Differential expression of liver and kidney proteins in a mouse model for primary hyperoxaluria type I Juan R. Herna ´ ndez-Fernaud 1 and Eduardo Salido 2 1 Max Planck Institute for Biochemistry, Department of Proteomics and Signal Transduction, Klopferspitz, Martinsried, Germany 2 Hospital Universitario Canarias, Center Biomedical Research on Rare Diseases (CIBERER) and Institute of Biomedical Technologies (ITB), Tenerife, Spain Introduction Primary hyperoxaluria type I (PHI) is a rare autosomal recessive disease caused by mutations in the alanine-gly- oxylate aminotransferase gene (AGXT). Alanine-glyoxy- late aminotransferase (AGT) (or alanine-glyoxylate aminotransferase 1, AGT1), the protein encoded by AGXT, plays an important physiological role in glyoxy- late detoxification by converting it into glycine. The enzyme is present in peroxisomes and ⁄ or mitochondria in different mammalian species, with peroxisomal AGT being mainly responsible for the detoxification of glyco- late-derived glyoxylate, and mitochondrial AGT playing a major role in the metabolism of hydroxyproline- derived glyoxylate [1]. In humans, insufficient AGT activity in peroxisomes leads to increased cytosolic conversion of glyoxylate to oxalate. Excessive renal excretion of oxalate causes calcium oxalate deposition (nephrocalcinosis and urolithiasis) and eventual loss of renal function. After renal failure, calcium oxalate depo- sition becomes widespread and life-threatening unless liver and kidney transplantation are performed. With a better understanding of glyoxylate metabolism, sub- strate depletion may potentially be a useful intervention in patients with PHI [2]. In order to further analyze the mechanisms of PHI disease, and to explore new therapeutic approaches, we have developed an Agxt knockout (AgxtKO) mouse that reproduces some key features of PHI [3]. Homozygous Agxt ) ⁄ ) mice show severe hyperoxaluria, Keywords hyperoxaluria; kidney; liver; mouse model; subcellular fractions Correspondence E. C. Salido, Hospital Universitario Canarias, Center Biomedical Research on Rare Diseases (CIBERER) and Institute of Biomedical Technologies (ITB), Tenerife 38320, Spain Fax: +34 922 647 112 Tel: +34 922 319 338 E-mail: esalido@ull.es (Received 29 July 2010, revised 3 September 2010, accepted 10 September 2010) doi:10.1111/j.1742-4658.2010.07882.x Mutations in the alanine-glyoxylate aminotransferase gene (AGXT) are responsible for primary hyperoxaluria type I, a rare disease characterized by excessive hepatic oxalate production that leads to renal failure. A deeper understanding of the changes in the metabolic pathways secondary to the lack of AGXT expression is needed in order to explore substrate depletion as a therapeutic strategy to limit oxalate production in primary hyperoxal- uria type I. We have developed an Agxt knockout (AgxtKO) mouse that reproduces some key features of primary hyperoxaluria type I. To improve our understanding of the metabolic adjustments subsequent to AGXT defi- ciency, we performed a proteomic analysis of the changes in expression lev- els of various subcellular fractions of liver and kidney metabolism linked to the lack of AGXT. In this article, we report specific changes in the liver and kidney proteome of AgxtKO mice that point to significant variations in gluconeogenesis, glycolysis and fatty acid pathways. Abbreviations AGT1, alanine-glyoxylate aminotransferase 1; AGT2, alanine-glyoxylate aminotransferase 2; AGXT, alanine-glyoxylate aminotransferase gene; Agxt ) ⁄ ) , alanine-glyoxylate aminotransferase homozygous knockout; ML, mitochondrial ⁄ lysosomal; PHI, primary hyperoxaluria type I; SPT, serine-pyruvate aminotransferase; 2-DE, two-dimensional electrophoresis. 