Biochem. J. (2013) 456, 373–383 (Printed in Great Britain)373

doi:10.1042/BJ20130825*Department of Matrix Medicine, Faculty of Medicine, Oita University, Yufu, Oita 879-5593, Japan, †Department of Clinical Nephroscience, Niigata University Graduate School of
Medical and Dental Sciences, Niigata 951-8120, Japan, ‡Electron Microscope Core Facility, Niigata University, Niigata 951-8120, Japan, §Laboratory of Animal Models for Human
Diseases, National Institute of Biomedical Innovation, Osaka 567-0085, Japan, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD
20892, U.S.A., and ¶Biochemical Laboratory, GlycoPharma Corporation, Oita 870-0822, Japan

Fabry disease is a lysosomal storage disorder in which neutral
glycosphingolipids, predominantly Gb3 (globotriaosylceramide),accumulate due to deﬁcient α-Gal A (α-galactosidase A)
activity. The GLAko (α-Gal A-knockout) mouse has beenused as a model for Fabry disease, but it does not have any
symptomatic abnormalities. In the present study, we generated
a symptomatic mouse model (G3Stg/GLAko) by cross-breeding
GLAko mice with transgenic mice expressing human Gb3
synthase. G3Stg/GLAko mice had high Gb3 levels in majororgans, and their serum Gb3 level at 5–25 weeks of age was
6–10-fold higher than that in GLAko mice of the same age.
G3Stg/GLAko mice showed progressive renal impairment, with

albuminuria at 3 weeks of age, decreased urine osmolality at
5 weeks, polyuria at 10 weeks and increased blood urea nitrogenat 15 weeks. The urine volume and urinary albumin concentration
were signiﬁcantly reduced in the G3Stg/GLAko mice whenhuman recombinant α-Gal A was administered intravenously.
These data suggest that Gb3 accumulation is a primary pathogenic
factor in the symptomatic phenotype of G3Stg/GLAko mice, andthat this mouse line is suitable for studying the pathogenesis ofFabry disease and for preclinical studies of candidate therapies.

INTRODUCTION

α-Gal A (agalsidase-α or agalsidase-β) [8,9]. However, thetreatment is expensive (approximately $200 000/patient per year[10]) and it has side effects related to the immunogenicity of
the recombinant enzyme [11]. Other therapies proposed for
Fabry disease include bone marrow transplantation [7], genetherapy [12], SRT (substrate reduction therapy) [13,14] andpharmacological chaperone therapy [15,16]. Preclinical studies
have examined the effect of these therapies [7,12,14] on the
Gb3 levels in GLAko mice; however, there is no guaranteethat reducing the Gb3 levels in GLAko mice will translate toa therapeutic effect in human Fabry disease. In a preclinical
SRT study using a mouse model for Sandhoff disease, aneuropathic glycosphingolipidosis caused by a lysosomal βhexosaminidase deﬁciency, the administration of SRT candidates
increased mouse survival and protected against neuronal damage
without reducing glycosphingolipid accumulations in the brain[17]. These ﬁndings indicate that we still do not completely
understand the pathogenesis of glycosphingolipidosis, and thata symptomatic mouse model is urgently needed for effective
preclinical studies of therapies for Fabry disease.
We previously generated TgG3S mice [human G3S (Gb3synthase)-transgenic mice] with elevated Gb3 levels in majororgans [18]. In the present study, we prepared a new mouse line
(G3Stg/GLAko) by cross-breeding TgG3S and GLAko mice toobtain a phenotypic model for Fabry disease. In the present paper
we report the characteristics of these symptomatic G3Stg/GLAko
mice in comparison with asymptomatic GLAko mice.

Fabry disease is an X-linked inherited lysosomal storage
disorder in which glycosphingolipids, predominantly Gb3(globotriaosylceramide), accumulate in visceral tissues and body
ﬂuids due to deﬁcient α-Gal A (α-galactosidase A) enzymatic
activity [1]. Gb3 accumulation results in a progressive disordercharacterized by cardiac disease, chronic renal insufﬁciency
and cerebrovascular stroke [2]. Without medical treatment,
patients eventually suffer from renal failure or cerebrovasculardisease in their 40s or 50s [3,4]. Affected hemizygous males
without detectable α-Gal A activity generally have severe clinicalsymptoms. Heterozygous females with intermediate α-Gal A
activity are usually asymptomatic, but may develop vascular heart
and kidney disease at a later age than males do [5].
The GLAko (α-Gal A-knockout) mouse has been used as amodel for Fabry disease [6]. Although GLAko mice accumulate
Gb3 in their organs, they have a normal lifespan and do not have a
Fabry disease phenotype. Gb3 accumulates in the GLAko mouse
liver and kidneys with age, but the kidney Gb3 concentration only
reaches 25 % of that found in patients with Fabry disease [7]. We
hypothesized that GLAko mice lack phenotypic manifestations
because mice have a lower capacity than humans for synthesizing
Gb3, and that increasing the organ Gb3 synthesis might provide
a phenotypic model mouse for Fabry disease.
Currently, Fabry disease can be treated with ERT (enzyme
replacement therapy) using infusions of recombinant human

Key words: enzyme replacement therapy, Fabry disease,
globotriaosylceramide synthesis, symptomatic mouse model.

