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Establishment of a Mouse IgA Nephropathy Model With the MBP-20-Peptide Fusion Protein.

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THE ANATOMICAL RECORD 293:1729–1737 (2010)
Establishment of a Mouse IgA
Nephropathy Model With the
MBP-20-Peptide Fusion Protein
LEI ZHANG,1 FEI YE,1 YAN HE,1 DAN KONG,2 CHANGSONG HAN,1 ZHIJIE ZHAO,1
JIANG ZHU,3 HONGXUE MENG,1 XINGHAN LIU,4 AND XIAOMING JIN1*
1
Department of Pathology, Harbin Medical University, Hei Longjiang Province,
People’s Republic of China
2
Molecular and Cellular Pathology, Graduate School of Medical Science,
Kanazawa University, Kanazawa, Japan
3
Department of Orthopedics, First Hospital of Harbin Medical University,
Hei Longjiang Province, People’s Republic of China
4
Department of Biochemistry, Harbin Medical University, Hei Longjiang Province,
People’s Republic of China
ABSTRACT
Here, we aimed to determine whether immunoglobulin-A nephropathy
(IgAN) could be induced in Balb/c mice by immunizing them with a fusion protein (MBP-20 peptide) comprising the maltose-binding protein (MBP) and a 20amino-acid peptide derived from Staphylococcus aureus. A recombinant plasmid
encoding the fusion protein was constructed and expressed in bacterial cells.
The synthetic 20-peptide was used to prepare the monoclonal antibody. Balb/c
mice were immunized with the MBP-20-peptide fusion protein over a 21-week
course before renal histology was examined at the light and electron microscopic
levels. Direct immunofluorescence staining with the anti-20-peptide monoclonal
antibody was also performed using renal biopsy tissue from human IgAN
patients as a comparison. IgA and IgG specific for the 20-peptide in human and
mice serum were detected. The IgAN experimental mice developed a clinical and
pathological profile that closely resembled that of human IgAN patients, including the induction of hematuria and numerous histopathological features. Levels
of IgA and IgG specific for the 20-peptide were significantly increased in serum
from the IgAN experimental mice and IgAN patients compared with control
mice and non-IgAN patients. In IgAN model mice, the anti-20-peptide antibody
labeled glomeruli, while the antibody strongly labeled glomeruli and weakly
labeled tubular epithelial cells in renal tissue from human IgAN patients. In conclusion, immunization with an MBP-20-peptide fusion protein is able to induce
clinical and pathological features closely resembling IgAN in Balb/c mice, indicating a potentially useful role for the model in the study of IgAN and related
C 2010 Wiley-Liss, Inc.
diseases. Anat Rec, 293:1729–1737, 2010. V
Key words: IgA nephropathy; Staphylococcus aureus; MBP-20peptide fusion protein; 20-peptide; IgA nephropathy
model
Grant sponsor: Heilongjiang Postdoctoral Grant; Grant number:
LRB-07-329; Grant sponsor: Harbin Special Fund for Technological
Innovation; Grant numbers: 2006RFXXS035, 2007RFLXS017; Grant
sponsor: The Innovation Foundation in Harbin Medical University.
Lei Zhang and Fei Ye are contributed equally to this work.
*Correspondence to: Xiaoming Jin, Department of Pathology,
Harbin Medical University, Baojian Road 157, Nangang DisC 2010 WILEY-LISS, INC.
V
trict, Harbin, China. Fax: 86-451-86669472.
E-mail: jinxm55@yahoo.com.cn
Received 10 November 2008; Accepted 15 May 2010
DOI 10.1002/ar.21225
Published online 20 August 2010 in Wiley Online Library
(wileyonlinelibrary.com).
1730
ZHANG ET AL.
Immunoglobulin-A nephropathy (IgAN) is the most
common primary and chronic glomerulonephritis worldwide (Sharmin et al., 2004). IgAN is typically diagnosed
in young adults and is more common in males. Approximately 40% of patients experience recurrent episodes of
macroscopic hematuria, frequently preceded by infection
1 or 2 days earlier (Van der Boog, 2005). Characteristic
histopathological features of the disease include mesangial deposition of IgA and other immunoglobulin isotypes, such as IgG and IgM, in addition to complement
components; leading to mesangial proliferation and ultimately glomerular fibrosis (Widstam-Attorps et al.,
1992). As many as 30% of patients progress to end-stage
renal failure (Montinaro et al., 1999).