4766 FEBS Journal 277 (2010) 4766–4774 Journal compilation ª 2010 FEBS. No claim to original German government works and males develop calcium oxalate crystalluria and cal- culi in the urine bladder, although no deposits in the renal parenchyma (nephrocalcinosis) are observed unless the animals are subjected to metabolic overload. To better characterize this model, and provide evidence useful in substrate depletion strategies, we report in this article a proteomic analysis of the changes in expression levels of various enzymes of liver and kidney metabolism linked to the lack of AGT. Results We first attempted to detect differences in protein expression between hyperoxaluric and control mice at the whole-organ proteome level, using either liver or kidney samples. This approach yielded insufficient protein spots and lower reproducibility than that based on subcellular fractionation, and was not pursued fur- ther. However, for each subcellular fraction studied, more than 300 protein spots were detected in each two- dimensional electrophoresis (2-DE) gel. For each frac- tion, three 2-DE silver- and Coomassie-stained gels were integrated and analyzed, and high reproducibility was achieved (Fig. 1). By image analysis, using the relative spot volume parameter, the comparison between gels of wild-type and knockout kidney proteomes revealed 22 spots whose protein levels were significantly different between groups (P < 0.01), with three exclusive of knockout mice. Twenty of these differentially expressed proteins were correctly matched to protein candidates in the database (Table 1) according to their peptide mass fingerprints analyzed by MALDI-TOF MS. pH3 10 AB CDE F GH pH3 10 Fig. 1. Comparison of 2-DE patterns among different extraction methods and cell fractions. (A, B) Total extraction protein method of kidney and liver organs, respectively. (C, D, E) Mitochondrial- lysosomal, peroxisomal and cytosolic fractions of kidney. (F, G, H) Mitochondrial- lysosomal, peroxisomal and cytosolic fractions of liver. Total protein (300 lg) was subjected to 2-DE (first dimension: glass capillaries; pH 3–10; 12 cm; second dimension: 10% polyacrylamide SDS ⁄ PAGE; 18 · 18 cm 2 ). Proteins were visualized by silver staining. J. R. Herna ´ ndez-Fernaud and E. Salido Proteome changes in primary hyperoxaluria FEBS Journal 277 (2010) 4766–4774 Journal compilation ª 2010 FEBS. No claim to original German government works 4767 Database search and functional exploration of these proteins revealed that they were associated with dif- ferent metabolic aspects, such as oxidoreductase activ- ity, glycolysis, glycine, glyoxylate, fatty acid and pyruvate metabolism. Hydroxyacid oxidase 3 was two-fold more abundant in knockout mice than in controls. In contrast, d-amino acid oxidase 1 was 2.3- fold downregulated in hyperoxaluric mice. Enolase 1 and malic enzyme were upregulated. Furthermore, acyl-coenzyme A dehydrogenase, mercaptopyruvate sulfotransferase and abhydrolase domain protein were only detected in knockout mouse kidneys (Table 1, Fig. 2A). In liver fractions, 18 spots were identified with protein levels significantly different between the groups (P < 0.01), and two were exclusively detected in knock- out mice. In 14 of the 18 spots, MALDI peptide mass fingerprints allowed the identification of the correspond- ing proteins in the database (Table 2). Database search and functional exploration of these proteins revealed that they were associated with gluconeogenesis and glycolysis. In this sense, fructose bisphosphatase was 2.4-fold upregulated in knockout mice. However, alde- hyde dehydrogenase, carbonic anhydrase, enolase and malic enzyme were downregulated (Table 2, Fig. 2B). In cytosolic fractions, the fumarylacetoacetate hydrolase and peroxiredoxin 6 appeared, with shifted pI from approximately 6.9 to 7 and 6 to 5.5, respectively. Western blot analysis was used to confirm the main differences