Abbreviations used: BUN, blood urea nitrogen; ERT, enzyme replacement therapy; α-Gal A, α-galactosidase A; Gb3, globotriaosylceramide;
GLAko, α-Gal A-knockout; GlcCer, glucosylceramide; G3S, Gb3 synthase; HPTLC, high-performance TLC; HRP, horseradish peroxidase; lyso-Gb3,
globotriaosylsphingosine; NB-DNJ, N -butyldeoxynojirimycin; SRT, substrate reduction therapy; Stx1B, Shiga toxin 1 B; TgG3S, human G3S-transgenic.
1
These authors contributed equally to this work.
2
This author is an employee of and a shareholder in GlycoPharma Corporation.
3
To whom correspondence should be addressed (email ishiis01@oita-u.ac.jp).
c 2013 Biochemical Society
c The Authors Journal compilation 

Biochemical Journal

Atsumi TAGUCHI*, Hiroki MARUYAMA†1 , Masaaki NAMETA‡, Tadashi YAMAMOTO‡, Junichiro MATSUDA§,
Ashok B. KULKARNI, Hidekatsu YOSHIOKA* and Satoshi ISHII*¶1,2,3

www.biochemj.org

A symptomatic Fabry disease mouse model generated by inducing
globotriaosylceramide synthesis

374

A. Taguchi and others

MATERIALS AND METHODSUrine osmolality

Animals

Urine osmolality was measured with an osmometer (Fiske MicroOsmometer Model 210).

Transgenic (TgG3S) mice expressing human G3S, generated
in our previous study [18], were maintained by breeding withwild-type C57BL/6 mice. The G3Stg/GLAko mouse line was
generated by cross-breeding male TgG3S mice and homozygous
female GLAko mice [6]. The G3S transgene was kept in asingle allele in the TgG3S and G3Stg/GLAko mice so that the
G3S expression could be consistently controlled. Studies wereconducted according to the principles and procedures outlined in
the Science Council of Japan’s Guidelines for Proper Conductof Animal Experiments, and were approved by the IACUC(Institutional Animal Care and Use Committee) at Oita University.

SDS/PAGE

Electrophoresis was performed using standard techniques. Eachurine sample was adjusted to an equal creatinine concentration
by adding distilled water. The sample was mixed with an equalvolume of sample buffer, boiled for 3 min and loaded on to
the gel. Protein bands were visualized with Coomassie Brilliant
Blue stain (Bio-Rad Laboratories). Albumin band intensities
were determined using Scion Image software and quantiﬁed bycomparison with authentic BSA (Sigma–Aldrich).

Genotyping

Mouse lines were genotyped by PCR ampliﬁcation of themouse α-Gal A (GLA) and human G3S (A4GALT) genes.
DNA samples were prepared from ear-punch samples digested
with protease K (TaKaRa Bio). The mouse Gla and knockout
alleles were detected by multiplex PCR as described previously[19]. The human G3S transgene was ampliﬁed with thefollowing primer set: 5 -TCAGTGCCACCTATGCTGTC-3 and5 -CATATGTCCTTCCGAGTGAG-3 .

Enzyme replacement study in G3Stg/GLAko mice

Recombinant human α-Gal A (agalsidase-β; Fabrazyme® ) was
purchased from Genzyme. An enzyme replacement study in 5week-old G3Stg/GLAko mice was designed to investigate theeffects of a clinically relevant dose of recombinant α-Gal A(1 mg/kg) injected into the tail vein once every other week for
15 weeks.

Sample collection

To determine the daily urine volume, mice were kept individuallyin metabolic cages (Tecniplast Spa) for 24 h with ad libitum
feeding. Fresh individual urine samples were collected by gently
applying abdominal pressure to the mouse. Blood collected fromthe postcaval vein was allowed to clot for 30 min at room
temperature (25 ◦ C), and serum samples were centrifuged at
3000 g for 10 min to remove any remaining blood cells. Urineand serum samples were stored at − 80 ◦ C.

Determination of Gb3 content and serum lyso-Gb3(globotriaosylsphingosine)

Neutral glycosphingolipids were extracted from each tissue
and serum and the Gb3 content was determined as described
previously [20]. Lyso-Gb3 extracted from serum was assayed as
described previously [21].

Biochemical assay

BUN (blood urea nitrogen) levels were measured using a ureaseindophenol assay kit (Wako Pure Chemicals). Urine creatinine
concentrations were determined by a quantitative colorimetricassay kit using the Jaffe method (Wako Pure Chemicals).
c 2013 Biochemical Society
c The Authors Journal compilation 

Western blot analysis

Western blot analysis was performed with an anti-albumin
antibody (R&D Systems) and an HRP (horseradish peroxidase)conjugated anti-mouse IgG antibody produced in goat (Thermo
Scientiﬁc Pierce), or with an anti-β 2 -microglobulin antibody(Abcam Japan) and an HRP-conjugated anti-rabbit IgG antibody
produced in donkey (Thermo Scientiﬁc Pierce). Urine samples
controlled by creatinine content were applied to a polyacrylamidegel. Following SDS/PAGE (12 % gel), the proteins weretransferred electrophoretically on to a Protran® nitrocellulose
membrane (Schleicher & Schuell) and visualized with
SuperSignal® Chemiluminescent Substrate (Thermo Scientiﬁc
Pierce).

Light microscopy

Kidneys removed from male 25-week-old G3Stg/GLAko and
TgG3S mice were ﬁxed immediately in 10 % Formalin Neutral
Buffer Solution (Wako Pure Chemicals) and embedded in parafﬁn.
Parafﬁn sections (4 μm) were stained with haematoxylin andeosin, and examined by light microscopy.

Gb3 staining with Stx1B (Shiga toxin 1 B) subunit

Frozen tissue sections (10 μm) of 4 % paraformaldehyde-ﬁxed
brain and kidneys were incubated with Stx1B (2.5 μg/ml), whichbinds speciﬁcally to Gb3, for 30 min at room temperature, after
quenching and blocking of sections. Slides were then incubatedwith an anti-Stx1B antibody (14 μg/ml) produced in rabbit [18].
Slides were visualized with the Cell & Tissue Staining kit (R&D
Systems) according to the manufacturer’s protocol. Sections weredeveloped with 3,3 -diaminobenzidine solution for 10 min andcounterstained with haematoxylin.