Some research groups have reported a relationship
between infection with methicillin-resistant Staphylococcus aureus (MSRA) and IgAN. For example, Koyama
et al. (2004) observed a correlation between the development of IgAN and a history of MSRA infection. These
and other MSRA-related glomerulonephritis have been
found to be associated with the glomerular deposition of
immune complexes containing IgA, C3, IgG, and sometimes IgM (Fridkin et al., 2005; Koyama et al., 1995;
Long and Cook, 2006; Satoskar et al., 2006; Shimizu
et al., 2005, 2007). Sharmin et al. (2004) demonstrated
that the IgAN pathological immune response was centered on Staphylococcus superantigens and found that S.
aureus cell membrane antigen, a 30–35 kDa protein,
was able to induce IgAN in Balb/c mice when administered subcutaneously. Otherwise, a 20-amino-acid peptide (NVGGDNVDIHSIVPVGQDPH) on a 30–35 kDa
protein was found to be the antigenic determinant responsible for the immune manifestations of the disease
(Koyama et al., 2004). Therefore, we speculated that this
20-amino-acid-peptide has a potential role in the pathogenesis of IgAN.
Several research groups have attempted to develop a
mouse model for IgAN based on an induced immune
response to BSA and Staphylococcus enterotoxin B (Liu
et al., 1989), outer membrane protein of Escherichia coli
(E. coli; Endo et al., 1993; Han et al., 1998) and dextran
G200 (Gesualdo et al., 1990; Isaacs et al., 1981). Our
group has attempted many IgAN models using methods
from the above references. Unfortunately, no study to
date has produced the stable and accurate model that
will be required for studying IgAN experimentally.
In this study, we established an IgAN animal model
with the 20-peptide as an antigen on the basis of S. aureus
antigens inducing IgA-type glomerulonephritis in Balb/c
mice (Sharmin et al., 2004). We investigated whether
using the 20-peptide as an antigen determinant of S. aureus can induce IgAN-like changes in Balb/c mice.
MATERIALS AND METHODS
Materials
A synthetic DNA fragment encoding the 20-aminoacid-peptide cell membrane antigen of S. aureus, the
pMAL-c2G/irrelevant 20 peptide plasmid and a synthetic
20-peptide was supplied by Shanghai Sangon Biological
Engineering Technology & Services. The pMAL-c2G
plasmid, which was used to generate the maltose-binding protein (MBP) fusion protein, was obtained from
New England Biolabs, Beverly, MA, as were the amylose
affinity columns, restriction endonucleases, T4 DNA
ligase, and T4 polynucleotide kinase. Taq DNA polymerase and DNA markers were purchased from TaKaRa
Bio. HiTrap Protein A HP was from Amersham Biosciences, Piscataway, NJ. Balb/c mice were obtained from the
Second Affiliated Hospital of Harbin Medical University.
Frozen renal biopsies from 250 patients who had
received a renal biopsy at the Pathology Department of
Harbin Medical University (Harbin, China) between
January 2009 and March 2010 were randomly selected.
The diagnosis of nephropathy was confirmed by histopathology. Sections from all biopsy specimens were also
stained routinely for IgA, IgG, IgM, and complement
component C3. Three investigators judged the fluorescence intensity of the staining independently; intensity
was graded semiquantitatively on a scale of 0 (no staining) to 4. Frozen renal IgAN biopsies were obtained from
50 patients (22 men and 28 women; average age 29.08 7.33 years). Two hundred biopsies from proven nonIgAN glomerulonephritis patients were included as controls, which included 50 cases of mesangial proliferative
glomerulonephritis (MsPGN), 50 cases of membranoproliferative glomerulonephritis (MPGN), 50 cases of membranous nephropathy (MGN), and 50 cases of focal
segmental glomerulosclerosis (FSGN). The protocol of
the study was approved by the ethics committee in Harbin Medical University, and informed consent was
obtained for sampling renal biopsy tissues.