in expression found in 2-DE gels, provided that antibodies were available. The results are summarized in Fig. 3A. In AgxtKO mice, kidney enolase was clearly overexpressed, as were liver fructose bisphosphatase and catalase, whereas liver enolase and carbonic anhydrase 3 were downregu- lated. Comparable amounts of b-actin were present in AgxtKO and wild-type cytosolic fractions, and the absence of AGT1 protein in AgxtKO samples was also confirmed by western blot. The changes in expression levels observed in these few proteins are in agreement with 2-DE results, which is consistent with the reliability of our comparative proteomic study. To assess the tissue specificity of the liver and kid- ney response, we also performed western blot analysis of skeletal muscle proteins. We observed high variabil- ity and could not reproduce the detected differences in liver and kidney samples (Fig. 3B). Discussion We have analyzed the changes in protein expression within the liver and kidney of Agxt ) ⁄ ) deficient mice compared with wild-type controls by 2-DE separation and MS. The analysis of specific subcellular fractions was necessary to obtain highly informative and repro- ducible 2-DE gels. The modified fractionation protocol adopted has been used previously in proteomic studies [4], but does not result in highly pure fractions, which is likely to be the reason for some inconsistencies between the fraction in which we detected a differen- tially expressed protein and their accepted subcellular localization. For instance, we detected d-amino acid oxidase in the mitochondrial ⁄ lysosomal (ML) fraction of kidney, whereas its accepted localization is either cytosolic or peroxisomal. Most likely, our ML fraction contained peroxisomes that cosedimented during the procedure used. Similarly, liver catalase was detected in our cytosolic fraction, indicating that peroxisomes and ⁄ or peroxisomal proteins were still present in the supernatant after the 7300 g centrifugation. Under standard purification procedures, peroxisomal proteins are known to contaminate other subcellular fractions because of peroxisomal fragility. With this limitation, our fractionation method was mainly useful as a sim- ple way to reduce the complexity of the proteome, facilitating the differential expression analysis between wild-type and AgxtKO mice. Agxt ) ⁄ ) mice have impaired glyoxylate detoxifica- tion, with subsequent oxalate overproduction by the liver and increased urinary oxalate excretion, similar to patients with PHI [3]. However, significant differences between mouse and human glyoxylate and glucose metabolism must be considered. Although human AGT1, the product of the AGXT gene, is predomi- nantly localized in the peroxisome, the mouse Agxt1 gene is transcribed into two different mRNA species, coding for mitochondrial and peroxisomal variants [5]. Indeed, rodent AGT1 is also known as serine-pyruvate aminotransferase (SPT) because the mitochondrial form participates in gluconeogenesis from serine, whereas the conversion of glyoxylate to glycine takes place largely in peroxisomes. No alterations of glucose metabolism have been described in patients with PHI. In AgxtKO mice, we detected an increase in liver fructose-1,6-bisphosphatase, an enzyme involved in the hydrolysis of fructose-1,6-bisphosphate, which plays an important regulatory role in gluconeogenesis [6]. In the same hepatic fractions, a decrease in cytosolic malic enzyme 1 was observed, pointing to a reduction in NADPH available for fatty acid biosynthesis. Taken together, these results seem to be indicative of an adap- tation in favor of liver gluconeogenesis in response to the lack of AGT1. Other downregulated enzymes, such as aldehyde dehydrogenase 2, enolase 1, UDP- glucose pyrophosphorylase 2 and fumarylacetoacetate Proteome changes in primary