Electron microscopy

Kidneys, brain and aorta were removed from mice and trimmed
into small blocks. The blocks were ﬁxed overnight with 2.5 %glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at 4 ◦ C, andpost-ﬁxed for 1 h in 1 % osmium tetroxide in 0.1 M phosphatebuffer. Samples were dehydrated in graded ethanol and embedded
in Epok-812 (Ohken Shoji). Ultrathin sections (90 nm) were cut
on an ultra microtome (Ultracut-N, Reichert Nissei), stainedwith uranyl acetate and lead citrate, and micrographed with atransmission electron microscope (Hitachi H-600A).

Symptomatic mouse model for Fabry disease
Table 1375

Gb3 content in organs and serum from 20-week-old miceMice (n > 5 in each group) were killed at 20 weeks of age, and serum and organs were collected immediately. Gb3 content was determined as described in the Materials and methods section. Values
are means +− S.D. ND, not detectable. The statistical signiﬁcance of differences in comparison with GLAko mice was determined by Student’s t test. *P < 0.05; **P < 0.01.
(a) Male miceGb3 content (μg/mg of protein in organs; μg/ml in serum)
Organ/serum

Wild-type

TgG3S

GLAko

G3Stg/GLAko

Heart
KidneySpleen
Liver
Brain
Serum

ND
0.89 +− 0.750.09 +− 0.07ND
ND
2.25 +− 3.000.84 +− 0.141.81 +− 0.870.63 +− 0.18ND
0.21 +− 0.093.30 +− 1.883.20 +− 1.2029.05 +
− 1.9114.47 +
− 0.863.04 +− 0.560.47 +− 0.096.96 +− 1.1733.13 +
− 9.95**57.31 +
− 8.95**111.95 +− 40.33*
44.22 +
− 18.74*
9.02 +− 1.48**75.52 +
− 39.32*(b) Female miceGb3 content (μg/mg of protein in organs; μg/ml in serum)
Organ/serum
Heart
KidneySpleen
Liver
Brain
Serum

Wild-type

TgG3S

GLAko

G3Stg/GLAko

G3Stg/GLAko ( + / − )ND
1.00 +− 0.440.08 +− 0.06ND
ND
3.34 +− 1.842.52 +− 0.37+ 0.452.13 −
0.72 +− 0.10ND
0.57 +− 0.11
5.18 +− 3.754.92 +− 1.02+ 6.2924.48 −
17.54 +
− 0.78
2.57 +− 0.86
1.22 +− 0.149.37 +− 3.1635.19 +
− 14.56**+ 9.53**45.92 −
74.53 +
− 8.20**27.50 +
− 9.68**29.57 +
− 0.98**23.76 +
− 1.06**16.22 +
− 0.7729.15 +
− 4.7511.29 +
− 3.871.08 +− 0.795.24 +− 2.41
5.83 +− 2.68

accumulations seen in the organs of G3Stg/GLAko mice result
from increased Gb3 synthesis and the lack of Gb3 degradation.

Effect of high Gb3 accumulations on the mouse condition andlifespanFigure 1

Representative HPTLC results of Gb3 in various mouse strains

Neutral glycolipids extracted from the same volume of serum or an equal protein content oforgans of 20-week-old mice were applied to HPTLC plates. Gb3 was separated and stained withorcinol–sulfuric acid.

Figure 2 shows the body weight and survival of wild-type,
TgG3S, GLAko and G3Stg/GLAko mice. Both male and female
G3Stg/GLAko mice appeared normal and healthy from birth
until 15 weeks of age, after which their body weight gradually
decreased (Figure 2A). G3Stg/GLAko mice died by 36 weeks ofage, with male and female mice surviving a median 27.6 and26.7 weeks respectively (Figure 2B). No gross abnormality or
early lethality was observed in wild-type, TgG3S or GLAko mice.
The G3Stg/GLAko mice began to develop spontaneous tremors,slow movements (Figure 2C), a rounded back (Figure 2D) andgait disturbances at 20 weeks of age. Both male and female
G3Stg/GLAko mice tucked their hind limbs to their body whensuspended by the tail (Figures 2E and 2G), whereas GLAko andheterozygous G3Stg/GLAko ( + / − ) mice spread their hind limbs(Figures 2F and 2H), as did wild-type and TgG3S mice (results
not shown).

Negative association between residual α-Gal A activity and Gb3
accumulation as well as phenotype
RESULTSHigh Gb3 accumulation in G3Stg/GLAko mouse organs

To determine how overexpressing G3S affects organ Gb3 levels,the neutral glycosphingolipids extracted from major organs ofage-matched mice were analysed by HPTLC (high-performanceTLC) (Figure 1 and Table 1). The Gb3 levels in the serum andmajor organs were markedly greater in G3Stg/GLAko mice thanin GLAko mice. Compared with wild-type mice, the organ Gb3
content was slightly elevated in TgG3S and markedly higher in
G3Stg/GLAko mice. These results indicate that the higher Gb3

We next compared female G3Stg/GLAko mice, which havehomozygous GLA-null alleles ( − / − ), female G3Stg/GLAko( + / − ) mice, which have heterozygous GLA alleles, and female
TgG3S mice, which have normal α-Gal A activity levels. All ofthese strains expressed the G3S transgene. Figure 3 summarizes
the α-Gal A activity and relative Gb3 levels in organs from thesethree mouse lines; we found that the residual α-Gal A activity
negatively correlated with the Gb3 level. The heart and kidney
Gb3 levels in the heterozygous G3Stg/GLAko ( + / − ) mice wereapproximately half those found in the G3Stg/GLAko mice, and theGb3 levels in the spleen, liver and brain of the heterozygous mice,c 2013 Biochemical Society
c The Authors Journal compilation 376

