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 ª2010 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
FGH
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 ª2010 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 pIfrom
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 gcentrifugation. 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 ª2010 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, anon-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 ª2010 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, anon-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 ª2010 FEBS. No claim to original German government works