Cloning of the pMAL-c2G/20 Peptide Plasmid
A synthetic DNA fragment encoding the 20 peptide
amino acid sequence (NVG GDNVDIHSIVPVGQDPH)
was synthesized (Koyama et al., 2004). It was ligated
between the Hind III and SnaBI sites of the pMAL-c2G
plasmid at a ratio of 3:1 using 1 lL T4 DNA ligase at
16 C overnight. The resulting plasmid was named
pMAL-c2G/20 peptide. The entire length of the recombinant plasmid was sequenced by the Shanghai Sangon
Biological Engineering Technology & Services, which
confirmed that the insert lay at the correct site and in
the correct orientation. The plasmid was extracted with
R Plus SV Minipreps DNA Purification Systhe WizardV
tem (Promega, Madison, WI) according to the manufacturer’s instructions. In addition, there is a 12aa
fragment between the Hind III and SnaBI sites of the
pMAL-c2G plasmid and we inserted another 8aa fragment on the Hind III site to form the pMAL-c2G/irrelevant 20 peptide plasmid. The pMAL-c2G/20 peptide and
pMAL-c2G/irrelevant 20 peptide plasmids were used to
transform E. coli BL21 cells, which were cultured in LB
medium with antiaminobenzyl penicillin overnight at
37 C. The pMAL-c2G/20 peptide and pMAL-c2G/irrelevant 20 peptide plasmids respectively express MBP-20peptide fusion protein and MBP-irrelevant-20 peptide
fusion protein. The sequence of irrelevant-20-peptide is
YVEFGSSRVDLQSFPRKASE.
Expression and Purification of Fusion Proteins
Induction of the Ptac promoter was accomplished by
incubating bacteria carrying the pMAL-c2G/20 peptide
plasmid or pMAL-c2G/irrelevant 20 peptide plasmid in
0.5 mM isopropylthiogalactoside (IPTG, Sigma-Aldrich,
St. Louis, MO) for 6 hr at 37 C. Following expression of
the plasmid in bacteria, the fusion protein was purified on
ESTABLISHMENT OF A MOUSE IgAN MODEL
1731
Fig. 1. Expression of the MBP-20-peptide fusion protein (A) SDSpolyacrylamide gel electrophoresis of amylose column fractions from
the purification of the MBP-20-peptide fusion protein using maltose.
Lane 1: purified protein; lane 2: hybrid protein; lane 3: precipitate after
ultrasound and centrifugation; lane 4: supernatant after ultrasound and
centrifugation; lane 5: induced bacterium liquid; lane 6: uninduced
bacterial liquid. (B) Western blot analysis of MBP-20-peptide fusion
protein and mouse monoclonal anti-20-peptide. Top two lanes: MBP20-peptide fusion protein; bottom two lanes: isolated 20-peptide. In
each case, both denatured (D) and nondenatured (ND) protein samples were loaded. The antibody detected a fragment of the correct
size in each case.
an affinity column containing maltose. In this expression
system, the protein or peptide of interest is normally
intended to be cleaved from the MBP moiety using a protease site engineered into the sequence of the vector. However, we predicted that the 20-peptide alone would not be
of sufficient size for an optimal immune response, so we
therefore recovered the MBP-20-peptide-fusion protein
intact by elution with free maltose (Fig. 1A).
We confirmed the affinity of the monoclonal antibody
for the 20-peptide by ELISA (data not shown). The sensitivity and specificity of the antibody for the 20-peptide
was confirmed by Western blotting (Fig. 1B).
Preparation of Mouse Monoclonal
Anti-20-Peptide
Keyhole limpet hemocyanin (KLH, Sigma-Aldrich) is a
commonly used carrier for peptide coupling in antibody
production. The monoclonal antibody was produced by the
following method: first, an injection was made of KLH
coupled synthetic 20-peptide (0.5 mg) and 250 lL deionized water, emulsified in 250 lL of complete Freund’s adjuvant. Balb/c mice were immunized by injection at two
sites and the surplus was administrated intraperitoneally.