Figure 2

A. Taguchi and others

Lifespan and behaviour of G3Stg/GLAko mice(A) Average body weight of male and female wild-type (䊊), TgG3S (䊉), GLAko (), G3Stg/GLAko (䉱) and heterozygous G3Stg/GLAko ( + / − ) mice (䊏). The statistical signiﬁcance of differencesin comparison with wild-type mice was determined by Student’s t test. *P < 0.05. (B) Survival curve of male wild-type, TgG3S, GLAko and G3Stg/GLAko mice (n = 10, 13, 19 and 24 respectively),and female wild-type, TgG3S, GLAko, G3Stg/GLAko and heterozygous G3Stg/GLAko ( + / − ) mice (n = 16, 14, 19, 24 and 11 respectively) by the Kaplan–Meier method. Occasional cannibalism
and accidental death before 4 weeks of age were not considered. Symbols are the same as in (A). (C) Representative images of 25-week-old GLAko and G3Stg/GLAko mice and (D) a G3Stg/GLAko
mouse. (E–H) Representative images of posture differences when suspended by the tail. (E) A male G3Stg/GLAko mouse, (F) a male GLAko mouse, (G) a female G3Stg/GLAko mouse and (H) aheterozygous G3Stg/GLAko ( + / − ) mouse.

which had relatively high α-Gal A activity, were less than 20 % of
those in the homozygous mice. The heterozygous G3Stg/GLAko( + / − ) mice appeared healthy, were indistinguishable fromfemale TgG3S mice and had a normal lifespan (Figure 2).

Serum lyso-Gb3 was detectable in GLAko, G3Stg/GLAko andheterozygous G3Stg/GLAko ( + / − ) mice, but not in TgG3S orwild-type mice (Table 2).

Gb3 levels in developing miceSerum lyso-Gb3 levels

Lyso-Gb3 has been suggested to be useful as a biomarker for
diagnosing Fabry disease and for monitoring ERT efﬁcacy [22].
c 2013 Biochemical Society
c The Authors Journal compilation 

We next measured age-related changes in Gb3 accumulation in
G3Stg/GLAko and GLAko male mice (Figure 4). Gb3 increased
with age in the serum and organs of both G3Stg/GLAko andSymptomatic mouse model for Fabry disease

Figure 3377

Tissue α-Gal A activity and Gb3 levels in G3S-expressing mouse strains(A) Assays of organ α-Gal A activity in 20-week-old female G3Stg/GLAko, heterozygous G3Stg/GLAko ( + / − ) and TgG3S mice (n = 4). (B) Tissue Gb3 levels relative to the level in female
G3Stg/GLAko mice (n = 4) are shown. Values are means +− S.D. The statistical signiﬁcance of differences in comparison with homozygous G3Stg/GLAko mice was determined by Student’s t test.
*P < 0.05; **P < 0.01.

Table 2

Serum lyso-Gb3 levels in 20-week-old miceSerum lyso-Gb3 levels (n = 4 for each group) were determined as described in the Materials
and methods section. Values are means +− S.D. ND, <10 nM. The statistical signiﬁcance ofdifferences in comparison with GLAko mice was determined by Student’s t test. **P < 0.01.
Serum lyso-Gb3 (nM)
GenderWild-type

TgG3S

GLAko

G3Stg/GLAko

G3Stg/GLAko ( + / − )MaleFemale

ND
ND

ND
ND

328 +
− 25
302 +
− 44

518 +
− 72**
505 +
− 209

–
65 +
−4

GLAko mice, with higher Gb3 levels in the G3Stg/GLAko thanin the GLAko mice throughout the experimental period. The
heart Gb3 levels measured in G3Stg/GLAko mice at 5, 10, 15,
20 and 25 weeks of age were higher than those in age-matched
GLAko mice, by 8.0-, 11.7-, 6.5-, 10.4- and 9.4-fold respectively.
Increased Gb3 levels were also seen in the kidney (1.8-, 1.3-,
1.3-, 1.9- and 2.1-fold), spleen (3.9-, 7.0-, 8.5-, 7.7- and 6.8-fold),liver (34.9-, 17.8-, 19.8-, 14.5- and 16.1-fold) and brain (14.9-,
19.3-, 10.7-, 11.5- and 14.2-fold). The serum Gb3 levels were also
consistently higher in the G3Stg/GLAko than in the age-matched
GLAko mice, with fold increases of 6.4, 7.2, 7.3, 10.1 and 8.6 at
5, 10, 15, 20 and 25 weeks respectively.

Organ weight

GlcCer (glucosylceramide) accumulation causes hepatosplenomegaly in Gaucher disease [23] and its mouse model [24]. Table 3
shows the weight of major organs relative to the body weightin 20-week-old mice. The liver weight in G3Stg/GLAko malemice and spleen weight in G3Stg/GLAko female mice wereslightly greater than those in their wild-type counterparts, butwere not statistically different from those in GLAko mice. The
kidney weight was signiﬁcantly higher (approximately 1.2-fold)in G3Stg/GLAko mice, both male and female, than in the otherthree mouse lines. The heart weight was the same in all four lines.
No weight change was observed in the organs of heterozygous
G3Stg/GLAko ( + / − ) mice compared with those of wild-type
mice.