hyperoxaluria J. R. Herna ´ ndez-Fernaud and E. Salido 4768 FEBS Journal 277 (2010) 4766–4774 Journal compilation ª 2010 FEBS. No claim to original German government works Table 1. Summary of kidney proteins that are differentially expressed (P < 0.01) in mitochondrial ⁄ lysozome (ML), peroxisomal (P) and cytosolic (C) fractions. Group number Protein name Short name NCBI no. Theorical MW (Da) ⁄ pI Matched peptide Sequence coverage (%) Mascot score Missed cleavage Fold ko expression Vol (%) Fraction 431 Enolase 1, a non-neuron Eno1 gi: 13278078 47 322 ⁄ 6.36 12 37 135 1 +2.27 – ML 577 D-Amino acid oxidase 1 Dao1 gi: 198572 39 017 ⁄ 7.19 10 35 130 1 )2.35 – ML 722 Hydroxyacid oxidase 3 Hao3 gi: 20379611 39 145 ⁄ 7.55 9 29 124 1 +2 – ML 976 a Mercaptopyruvate sulfotransferase Mpst gi: 13278579 33 100 ⁄ 6.12 11 33 123 1 Only ko 0.25 ML 1280 a Acyl-coenzyme A dehydrogenase, short chain Acads gi: 16740777 45 146 ⁄ 8.68 13 33 115 2 Only ko 0.225 ML 96 PDZ domain containing 1 Pdzk1 gi: 15488745 56 862 ⁄ 5.34 10 25 133 1 +15.15 – P 227 Hydroxyacid oxidase 3 Hao3 gi: 20379611 39 145 ⁄ 7.55 7 23 71 2 +18.65 – P 257 Endoplasmic reticulum protein 29 Erp29 gi: 16877776 28 862 ⁄ 5.90 10 37 120 2 )3.3 – P 67 Aconitase 1 Aco1 gi: 110347487 98 705 ⁄ 7.23 10 19 103 1 )3.61 – C 129 Malic enzyme 1, NADP(+)-dependent, cytosolic Me1 gi: 13096987 64 426 ⁄ 6.87 9 13 91 2 +3.22 – C 165 DnaJ (Hsp40) homolog, subfamily A, member 1 DnaJA1 gi: 81894107 45 581 ⁄ 6.65 7 31 74 2 +1.56 – C 202 Apolipoprotein A-IV ApoA4 gi: 14789706 44 545 ⁄ 5.48 13 40 128 1 +1.68 – C 216 Actin, b, cytoplasmic ActB gi: 387083 39 446 ⁄ 5.78 8 33 109 1 +1.92 – C 224 NSFL1 (p97) cofactor (p47) Nsfl1C gi: 12850132 40 685 ⁄ 5.04 15 44 178 1 +1.92 – C 240 Aminoacylase 1 Acy1 gi: 13542872 45 980 ⁄ 5.89 9 29 82 1 +3.76 – C 300 Phosphoglycerate mutase 1 Pgam1 gi: 12805529 28 797 ⁄ 6.75 7 21 89 1 )3.28 – C 303 Indolethylamine N-methyltransferase Inmt gi: 15488762 30 068 ⁄ 6.0 4 30 64 0 )4.73 – C 311 Peroxiredoxin 6 Prdx6 gi: 15488685 24 838 ⁄ 5.72 7 37 90 1 )9.28 – C 313 Apolipoprotein A-I ApoA1 gi: 109571 30 358 ⁄ 5.52 7 25 77 1 +7.69 – C 337 a Abhydrolase domain containing 14b Abhd14b gi: 18043201 22 551 ⁄ 6.82 5 30 63 1 Only ko 0.191 C a The figures show the magnified comparison maps between wild-type (wt) and knockout (ko) of spots 1280, 976 and 337. 337 wt ko 1280 wt ko 976 wt ko J. R. Herna ´ ndez-Fernaud and E. Salido Proteome changes in primary hyperoxaluria FEBS Journal 277 (2010) 4766–4774 Journal compilation ª 2010 FEBS. No claim to original German government works 4769 hydrolase, appear to support this observation. These results are consistent with our previous observation that AgxtKO mice did not seem to show a deficit in gluconeogenesis despite the absence of the AGXT1 gene product [3]. There is also a significant level of another aminotransferase, AGT2, in mouse liver [7], although kinetic studies [8] indicate that its alanine- glyoxylate aminotransferase activity is not favored over aminobutyrate-pyruvate, b-alanine-pyruvate and dimethylarginine-pyruvate aminotransferase activities. In the rat, gluconeogenesis from l-serine takes place mainly through l-serine dehydratase, whereas the flux through SPT ⁄ AGT in gluconeogenesis from serine has been shown to be significant only after the liver mito- chondrial form of the AGT1 enzyme had been induced by glucagon [9]. However, the peroxisomal form of SPT ⁄ AGT predominates during constitutive expression of rat and mouse AGXT genes, and the gluconeogenic flux from serine also takes place in this organelle to some extent [10]. Amino acid metabolism is considered to be a major contributor to endogenous oxalate syn- thesis, justifying the study of changes in liver enzymes in the context of primary hyperoxaluria. It could be speculated that our finding of enhanced liver