Second, Mice were immunized after 3 weeks with the
same dose of KLH coupled 20-peptide which was emulsified in incomplete Freund’s adjuvant. Balb/c mice were
administered by intraperitoneal immunization, with
increasing doses every 2 weeks. Third, from the third
increased dose immunization, tail blood was collected to
measure the antibody titer (valence) every 7 days. When
valence was accorded with standards, nonemulsified KLH
coupled 20 peptide was used for the increasing dose
immunizations by intraperitoneal administration. Spleen
cells were fused after 3 days with traditional methods to
prepare monoclonal antibodies against KLH coupled 20peptide. Monoclonal antibodies were identified using indirect enzyme-linked immunosorbent assay (ELISA) methods, 1 lg mL1 BSA coupled 20-peptide coated ELISA
flask to select monoclonal hybridomas. Balb/c mice were
administered by intraperitoneal injection of the hybridoma and monoclonal antibody was purified with
HiTrapTM Protein A HP Columns (Amersham Biosciences) for use in the following experiments.
Western Blot Analysis
The MBP-20-peptide fusion protein and 20-peptide
concentration was quantitated using the Bio-Rad protein
assay (Bio-Rad Laboratories, Hercules, CA). Denatured
MBP fusion protein was resolved on 12% gradient SDSpolyacrylamide gels and nondenatured MBP fusion protein on 12% gradient polyacrylamide gels and blotted
onto nitrocellulose membrane for Western blot analysis.
Denatured 20 peptide resolved on 16% gradient SDSpolyacrylamide gels and nondenatured 20 peptide on
16% polyacrylamide gels for Western Blot analysis. The
blot was probed with suitable monoclonal anti-20-peptide
and goat antimouse antibodies and was developed by the
ECL chemiluminescent method (Amersham Biosciences)
according to the manufacturer’s instructions.
Immunization Mice
One hundred and seven four-week old male Balb/c
mice (weighing 20-–22 g) were acclimatized to standard
laboratory conditions for 7 days prior to experimentation. A total of 30 mice received no treatment. Mice in
the remaining groups were injected subcutaneously every 14 days. Of these, 30 mice received column elution
buffer, 30 received 3 mg/kg of the MBP-irrelevant-20peptide fusion protein (derived from the expression of
pMAL-c2G/irrelevant 20 peptide plasmid), and 80 mice
received 3 mg/kg of the MBP-20-peptide fusion protein
(IgAN experimental group). Injections were in incomplete Freund’s adjuvant (Sigma-Aldrich) in all groups.
Injections continued for 21 weeks, during which time
urine was collected weekly and analyzed for the presence of protein and red blood cells. Mice in each group
were sacrificed at the end of 21 weeks and sections of
1732
ZHANG ET AL.
the major organs were processed for light and electron
microscopy. All morphological findings from experimental animals were observed independently by three pathology researchers (LZ, FY, and XM, J). Ten
microscopic fields of two different areas (total 20 fields
per animal at 400 magnification) were randomly chosen. The microscopic scores were set based on the sizes
of the lesion involved. In our study, the IgAN animal
model only showed proliferation in the mesangial region,
thus we used the extent of mesangial proliferation as
the evaluation standard of tissue injury. Normal histology with no mesangioproliferation was 0; mild mesangioproliferation, 1; moderate mesangioproliferation, 2;
strong mesangioproliferation, 3. The severity of each
variable was also graded as from 0 to 3. The overall histological injury scores were calculated by a summation
of the scores relating to size and other variables.
Urinalysis
Blood and urine specimens were collected weekly and
at the time of autopsy. Urinalysis was undertaken before
freezing at 80 C. Urine erythrocytes were detected
using a dipstick system (MULTISTIX (SBA)-610 multifunctional half-automatic biochemical analysis apparatus; Ji lin, China). Urine erythrocytes were also
observed under light microscopy to distinguish between
erythrocytes and hemoglobin. Therefore, our results
demonstrated the level of erythrocytes in urine of the
IgAN animal model, but not hemoglobin. Urine protein
and creatinine were measured respectively using the
Bradford protein assay and the Jaffe Creatinine Assay.