Figure 4 Developmental changes in Gb3 content in the serum and organsof GLAko and G3Stg/GLAko miceGb3 content in the serum and organs of male GLAko (䊊) and G3Stg/GLAko (䊉) mice at theages indicated (n = 4). Values are means +− S.D. Statistical signiﬁcance was determined byStudent’s t test. *P < 0.05; **P < 0.01.


c 2013 Biochemical Society
c The Authors Journal compilation 378
Table 3

A. Taguchi and others
Organ weight of 20-week-old miceMice (n > 5 for each group) were killed at 20 weeks of age, and organs were immediately collected and weighed. Values are means +− S.D. The statistical signiﬁcance of differences in comparison
with wild-type mice was determined by Student’s t test. *P < 0.05; **P < 0.01.
(a) Male mice
Organ weight in male mice (% of body weight)
Organ

Wild-type

TgG3S

GLAko

G3Stg/GLAko

Heart
KidneySpleenLiver

0.375 +
− 0.028
1.238 +
− 0.0880.252 +
− 0.029
4.286 +
− 0.4110.397 +
− 0.018
1.277 +
− 0.184
0.213 +
− 0.032
4.453 +
− 0.548

0.392 +
− 0.058
1.259 +
− 0.1080.264 +
− 0.0364.900 +
− 0.153

0.390 +
− 0.064
1.461 +
− 0.080**0.278 +
− 0.035
5.225 +
− 0.394*

Organ weight in female mice (% of body weight)
Organ

Wild-type

TgG3S

GLAko

G3Stg/GLAko

G3Stg/GLAko ( + / − )Heart
KidneySpleenLiver

0.449 +
− 0.014
1.192 +
− 0.0390.320 +
− 0.037
4.941 +
− 0.304

0.425 +
− 0.043
1.171 +
− 0.175
0.296 +
− 0.072
5.081 +
− 0.5900.420 +
− 0.045
1.266 +
− 0.097
0.398 +
− 0.092
5.056 +
− 0.549

0.421 +
− 0.040
1.558 +
− 0.450**
0.389 +
− 0.051*5.298 +
− 0.456

0.402 +
− 0.026
1.242 +
− 0.035
0.380 +
− 0.0715.468 +
− 0.355

Table 4

Effect of ERT on Gb3 content and renal function

G3Stg/GLAko male mice treated with ERT (n = 5) were killed at 20 weeks of age, and organs were immediately collected and assayed. Values are means +− S.D. The statistical signiﬁcance ofdifferences in comparison with age-matched G3Stg/GLAko male mice was determined by Student’s t test. *P < 0.05; **P < 0.01. ND, not detectable. Values in parenthesis are the percentage of thecontrol.
(a)Organα-Gal A activity (units/mg of protein)

Gb3 content (μg/mg of protein)Heart
KidneySpleen
Liver
Brain

1.7 +
− 1.4
3.1 +
− 1.3
39.6 +− 12.8**97.5 +− 34.7**ND

18.31 +
− 1.38** (55.3)
23.43 +
− 14.43** (40.9)
2.38 +− 1.56** (2.1)
0.13 +− 0.23** (0.3)
7.19 +− 3.40 (79.7)

α-Gal A activity (units/ml)

Gb3 level (μg/ml)

Lyso-Gb3 level (nM)ND

0.94 +− 1.27** (1.2)

126 +− 61** (24.3)

Age

BUN (mg/dl)

Urine volume (ml)Urinary albumin concentration (mg/mg of creatinine)

Urine osmolality (mOsm/kg)

10-Week-old
15-Week-old
20-Week-old––
50.9 +− 8.1 (79.5)

1.74 +− 0.38** (68.4)
2.24 +− 0.61** (60.7)
2.79 +− 0.91 (83.7)

0.73 +− 0.29* (61.7)
0.51 +− 0.26** (53.6)
0.43 +− 0.20 (66.3)

1479.4 +− 352.5 (112.9)
1310.6 +− 307.6 (119.3)
945.0 +
− 170.6 (100.1)

(b)

Serum
(c)

Renal function

To evaluate how Gb3 accumulation affected renal function, we
measured the BUN, urine volume and osmolality (Figure 5), andalbuminuria (Figure 6). Compared with age-matched wild-type
mice, the BUN levels were not signiﬁcantly altered in the TgG3S
or GLAko mice, but were signiﬁcantly higher in 20-week-old
G3Stg/GLAko mice, both male and female (Figure 5A). BUN
was normal in 5-week-old male G3Stg/GLAko mice, but rose
progressively and differed signiﬁcantly from the BUN levels in
GLAko mice from 15 weeks of age onwards (Figure 5B).
To determine 24-h urine volume and urine osmolality, mice
were housed individually in metabolic cages. In both maleand female 20-week-old G3Stg/GLAko mice, the urine volume(approximately 4 ml/day) was signiﬁcantly higher than that of
c 2013 Biochemical Society
c The Authors Journal compilation other lines (approximately 1 ml/day) (Figure 5C). We were unable
to determine the urine volume of 5-week-old G3Stg/GLAko mice
because they died from stress in the metabolic cages. At 10 weeks
of age, the G3Stg/GLAko mice produced a signiﬁcantly higher
volume of urine than GLAko mice, and they produced theirhighest urine volume at 15 weeks of age (Figure 5D). The average
daily water intake of a male 15-week-old G3Stg/GLAko mouse(9 ml/day) was greater than that of an age-matched GLAko
mouse (6.4 ml/day). The urine osmolality was signiﬁcantly lower
in the G3Stg/GLAko mice than in the other mice (Figure 5E) from
10 weeks of age onwards (Figure 5F). These data indicate that thekidneys of G3Stg/GLAko mice are less able to concentrate urine.
The total urinary creatinine excretion (Figures 5G and 5H) was thesame in the G3Stg/GLAko mice and other mouse lines. Although
the serum creatinine concentrations were too low to determineSymptomatic mouse model for Fabry disease379

analysis conﬁrmed that this protein band was albumin (Figure 6B).
Since β 2 -microglobulin, which is approximately 12-kDa, passes
easily through the glomerular basement membrane and is mostly
reabsorbed through the proximal tubules, the amount of β 2 microglobulin in the urine provides an index for proximal tubule
impairment [26]. Urine from G3Stg/GLAko mice contained
detectable amounts of β 2 -microglobulin (Figure 6C).
Since low urine osmolality and albuminuria were observedin 10-week-old G3Stg/GLAko mice, we assayed these factors in
raw urine samples collected from younger mice. Albuminuria wasalready present in 3-week-old G3Stg/GLAko mice (Figure 6D).
The urinary albumin concentration increased until 9 weeks of age,and decreased slightly thereafter. The highest level of albumin
excreted by G3Stg/GLAko mice was 1.28 mg/mg of creatinine,
which is still lower than the levels reported in other glomerularinjury mouse models ( > 100 mg/mg of creatinine) [27]. Urine
osmolality, which was normal in 4-week-old G3Stg/GLAko micebut decreased gradually thereafter, was signiﬁcantly different
from that of GLAko mice from 5 weeks of age onwards(Figure 6E).
Lens