gluconeo- genesis in the PHI mouse model is an adaptation to the lack of serine flux through AGT, and modifications that potentiate neoglucogenesis might be beneficial in primary hyperoxaluria, reducing the oxalate contribu- tion from amino acid metabolism. These modifications might be seen as a form of substrate depletion. How- ever, the above-mentioned differences in AGT subcel- lular localization between humans and laboratory Fig. 2. Metabolic kidney (A) and liver (B) enzymes upregulated (+) and downregulated ()) in knockout mice, or spots only present in knockout mice. Acads, acyl-coenzyme A dehydrogenase, short chain; Aco1, aconitase 1; Agt ) , alanine-glyoxylate aminotransferase knockout; Aldh2, aldehyde dehydrogenase 2; Car3, carbonic anhydr- ase 3; Cat, catalase; Dao1, D-amino acid oxidase 1; Eno1, enolase 1, a non-neuron; Fah, fumarylacetoacetate hydrolase; Fbp1, fructose bisphosphatase 1; Hao3, hydroxyacid oxidase 3; Me1, malic enzyme 1, NADP(+)-dependent; Mpst, mercaptopyruvate sulfotransferase; Pgam1, phosphoglycerate mutase 1; Prdx6, peroxiredoxin 6; Ugp2, UDP-glucose pyrophosphorylase 2. Proteome changes in primary hyperoxaluria J. R. Herna ´ ndez-Fernaud and E. Salido 4770 FEBS Journal 277 (2010) 4766–4774 Journal compilation ª 2010 FEBS. No claim to original German government works Table 2. Summary of liver proteins that are differentially expressed (P < 0.01) in mitochondrial ⁄ lysosomal (ML), peroxisomal (P) and cytosolic (C) fractions. Group number Protein name Short name NCBI nr Theorical MW (Da) ⁄ pI Matched peptide Sequence coverage (%) Mascot score Missed cleavage Fold ko expression Vol (%) Fraction 250 Aldehyde dehydrogenase 2, mitochondrial Aldh2 gi:13529509 57 015 ⁄ 7.53 14 29 149 1 )1.90 – ML 265 Eukaryotic translation elongation factor 2 Eef2 gi:192989 30 212 ⁄ 6.2 6 32 76 1 +2.27 – ML 333 Fructose bisphosphatase 1 Fbp1 gi:9506589 37 288 ⁄ 6.15 8 33 99 1 +2.44 – ML 219 Carbonic anhydrase 3 Car3 gi:15029812 29 502 ⁄ 6.97 10 55 130 1 )2.99 – P 220 Carbonic anhydrase 3 Car3 gi:15029812 29 502 ⁄ 6.97 10 55 130 1 )3.69 – P 109 Malic enzyme 1, NADP(+)-dependent Me1 gi:13096987 64 426 ⁄ 6.87 7 14 84 0 )2.11 – C 120 Catalase Cat gi:157951741 59 882 ⁄ 7.72 8 21 97 0 +4.54 – C 145 UDP-glucose pyrophosphorylase 2 Ugp2 gi:21314832 55 634 ⁄ 6.92 9 27 116 1 )1.79 – C 203 Fumarylacetoacetate hydrolase Fah gi:14789698 46 416 ⁄ 6.92 11 28 120 0 )1.67 – C 146 a Fumarylacetoacetate hydrolase Fah gi:14789698 46 416 ⁄ 6.92 8 21 86 0 Only ko 1.18 C 215 Enolase 1, a non-neuron Eno1 gi:55491 47 322 ⁄ 6.36 7 18 79 0 )2.50 – C 258 Carbonic anhydrase 3 Car3 gi:15029812 29 502 ⁄ 6.97 8 45 121 0 )3.86 – C 267 Peroxiredoxin 6 Prdx6 gi:15488685 24 925 ⁄ 5.98 9 53 127 1 )2.49 – C 201 a Peroxiredoxin 6 Prdx6 gi:15488685 24 925 ⁄ 5.98 7 43 96 1 Only ko 0.561 C a The figures show the magnified comparison maps between wild-type (wt) and knockout (ko) of spots 146 and 201. 146 wt ko 201 wt ko J. R. Herna ´ ndez-Fernaud and E. Salido Proteome changes in primary hyperoxaluria FEBS Journal 277 (2010) 4766–4774 Journal compilation ª 2010 FEBS. No claim to original German government works 4771 rodents pose a major limitation to our study and the inferences that can be based on our findings. Further studies of the sources of endogenous oxalate synthesis in humans are needed. The phenotypic features of Agxt ) ⁄ ) mice are proba- bly the direct consequence of impaired glyoxylate detoxification, with subsequent oxalate overproduction by the liver and increased urinary oxalate excretion. As AGT1 is not expressed at significant levels in the kidney, the changes observed in the kidney proteome could be a consequence of variations in filtered metab- olites, such as oxalate, present at high levels in AgxtKO mice. Nephrocalcinosis is essentially absent in Agxt ) ⁄ ) mice, despite high urinary oxalate