Enzyme-Linked Immunosorbent Assay (ELISA)
The concentrations of IgG and IgA in mice and human
sera were assayed by ELISA. Each well of a polystyrene
microtiter plate (Corning, NY) was coated with 20-peptide in carbonate buffer (0.05 M, pH 9.6) overnight at
4 C. After washing with PBS containing 0.05% Tween
20 (PBS-T), the plates were incubated with 1% fetal bovine serum for 60 min to block nonspecific reactivity of
the sera. Plates were then incubated with mouse serum
samples from control and IgAN experimental mice and
serum from IgAN and non-IgAN patients at room temperature for 2 hr, and then washed with PBS-T. Peroxidase-conjugated goat antimouse and antihuman IgG and
IgA were added to the plates and incubated at RT for
1 hr. After washing with PBS-T, TMB peroxidase substrate was added, and the reaction stopped with 2 N
H2SO4. Absorbance was measured with BIO-RAD 550
microplate reader at a wavelength of 450 nm.
Immunofluorescence Examination
Frozen slices from renal tissue of experimental mice
were fixed in acetone for 1 min. After fixation, nonspecific protein binding sites were blocked with 5% normal
goat serum in PBS (pH 7.4). IgA, IgG, IgM, and C3 in
mouse renal tissues were detected with Fluorescein-labeled goat antimouse IgA, IgG, IgM (all Invitrogen, CA),
and FITC antimouse complement component C3 (Cedarlane, Canada). A total of 20 glomeruli per mouse at
400 magnification were randomly chosen and images
were acquired with a fluorescence microscope (Nikon
E800) using a digital camera (1200F; Nikon) and software (ACT-1; Nikon).
Frozen slices from human renal biopsy specimens (50
cases of IgAN and 200 cases non-IgAN) and renal tissue
of experimental mice after fixation were blocked, and
the slices were then incubated with the mouse monoclonal anti-20-peptide (1:100 dilution for 12 hr at 4 C),
washed three times in PBS and incubated with FITC-labeled antimouse IgG antibody (Vector Laboratories, CA).
All results were observed using a fluorescence microscope (Nikon E800).
Statistical Analysis
Data are expressed as the mean SD. Serological statistical analysis of differences between the IgAN experimental group and controls were calculated using a oneway ANOVA. Immunofluorescence statistical analysis
was calculated using Kruskal-Wallis test for IgM and C3
and t-tests for IgA and IgG. Different statistical analyses
were used to evaluate the immunofluorescence differences between the IgAN experimental group and controls
because the mean square of the IgA and IgG immunofluorescence value was irregular. Histological injury statistical analysis was performed using a t-test. Statistical
analysis of the differences in 20-peptide between IgAN
patients and non-IgAN patients were performed using
v2 analysis. P < 0.05 was considered statistically
significant.
RESULTS
Urinalysis
Figure 2 shows that mice in the IgAN experimental
group began to develop hematuria during the 11th week.
It rose sharply on the 15th week before reaching a plateau
on the 20th week (the IgAN experimental group; n ¼ 80,
control groups; n ¼ 90) (P < 0.05). The urine P/C ratio
began to remarkably increase after the 15th week, and
then climbed again at 20 weeks to reach a maximum of
approximately 4.2 0.46 (the IgAN experimental group;
n ¼ 80, control groups; n ¼ 90) (P < 0.05). Neither hematuria nor proteinuria was seen in either of the control
groups.
Light and Electron Microscopy
Light microscopy revealed a mild and moderate
increase in the amount of mesangial matrix as well as
proliferation of mesangial cells in the IgAN experimental
mice while showing no other pathological changes (Fig.
3B). In contrast, in the other three control groups, mesangial matrix expansion and mesangial cell proliferation
were not observed (Fig. 3A) (P < 0.05). Electron microscopy showed numerous large, electron-dense deposits in
the mesangium and subendothelium in the immunized
group (Fig. 3D,E), whereas there were no electron-dense
deposits in the other three control groups (Fig. 3C).
Immunofluorescence Findings
Immunofluorescence microscopy, using anti-IgA, -IgG,
-IgM, and -C3 antibodies showed deposits in the glomeruli in the IgAN experimental group, with particularly
intense deposition of IgA and IgG only in the mesangium, there is no specific immunofluorescence in the
ESTABLISHMENT OF A MOUSE IgAN MODEL
1733
Fig. 2. Measurement of hematuria and proteinuria in mice chronically administered with MBP-20-peptide fusion protein (A) Hematuria. A small degree of hematuria was seen in the IgAN experimental
group until the 15th week, when it increased sharply to reach a
maximum at 17 weeks (P < 0.05, IgAN experimental group, n ¼
80; the control groups, n ¼ 90). (B) Proteinuria. The urine P/C ratio
began to remarkably increase after the 15th week and then climbed
again at the 20th weeks to reach a maximum of approximately 4.2
0.46 (P < 0.05, IgAN experimental group, n ¼ 80; the control
groups, n ¼ 90).
