been identified in previous nitration studies and induces ER-associated stress in renal proximal tubular cells [56]. In common with previous reports concerning oxidative/ nitrosative stress, enzymes involved in cellular energy production and intermediary metabolism were identified in the present study: enolase 1 [57]; malate dehydrogenase [21, 35, 57]; phosphoenolpyruvate carboxykinase 1 [39], dihy© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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drolipoamide dehydrogenase and glycerol-3-phosphate dehydrogenase. Restricting glycolysis may be a common mechanism for preventing excessive ROS or RNS generation, but could compromise basic energy metabolism. Such a strategy seems to be in place in the hypoxic regions of kidney but may be perturbed in SHR. In our study, SHR medulla exhibited enhanced carbonylation, and increased carbonylation and decreased antioxidant capacity are global features within SHR kidney [58]. In our LC-MS/MS study 3NT peptides were not detected, presumably because of being below detection limits. Miyagi et al. [35] encountered a similar problem in their study of endogenous 3NT formation in rat retinas, and other groups have encountered difficulty in mapping 3NT formation due to the low yield of nitrated proteins generated via endogenous processes [36]. Individual 2-DE gel spots may contain multiple protein isoforms, and the relative abundance of a nitrated protein could be low within each spot. The 3NT modification is both labile under strongly reducing conditions and quite rare even in pathology. Peptides containing 3NT are susceptible to photodecomposition during MALDI, which decreases the mass of the intact peptide, and causes 3NT-containing residues to be overlooked due to underestimation of their characteristic mass [59]. Groups have attempted to address these problems with methods such as residue-specific antinitrotyrosine antibodies [20, 60], dansyl chloride labelling of 3NT residues [61], reduction to aminotyrosine followed by acetylation to enhance detection via MS [59] and sample enrichment using a nitrotyrosine affinity column [62]. Our observed increase in nitration was detected against a background of extensive protein oxidation, suggesting that, in addition to 3NT formation, oxidation of residue sidechains to protein carbonyls could also contribute to alteration of activity. Xu et al. [20] attributed a 50% decrease in Mn-SOD activity to nitration of Tyr-34, but studies by Ghosh et al. [21] into catalase inactivation found that tyrosine chlorination was 20-fold greater than nitration. The principal RNS involved in ONOO2 based protein nitration under physiological conditions are not definite [63]. SHR kidney represents a singular scenario with respect to nitrosative stress, given the presence of increased ROS in a nominally hypoxic environment and the unusually high NO generating potential of the medulla. This may create the ‘supraphysiological’ conditions necessary to cause enhanced nitrosative stress. Further work will be necessary to explore our findings’ deeper implications. Several of the identified proteins have the potential to contribute to renopathy observed in SHR but they could also be biomarkers rather than effectors of the pathophysiology. The authors would like to acknowledge the contribution of the Proteomics Unit, University of Aberdeen, Scotland in preparing this manuscript. Our laboratory (RT and DS) is funded by the Higher Education Authority of Ireland Programme for Research in Third Level Institutions, Cycle 3. www.proteomics-journal.com

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