excretion, unless glyoxylate precursors are administered. Thus, the response in the kidney proteome to AGT1 deficit is unlikely to be secondary to serious tissue damage. The increase in kidney enolase points to an enhanced gly- colysis, whereas higher levels of hydroxyacid oxidase 3 could represent adjustments in medium-chain hydroxy- fatty acid metabolism. The overexpression of enolase 1 and malic enzyme 1 supports the induction of fatty acid metabolism. The reduction in d-amino acid oxi- dase 1 expression in the kidney proteome is interesting, in view of the contribution of this enzyme to glyoxy- late production from glycine. Thus, it could be specu- lated that a decrease in d-amino acid oxidase 1 expression might be aimed at reducing the glyoxylate overload in the kidney of AgxtKO mice. In conclusion, changes in the proteome contents of hyperoxaluric liver and kidney subcellular fractions should improve our understanding of the metabolic adjustments subsequent to AGXT deficiency, and might provide relevant clues for future developments of substrate depletion approaches in the treatment of primary hyperoxaluria. Experimental procedures Animals Mice were bred and maintained in a pathogen-free facility, with free access to standard chow (A04, SAFE, Augy, France) and water. Male mice from heterozygous (Agxt + ⁄ ) ) breeders (B6.129SvAgxt tm1Ull ) were used and genotyped as reported previously [3]. All the experiments were performed in accordance with Spanish and European law regarding the use of animals in research (European Community Council Directive of 24 November 1986, 86 ⁄ 609 ⁄ EEC), following a protocol that had been approved by our Institutional Committee on Ethics in Animal Experi- mentation (CEBA-HUC). Mice were sacrificed by cervical dislocation, performed by trained personnel, in accordance with CEBA-HUC-approved protocols. Immediately after brain death, laparotomy and bilateral thoracotomy were performed and the organs were harvested, making sure that the animals did not suffer at any stage of the procedure. Tissues from eight, 3-month-old male mice allocated to 0 1 2 3 4 5 6 7 8 9 Eno1-K Fbp1-L FML Car3-L FP Car3-L FC Cat-L FC Eno1-L Agt-L FP Actin-L FC Grhpr-L FML Grhpr-K FML Eno1-M FML Fbp1-M FML FP FC FC Eno1-M FC 0 1 2 3 A B Fig. 3. (A) Western blot analyses of enolase (Eno1-K) and glyoxylate reductase ⁄ hydroxy- pyruvate reductase (Grhpr-K) from kidney and fructose bisphosphatase (Fbp1-L), carbonic anhydrase 3 (Car3-L), catalase (Cat-L), enolase (Eno1-L), alanine-glyoxylate aminotransferase (Agt-L), actin (Actin-L) and glyoxylate reductase ⁄ hydroxypyruvate reductase (Grhpr-L) from liver. (B) Western blot analyses of enolase (Eno1-M), fructose bisphosphatase (Fbp1-M), carbonic anhydr- ase 3 (Car3-M) and catalase (Cat-M) from muscle. The analyses are in mitochon- drial ⁄ lysosomal (FML), peroxisomal (FP) and cytosolic (FC) fractions. Densitometry of western blot bands was performed and units of selected proteins were calculated. A representative western blot band for each protein of each experimental group is shown. Relative molecular weights were 47, 74, 29, 60, 45, 40 and 36 kDa for Eno1, Fbp1, Car3, Cat, Agt, Actin and Grhpr, respectively. Proteome changes in primary hyperoxaluria J. R. Herna ´ ndez-Fernaud and E. Salido 4772 FEBS Journal 277 (2010) 4766–4774 Journal compilation ª 2010 FEBS. No claim to original German government works Agxt ) ⁄ ) (hyperoxaluric) and from homozygous wild-type (Agxt + ⁄ + ; control) mice were harvested, sliced and thor- oughly rinsed in ice-cold saline before freezing. Sample preparation Tissues for whole-protein extraction were frozen and crushed in liquid nitrogen. The powder was lyophilized and 10 mg were extracted, during 1 h at 4 °C, in 350 lLof extraction buffer with 8 m urea, 4% Chaps, 40 mm Tris, 65 mm 