Fig. 3. Histology and electron microscopy of glomeruli in the experimental mice. (A, B) Light micrographs of sections from a control mouse
treated with the MBP-irrelevant-20-peptide fusion protein and the immunized mouse treated with MBP-20-peptide fusion protein stained with
H&E (glomeruli indicated by arrows) (magnification: 400). (A) MBP-irrelevant-20-peptide fusion protein did not induce any pathological change.
(B) The MBP-20-peptide fusion protein induced mild mesangial cell proliferation and mesangial matrix expansion in glomeruli. (C, D, E) Electron
micrograph of glomeruli from a control mouse treated with the MBP-irrelevant-20-peptide fusion protein and the immunized mouse treated with
MBP-20-peptide fusion protein (electron dense deposits indicated by
arrows; 8000 magnification:). (C) In the MBP-irrelevant-20-peptide
fusion protein control group, there was no pathological change. (D) An
IgAN model mouse showing numerous large, electron-dense deposits in
the mesangium. (E) An IgAN model mouse showing numerous large,
electron-dense deposits in the subendothelium.
capillary walls, blood vessels or interstitium (P < 0.001)
(the IgAN experimental group; n ¼ 80, control groups; n
¼ 90). In contrast, the glomeruli in the control group
where mice were immunized with the MBP-irrelevant20-peptide fusion protein showed slight IgG and IgM
deposits (Fig. 4). The other two control groups of mice
showed an absence of immunofluorescence.
We next examined renal tissue from both experimental mice and renal biopsies of IgAN and non-IgAN
patients using the mouse monoclonal anti-20-peptide
antibody in this study. The 20-peptide antigen was primarily associated with glomeruli in the IgAN experimental group but was not expressed in the other three
control groups (Fig. 5A,B). In patient tissue biopsies,
1734
ZHANG ET AL.
Fig. 4. Deposition of immunoglobulins and complement in glomeruli.
A. Immunofluorescence microscopy, using anti-IgA, -IgG, -IgM and -C3
antibodies showed glomerular deposits in the IgAN experimental group.
Of these, glomerular deposition of IgA and IgG in the mesangium was
particularly intense. In contrast, the glomeruli in the MBP-irrelevant-20peptide fusion protein control group showed slight IgG and IgM deposits.
The other two control groups of mice were negative (immunized; n ¼ 80,
control; n ¼ 30 in each group) (magnification: 200). B. The immunofluorescence signal of IgA, IgG, IgM and C3 in the IgAN experimental group
is stronger than in the control groups (IgAN experimental group, n ¼
80;the control groups, n ¼ 90) (P < 0.001). Images were captured using a
Nikon E800, and photomicrographs were quantified using ACT-1.
mild and moderate 20-peptide antigen deposition was
detected in 82% of IgAN, 16% of MsPGN, 14% of MPGN,
18% of MGN and 20% of FSGN patients. The expression
of the 20-peptide in IgAN is significantly higher than
that in non-IgAN (P < 0.001) (n ¼ 50 in each group).
The 20-peptide antigen was mainly deposited in glomeruli, with very little 20-peptide detected in renal tubular
epithelial cells in biopsy tissue from IgAN patients (Fig.
5C,D).
ferative glomerulonephritis with a severe glomerular
IgA deposition in later life was described (Launay
et al., 2000). Researchers then selected a strain from
the ddY mice with a high incidence and an early onset
of glomerular IgA deposition to develop a model of
IgAN (Miyawaki et al., 1997). Now, some researchers
focus on the relationship between respiratory tract
infections and IgAN, so the outer membrane antigens
of Haemophilus parainfluenzae (OMHP; Yamamoto
et al., 2002) and S. aureus (Sharmin et al., 2004) antigens were used to establish an experimental model of
IgAN in C3H/HeN mice and Balb/c mice (Sharmin
et al., 2004) respectively. On the basis of the IgAN
model induced by S. aureus, the 20-peptide of S. aureus
was used to establish IgAN model in Balb/c mice for
the first time in our study.