Lens opacities were observed by slit-lamp examination. Moderate‘whitish’ granular deposits and diffuse posterior cataract wereobserved in 7-week-old G3Stg/GLAko mouse eyes (Figure 7).
Some deposits were found in the left eye but not in the right eyeof a 7-week-old GLAko mouse.
Morphological observation

Although enlarged tubular cells were observed in theG3Stg/GLAko mouse kidneys, there were no structuralabnormalities of the Bowman’s capsule as are often seen inpatients with Fabry disease (Figure 8A). No abnormalities
were observed in the kidneys of the other mice, including
the TgG3S mice (Figure 8B). An obvious difference between
symptomatic G3Stg/GLAko and asymptomatic GLAko mice wasobserved in the kidney and brain Gb3 immunostaining with Stx1B(Figures 8C–8F). Strong Gb3 staining was seen in the medulla ofthe kidney and in the cerebral cortex, hippocampus, thalamus andhypothalamus from a 25-week-old G3Stg/GLAko mouse.
Electron microscopy

Figure 5

BUN and urine volume of G3Stg/GLAko mice

(A and B) BUN levels were determined as described in the Materials and methods section. Daily
urine volume (C and D), urine osmolality (E and F) and urine creatinine excretion (G and H)
were assayed by keeping 20-week-old mice (n > 4) in metabolic cages for 24 h (A, C, E and
G). Developmental changes in male GLAko (䊊) and G3Stg/GLAko (䊉) mice (B, D, F and H).
(A), (C), (E) and (G) Lanes 1 and 5, wild-type mice; lanes 2 and 6, TgG3S mice; lanes 3 and7, GLAko mice; and lanes 4 and 8, G3Stg/GLAko mice. Lanes 1–4, male mice; and lanes 5–8,
female mice. Values are means +− S.D. Statistical signiﬁcance was determined by Student’s ttest. *P < 0.05; **P < 0.01; NS, not signiﬁcant.

Electron microscopy revealed large lipid inclusions with electrondense concentric lamellar structures in the proximal and distal
convoluted tubules and collecting ducts from a 25-week-old
G3Stg/GLAko mouse (Figures 9A–9C). A small number of lipid
inclusions was observed in podocytes, but not in glomelularmesangial cells or endothelial cells (Figure 9D). In brain, lipid
inclusions were observed in neurons (Figure 9E) and cells aroundblood vessels (Figure 9F). We also examined aortic endothelial
cells from this mouse, but no lipid inclusion was detected (results
not shown).
Effect of ERT on G3Stg/GLAko micethe creatinine clearance precisely, the creatinine clearance may
be normal in G3Stg/GLAko mice.
Albuminuria was determined by subjecting mouse urine
samples to SDS/PAGE, since a measurable level of lowmolecular-mass protein is excreted in mouse urine [25]. Highlevels of a 67-kDa protein were observed in the urine of both maleand female G3Stg/GLAko mice (Figure 6A), and Western blot

We next examined whether reducing the Gb3 accumulation by
ERT could prevent the renal impairment in the G3Stg/GLAko
mice. Male G3Stg/GLAko mice were given injections ofrecombinant human α-Gal A (1 mg/kg of body weight) at 5, 7, 9,
11, 13, 15, 17 and 19 weeks of age, and were killed at 20 weeks
of age. One week after the last injection, the recombinant αGal A activity was still high in the spleen and the liver, withc 2013 Biochemical Society
c The Authors Journal compilation 

380

Figure 6

A. Taguchi and others

Albuminuria and urine osmolality in G3Stg/GLAko mice

SDS/PAGE analysis (10 % gel) of urine from 15-week-old mice; proteins were stained with Coomassie Brilliant Blue (A), and Western blot analysis was performed with an anti-albumin monoclonal
antibody (B) and an anti-β 2 -microglobulin monoclonal antibody (C). Lanes 1 and 5, wild-type mice; lanes 2 and 6, TgG3S mice; lanes 3 and 7; GLAko mice; and lanes 4 and 8, G3Stg/GLAko mice.
Lanes 1–4, male mice; and lanes 5–8, female mice. The molecular mass in kDa is indicated. (D and E) Developmental changes in albuminuria (D) and urine osmolality (E) in male G3Stg/GLAko(䊉) and GLAko (䊊) mice were determined from raw urine. Values are means +− S.D. The statistical signiﬁcance of the difference in comparison with age-matched GLAko mice was determined byStudent’s t test. *P < 0.05.

in the G3Stg/GLAko mice. This mouse line may be useful indetermining an effective ERT clinical dosage.