1,4-dithioerythritol, 0.05% SDS and 2% ampho- lytes. Next, the sample was centrifuged at 13 000 g for 30 min at 4 °C to form a pellet of insoluble material. Subcellular fractionation by differential centrifugation was performed as described previously [4]. The tissue was imme- diately minced in ice-cold isotonic buffer (5 : 1, v ⁄ w) contain- ing 250 mm sucrose, 10 mm Tris ⁄ HCl, pH 7.5, and 1 mm EDTA. The cells were ruptured by 20 strokes in a glass homogenizer, and the lysate was centrifuged at 200 g for 10 min to sediment the nuclei. The supernatant was centrifuged again at 2000 g for 10 min to sediment a mitochondria-containing pellet that was homogenized in 1 mL of isotonic buffer and centrifuged at 7300 g for 10 min to obtain a crude mitochondrial pellet. The 2000 g superna- tant was centrifuged at 7300 g for 10 min to obtain a crude peroxisomal fraction. The peroxisome pellet was homoge- nized and centrifuged once again, and the supernatant was centrifuged for 60 min at 7300 g to remove additional organ- elles from the cytosolic fraction. Proteins in the cytosolic fraction were precipitated with cold acetone (1 : 1, v ⁄ v) for 60 min at )20 °C and centrifuged at 13 000 g for 30 min at 4 °C. The pellet was washed with cold acetone, centrifuged once and air dried. Organelle and protein pellets were solubilized in the extraction buffer and centrifuged at 13 000 g for 30 min at 4 °C. The supernatants were recov- ered and stored at )20 °C. The protein concentration was determined by the Bradford method with BSA as the pro- tein standard. 2-DE Isoelectric focusing was performed using glass capillary tubes (inside diameter, 1.5 mm; length, 12 cm). For separa- tion in the pH 5–8 range, capillary tubes were filled with solution containing 3% acrylamide, 7 m urea, 0.6% Triton X-100, 0.75% ampholytes pH 5–8, 0.22% ampholytes pH 3–10 and 0.22% ampholytes pH 7–9, 0.045% N,N,N¢,N¢-tetramethylethylenediamine and 0.08% ammo- nium persulfate. For separation in the pH 3–10 range, 0.75% ampholytes pH 3–10 and 0.22% ampholytes pH 5–8 were used instead. Samples containing approximately 300 lg of total protein were applied to the basic end of the tube gel. Cathodic and anodic buffers were 20 mm NaOH and 8.7 mm H 3 PO 4 , respectively. Isoelectric focusing steps consisted of 1 h at 100 and 1 h at 300 V, followed by 17.5 h at 1000 V and 30 min at 2000 V. Next, the capillar- ies were equilibrated for 15 min in reducing buffer contain- ing 50 mm Tris ⁄ HCl, pH 8.8, 30% glycerol, 6 m urea, 2% SDS and 1% dithiothreitol, followed by a blocking step in similar buffer containing 2.5% iodoacetamide instead of dithiothreitol for another 15 min. The capillary gels were then transferred to the top of 18 · 18 cm 2 , 1.5-mm-thick, 10% polyacrylamide gels (SDS ⁄ PAGE) and embedded in 0.5% low-melting agarose containing a trace of bromophe- nol blue. SDS ⁄ PAGE was run at 15 °C, initially at 20 mA for 15 min and then at 50 mA per gel until the blue front reached the bottom. For external calibrations, molec- ular mass markers (Sigma, St. Louis, MO, USA) were loaded onto the second dimension. The protein spots were visualized by staining with either Coomassie blue R-250 for preparative gels [11] or silver nitrate for analyti- cal gels [12]. Image capture and analysis Gels were scanned using a UMAX scanner (Amersham Biosciences, Barcelona, Spain) and the images were ana- lyzed with melanie version 5.0 software (GeneBio, Geneva, Switzerland), including spot detection, quantifica- tion, normalization, data analysis and statistics. Compara- tive analysis of protein spots was performed by matching corresponding spots across different gels. Each of the matched protein spots was rechecked manually. Intensity volumes of individual spots were normalized with the total intensity volume of all spots present in each gel