Our study used the MBP-irrelevant 20-peptide fusion
protein as a control for the MBP-20-peptide fusion protein to immunize Balb/c mice. MBP-20-peptide fusion
protein induced glomerular deposition of IgA, IgG, IgM,
and C3, whereas MBP-irrelevant-20-peptide fusion protein only induced glomerular deposition of IgG and IgM.
Immunofluorescence showed IgA antibody and 20-peptide antigen co-deposition in the glomeruli of the IgAN
experimental mice, demonstrating that using the 20-peptide as an antigenic determinant of S. aureus can induce
experimental IgAN in Balb/c mice. The antibody to the
20-peptide antigen also mainly labeled glomeruli of
human IgAN patients (82% in IgAN). Therefore, we
believe this study illustrates that a close relationship
Serological Findings
Levels of serum anti-20-peptide IgA and IgG antibodies
in the IgAN experimental group were significantly higher
than in the control groups (P < 0.001) (the IgAN experimental group; n ¼ 30, control; n ¼ 30 in each group), and
the IgA and IgG concentration in serum from IgAN
patients were higher than those from non-IgAN patients
(P < 0.001) (n ¼ 30 in each group) (Fig. 6).
DISCUSSION
In the previous study, we tried to establish the
mouse models using several previously described methods (Endo et al.,1993; Gesualdo et al., 1990; Han et al.,
1998; Isaacs et al., 1981; Liu et al., 1989). However, we
found that the result was not satisfactory. With the
exception of the above-mentioned models, existing animal models of IgA are described below. Initially, a ddY
mouse that can spontaneously develop mesangioproli-
ESTABLISHMENT OF A MOUSE IgAN MODEL
1735
Fig. 5. Immunofluorescence detection in renal tissue from IgAN model
mouse and patients using the mouse monoclonal anti-20-peptide antibody (A) Section of kidney from a control mouse treated with the MBPirrelevant-20-peptide fusion protein. No expression of 20-peptide was
detected (magnification: 400). (B) Section of kidney from an IgAN
mouse. The glomeruli were intensely stained (glomeruli indicated by
arrows; 400 magnification). (C) Renal biopsy section from a non-IgAN
patient. No expression was detected in the glomeruli and tubular epithelial
cells (200 magnification) of most non-IgAN cases. (D) Renal biopsy section from an IgAN patient. There is strong labeling of glomeruli and weak
labeling of tubular epithelial cells in 82% cases from IgAN (glomeruli indicated by arrows; 200 magnification) (n ¼ 50 in each group) (P < 0.001).
exists between the 20-peptide antigen and the pathogenetic development of IgAN.
The IgAN experimental mice showed mild and moderate mesangial cell proliferation and mesangial matrix
expansion, exhibiting electron-dense mesangial deposits
after immunization with the MBP-20-peptide fusion
antigens which have similarities to the pathological
changes of human IgAN. IgA immune complexes deposited in glomeruli can induce leukocyte infiltration and
inflammatory reactions, leading to damage of the nephric tubule and interstitium but not the glomeruli. Tubulointerstitial
damage
can
influence
glomerular
hemodynamic changes resulting in hematuria and proteinuria (Sánchez-Lozada et al., 2003). In our study, hematuria was detected by the 11th week, which rose
sharply on the 15th week before reaching a plateau by
the 20th week. The urine P/C ratio began to remarkably
increase after the 15th week, and then peaked at the
20th week. This result demonstrated that glomerular hemodynamic damage is aggravated when immunization
times were increased. These findings were significantly
different from those of mice treated with the MBP-irrele-
vant-20-peptide fusion protein and the other two control
groups.
In addition, our study demonstrated that anti-20 peptide IgA and IgG levels in mouse serum increased in the
IgAN experimental Balb/c mice, resembling our observations in human IgAN. IgA in serum may conjugate the
20-peptide to form immune complexes which deposit in
the glomeruli resulting in IgAN-like changes.