DISCUSSION

Figure 7

Lenticular opacity

Posterior capsular cataract, visible with slit-lamp microscopy, was observed in male 7-week-old
G3Stg/GLAko and GLAko mice. Typical abnormalities are indicated by arrows.

strong clearance of the accumulated Gb3 (Table 4). The clearanceof Gb3 in the heart and kidney was relatively small because of
the low enzyme distribution to these organs, as described by
Ioannou et al. [28]. The serum Gb3 and lyso-Gb3 levels weresigniﬁcantly decreased, and the effect of 1 mg of recombinant αGal A/kg on the serum lyso-Gb3 level in the G3Stg/GLAko mice(24.3 % of that in untreated control) corresponded to the effect of
ERT on plasma lyso-Gb3 levels in male patients (approximately
25 % of that untreated male patients) [29]. Signiﬁcant reductions
were observed in the urine volume and albumin concentration
at 10 and 15 weeks of age, but the urine volume gradually
increased. Treatment with 1 mg of recombinant α-Gal A/kg
did not change the BUN or urine osmolality. However, in apreliminary experiment with a dose of 3 mg of recombinant αGal A/kg, the urine osmolality was much higher in the treatedmice (60 % of normal) than in untreated mice (less than 40 %of normal). This indicates that 1 mg of recombinant α-Gal
A/kg is not sufﬁcient to block the renal impairment completelyc 2013 Biochemical Society
c The Authors Journal compilation 

The results of the present study show that increasing theGb3 synthesis by overexpressing G3S in the organs promotes
phenotypic manifestations in GLAko mice. Both male and female
G3Stg/GLAko mice showed early lethality associated with lossof body weight, neurological abnormalities and progressive renal
impairment. A decreased ability to concentrate urine, leading topolyuria, is thought to be the ﬁrst symptom of Fabry disease[4]. The initial symptom in G3Stg/GLAko mice was albuminuria,
seen at 3 weeks of age, followed by polyuria at 10 weeks of age.
The BUN increased signiﬁcantly in these mice after 15 weeks ofage. In contrast, we saw no abnormalities in GLAko mice during
the experimental period. Although there was little differencein the kidney Gb3 level between 5-week-old G3Stg/GLAko and
GLAko mice, the serum Gb3 level was 6.4-fold higher in theG3Stg/GLAko than in the GLAko mice. The high Gb3 serum levelmay increase the accumulation of Gb3 in tubular cells, causing
renal impairment in the G3Stg/GLAko mice.
It has been suggested that distal tubular dysfunction, which
is most commonly seen in patients with Fabry disease, impairs
the kidney’s ability to concentrate urine and results in polyuria[4]. The G3Stg/GLAko mice accumulated lamellar inclusion
bodies in the proximal and distal convoluted tubules and incollecting ducts. Polyuria and decreased urine osmolality in boththe G3Stg/GLAko mice and patients with Fabry disease may result
from dysfunctional distal convoluted tubules and collecting ducts.
On the other hand, albuminuria might be caused by decreased
protein reabsorption at the proximal tubules, since G3Stg/GLAko
mice excreted β 2 -microglobulin, a sensitive marker for protein

Symptomatic mouse model for Fabry disease

381

Figure 8 Changes in kidney morphology and Gb3 staining of kidney andbrain in G3Stg/GLAko mice
Light microscopy of kidney tissue from 25-week-old male (A) G3Stg/GLAko and (B) TgG3S mice.
Scale bars in (A) and (B) are 50 μm. The stain is haematoxylin & eosin. Immunohistochemical
Gb3 staining with Stx1B in the kidney (C and D) and the brain (E and F) from 25-week-old male
(C and E) G3Stg/GLAko and (D and F) GLAko mice. Scale bars in (C–F) are 300 μm.

reabsorption into the urine through the proximal tubules [26].
G3Stg/GLAko mice between 5 and 20 weeks of age excreted lessalbumin into the urine than do glomerular-injury model mice [27].
The G3Stg/GLAko mice may have normal glomerular ﬁltration
throughout their lifespan, since the level of creatinine excreted
into the urine was constant over time and there was no Bowman’s
capsule abnormality. Patients with Fabry disease often have mildalbuminuria and a decreased ability to concentrate urine, evenwhen glomerular ﬁltration rates are normal [30]. These ﬁndings
indicate that the renal impairment seen in G3Stg/GLAko mice
may correspond to the initial steps of renal involvement in patientswith Fabry disease. Mild albuminuria at 3 weeks, low urine
osmolality at 5 weeks, polyuria at 10 weeks and an increase in
BUN at 15 weeks of age should be considered as disease markers
for progressive renal impairment in this mouse line.
The plasma lyso-Gb3 levels are markedly abnormal in Fabry
patients, especially in females, making it a more sensitive
biomarker than plasma Gb3 [31]. In our mouse model, the serumlyso-Gb3 level differed signiﬁcantly between the heterozygous
G3Stg/GLAko ( + / − ) and female TgG3S mice (65 +− 4 nM

Figure 9 Electron microscopy of the kidney and brain in a 25-week-old
G3Stg/GLAko male mouse(A) Proximal tubule, (B) distal tubule, (C) collecting duct, (D) glomerulus (P, podocyte; E,
endothelial cell; M, mesangial cell), (E) neuronal cell and (F) blood vessel in brain. Scale bars
are 5 μm. Typical inclusion bodies are indicated by arrows.

and <10 nM respectively) (Table 2), although their serum
Gb3 level was in the same range (5.83 +− 2.68 μg/mg ofprotein and 5.18 +
3.75
μg/mg
of
proteinrespectively)(Table 1).
−
The differences in serum Gb3 levels between symptomatic
G3Stg/GLAko mice and asymptomatic GLAko mice (10.9-foldand 2.5-fold in males and females respectively) were more
pronounced than the differences in serum lyso-Gb3 (1.6-foldand 1.7-fold in males and females respectively). These data
indicate that although serum lyso-Gb3 is a sensitive marker for
deﬁcient α-Gal A activity in mice, it may not correlate wellwith the symptomatic condition. Urinary albumin excretion andurine osmolality are good symptomatic biomarkers for the renal
condition in patients with Fabry disease.
Although hypertrophic cardiomyopathy is a prominent
characteristic of Fabry disease, the heart weight was not increasedin the G3Stg/GLAko mice. Diastolic and systolic dysfunctions
are characteristics of Fabry cardiomyopathy and can usually
be detected before the onset of left ventricular hypertrophy[32]. In our preliminary study, G3Stg/GLAko mice (30 weeksc 2013 Biochemical Society
c The Authors Journal compilation 

