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

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

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-

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,

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
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

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

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

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

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

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,

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

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