However, the IgA system of mice significantly differs
from that of the human. In humans, several research
groups have found that glomerular IgA deposition might
occur not only due to IgA immune complexes but also
due to the nonimmunological formation of macromolecular IgA1 induced by abnormal O-glycosylation in the
IgA1 hinge (Sano et al., 2002). Altered O-glycosylation
might favor self-aggregation of IgA1 (Kokubo et al.,
1997) or act as an autoantigen in immune complexes
with IgG (Tomana et al., 1999). Besides, the abnormal
physiochemical properties of circulating IgA1, such as
size, charge and glycosylation, might be one of the key
pathogenesis factors of IgAN. (Hashim et al., 2001;
Iwase et al., 2002; Leung et al., 2001, 2002; Sano et al.,
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ZHANG ET AL.
Fig. 6. Anti-20-peptide immunoglobulins are increased in IgAN
patient and the immunized mice sera The concentration of IgA (A)
and IgG (B) antibodies against the 20-peptide in the serum of
IgAN patients was higher in the non-IgAN patients (P < 0.001) (n
¼ 30 in each group). The levels of IgA (C) and IgG (D) antibodies
against 20-peptide in the serum of IgAN experimental mice was
significantly higher than in control mice (n ¼ 30 in each group) (P
< 0.001).
2002). The interaction between IgA1 and human mesangial cells (HMC) via some special receptors (Tamouza
et al., 2007; Wang et al., 2004) is one of the most important aspects in the pathogenesis of IgAN, resulting in
the enhancement of HMC proliferation, inflammation,
sclerotic cytokine release and extracellular matrix production to induce the renal injury of IgAN (Wang et al.,
2004). Mice have only one form of IgA and lack the
hinge region. Human IgA is mostly monomeric, whereas
murine IgA is mostly polymeric. Moreover, human IgA
has O- and N-glycans, whereas murine IgA has only Nglycans (Suzuki et al., 2005). Therefore, we propose that
the 20-peptide antigen itself and/or this immune complex may be more important for the glomerular deposition of IgA than the nature of IgA in the immunized
mice.
Some studies demonstrated that the genetic background of IgAN patients could contribute to disease susceptibility (Galla, 2001; Hsu et al., 2000; Schena, 1995;
Scolari, 2003). Resembling the human situation, the ddY
mouse is a spontaneous animal model of human IgAN
with a highly variable incidence and extent of mesangial
proliferation and extracellular matrix expansion with
paramesangial IgA depositions as a result of the heterogeneous background (Imai et al., 1985; Suzuki et al.,
2005). Whether the induction of IgAN induced by the
20-peptide antigen is somehow regulated by specific
genetic factors, or if the 20-peptide has changed the disease susceptibility in our study is unknown, but is worth
investigating in the future.
We chose to express the 20-peptide antigen as a fusion
protein with MBP. One potential problem with this
approach was that the antigen might have become hid-
den within the foreign protein sequences during folding,
thus becoming inaccessible to the immune system. However, our immunoblot data demonstrated that the monoclonal antibody against the 20-peptide was able to label
the fusion protein under both denaturing and nondenaturing conditions. In addition, the fusion protein could
be used to produce a faithful mouse model of the human
disease, and the monoclonal antibody against 20-peptide
distinguished IgAN from non-IgAN biopsy tissues. Nonetheless, the potential for the antigen to be obscured
remains a consideration for other studies and should not
be ruled out.
A major difference between our approach and those
used historically is that previous studies used whole
membrane antigens of S. aureus in establishing an
immunological IgAN model (Sharmin et al., 2004). The
advantage of our model is that using the 20-peptide
removes the influence of other S. aureus membrane antigens that do not normally participate in IgAN
pathogenesis.
We selected 21 weeks as the observation time to establish the IgAN animal model in our experiments,
although this is more prolonged than in the previously
established model (normally 15–16 weeks; Sharmin
et al., 2004). The IgAN experimental mice showed mild
and moderate mesangial cell proliferation and mesangial
matrix expansion while showing no other morphological
pathological changes.
In summary, the 20-peptide of S. aureus induced mesangial deposition of IgA and C3, mesangial cell proliferation and mesangial matrix production in Balb/c mice.
Our study has demonstrated that the 20-peptide-IgA
complex induced glomerular and tubulointerstitial
ESTABLISHMENT OF A MOUSE IgAN MODEL
damage resulting in hematuria and proteinuria. Our
study is the first to establish an experimental model of
IgAN with the 20-peptide of S. aureus.
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