before per- forming differential expression analysis. The Kolmogorov– Smirnov test was used to assess the statistical significance of the differences between the normalized intensity volumes of individual spots of the control (Agxt + ⁄ + ) and those of the hyperoxaluric (Agxt ) ⁄ ) ) group. Only differentially expressed proteins were excised and subjected to subsequent identification by MS. MALDI-TOF MS Protein spots were manually excised from Coomassie- stained gels, and tryptic in-gel digestion and desalting steps were performed using 96-well ZipPlates (Millipore, Bedford, MA, USA) according to the manufacturer’s instructions. The resulting peptides were mixed with 1 lLofa-cyano-4- hydroxycinnamic acid (1 mgÆmL )1 ) and spotted on Anchor- chip plates as described by the manufacturer (Bruker Daltonics, Bremen, Germany). Peptide mass fingerprint spectra were measured on an Autoflex MALDI-TOF mass spectrometer (Bruker-Daltonics) in a positive ion reflection mode at an accelerating voltage of 20 kV, and spectra in the 900–3200 Da range were recorded. For one main spec- trum, 30 subspectra with 30 shots per subspectrum were accumulated. A pepmix calibration kit (Bruker-Daltonics) was used for calibration and the standard mass deviation J. R. Herna ´ ndez-Fernaud and E. Salido Proteome changes in primary hyperoxaluria FEBS Journal 277 (2010) 4766–4774 Journal compilation ª 2010 FEBS. No claim to original German government works 4773 was < 10 ppm. The peak lists were created with flex analysis (v 2.4) software. The selected settings were as fol- lows: SNAP peak detection algorithm; signal-to-noise ratio, 10; quality factor threshold, 30; maximal 100 peaks per spot. The peptide mass fingerprints were rechecked manu- ally. Peptide mass fingerprint data were submitted to the MASCOT search engine for protein identification using the Mascot database. The search parameters were set according to the following criteria: Mus musculus for taxonomy; carb- amidomethyl (C) for fixed modifications; oxidation (M) for variable modifications; and ±100 ppm for peptide ion mass tolerance. Western blot Subcellular fractions from six mice were obtained as described above. Protein concentration was measured using the Bradford method, and 50 lg of protein were analyzed by immunoblotting [13] with anti-AGT affinity-purified rab- bit serum, anti-carbonic anhydrase 3 (1:1000 dilution) goat serum or anti-fructose-1,6-bisphosphatase (1:1000) rabbit serum (Santa-Cruz Biotechnologies, Santa Cruz, CA, USA), anti-enolase (1:1000 dilution) rabbit serum (ABCAM, Cambridge, UK) and anti-catalase (1:5000 dilu- tion) mouse IgG1 (Sigma-Aldrich, St Louis, MO, USA). Peroxidase-conjugated anti-rabbit IgG, anti-mouse IgG or anti-goat IgG (Jackson Immunoresearch, West Grove, PA, USA) was used as secondary antibody and the chemilumi- nescence substrate was obtained from Pierce (Rockford, IL, USA). Controls to ensure that equal amounts of protein were loaded per lane involved Coomassie staining of parallel gels, and reprobing the membranes with mouse anti-b-actin (1 : 10 000; Sigma) and rabbit anti-glyoxylate reductase ⁄ hydroxypyruvate reductase (1 : 10 000, raised against recombinant protein) antibodies. Inmunoreactive specific bands were quantified using a UMAX scanner (Amersham Biosciences) and melanie version 5.0 software (GeneBio). The signal was normalized against each group to obtain the protein changes as expression ratios. Acknowledgements We are grateful to Cristina Paz for excellent technical help. The study was supported by grant 2007-62343 (Spanish Ministry of Science). References 1 Behnam JT, Williams EL, Brink S, Rumsby G & Danpure CJ (2006) Reconstruction of human hepatocyte glyoxylate metabolic pathways in stably transformed Chinese-hamster ovary cells. 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