382

A. Taguchi and othersof age) showed reductions in both diastolic and systolic functions
compared with wild-type mice by cardiac catheterization. Cardiac
failure, renal failure and stroke are the major causes ofmortality in Fabry disease [33]. The cause of premature deathin G3Stg/GLAko mice is still unknown; at present, we do nothave any evidence that their death was associated with heart orkidney defects.
The G3Stg/GLAko mice had neurological abnormalities.
Similar phenotypic manifestations have been reported in mousemodels for other neuronal disease models, such as GM1gangliosidosis [34], saposin-B knockout [35] and Huntington’sdisease [36]. It is very likely that Gb3 accumulation causes
neuronal damage that may affect the mortality of theG3Stg/GLAko mice since lamellar inclusions were observed inneuronal cells in this mouse.
All of the symptomatic manifestations in G3Stg/GLAko micecorrelated with a loss of α-Gal A activity. We did not observe any
symptoms in heterozygous G3Stg/GLAko ( + / − ) mice, whichaccumulated intermediate amounts of Gb3 in their organs and had
approximately half of the α-Gal A activity seen in normal mice.
These data indicate that low levels of α-Gal A enzyme activity
reduce the symptom severity in G3Stg/GLAko mice. We showedthat recombinant α-Gal A treatment at a dosage of 1 mg/kg
starting at 5 weeks of age could decrease the urine volume andalbumin excretion at 10 and 15 weeks of age. It has been suggested
that therapeutic intervention should begin early to protect renal
function, since ERT has not been effective in patients with latestage renal disease [37]. However, the best time to start ERT
should be determined based on symptoms, since a strict genotype–phenotype relationship is difﬁcult to deﬁne in Fabry disease [38].
G3Stg/GLAko mice should prove useful in experimental ERT
studies to determine, for example, the minimum dosage andoptimal timing of treatments.
In our present study, we compared G3Stg/GLAko mice and
GLAko mice. The mice were often siblings, since G3Stg/GLAko
mice carrying the G3S transgene in a single allele were matedwith GLAko mice, and the offspring differed only in G3S
expression. Thus our mouse data clearly showed that phenotypic
manifestations were related to the level of Gb3 produced. Gb3
production can be modiﬁed by inﬂammatory conditions, since
inﬂammatory mediators are known to enhance the Gb3 [39] andG3S expression [40]. G3S expression appears be a critical factor
for Gb3 production; this may help to explain the complicatedrelationship of genotypes to phenotypes in Fabry disease.
One option for treating lysosomal storage diseases is SRT [41].
NB-DNJ (N-butyldeoxynojirimycin) is the only SRT approved for
Gaucher’s disease type 1 [42]. NB-DNJ potently inhibits GlcCer
synthase, which is the ﬁrst step in glycosphingolipid synthesis.
Our present data indicate that Gb3 synthase may be another
SRT target in Fabry disease. Inhibiting the Gb3 production in
G3Stg/GLAko mice may diminish or slow the onset of phenotypic
manifestations, since GLAko mice with low Gb3 expression
are asymptomatic. Thus the G3Stg/GLAko mouse will be auseful experimental model for preclinical studies of SRT drugcandidates.
Although GLAko mice lack a spontaneous vascular phenotype,they have been used as experimental models of thrombosis [43]and atherosclerosis [44]. In our preliminary determination, Gb3
content in aorta from G3Stg/GLAko mice was 4-fold higher
than that from GLAko mice. G3Stg/GLAko mice will also be
useful for studying the pathogenesis of vascular manifestations,because abnormalities of the aortic endothelial cells are observed
depending on the Gb3 accumulation [45].
In conclusion, we have generated a mouse model that mimics
Fabry disease with renal impairment, and this progressive renalc 2013 Biochemical Society
c The Authors Journal compilation 

impairment was achieved by increasing Gb3 accumulation ina GLA-null background. This is the ﬁrst clear evidence thatsigniﬁcant Gb3 accumulation is the primary cause of Fabry
disease. Our G3Stg/GLAko mouse model will be useful forstudying the pathogenesis of Fabry disease and for preclinicalstudies of drug candidates.
AUTHOR CONTRIBUTION
Atsumi Taguchi, Hiroki Maruyama, Hidekatsu Yoshioka and Satoshi Ishii conceived anddesigned experiments, and wrote the paper. Atsumi Taguchi and Satoshi Ishii performed
all of the experiments, except for Figure 9, which was undertaken by Hiroki Maruyama,
Masaaki Nameta and Tadashi Yamamoto. Junichiro Matsuda and Ashok Kulkarni provided
transgenic mouse lines and insightful advice for animal experiments. All of the authors
were involved in the critical review of the paper prior to submission.

ACKNOWLEDGEMENTS
We thank Ryoji Hamanaka, Noritaka Matsuo, Sawako Adachi, Kenichi Kimoto and theanimal centre staff (Oita University, Oita, Japan) for helpful advice and support for animal
experiments.

FUNDING
This work was supported by a Grant-in-Aid for Scientiﬁc Research (B) from the Ministry of
Education, Science and Culture of Japan [grant number KM23390223 (to H.M. and S.I.)].

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Received 20 June 2013/27 September 2013; accepted 7 October 2013
Published as BJ Immediate Publication 7 October 2013, doi:10.1042/BJ20130825

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c 2013 Biochemical Society
c The Authors Journal compilation 