Human Pathology (2010) 41, 12761285

www.elsevier.com/locate/humpath

Original contribution

Localization of nephritis-associated plasmin receptor in

acute poststreptococcal glomerulonephritis
Takashi Oda MD a,, Nobuyuki Yoshizawa MD b , Kazuo Yamakami PhD c ,
Kikuko Tamura MD d , Aki Kuroki MD e , Tetsuzo Sugisaki MD e , Emi Sawanobori MD f ,
Kohsuke Higashida MD f , Yoshiyuki Ohtomo MD g , Osamu Hotta MD h ,
Hiroo Kumagai MD a , Soichiro Miura MD a
a
Department of Internal Medicine, National Defense Medical College, Saitama 359-8513, Japan
b
Hemodialysis Center, Chofu Hospital, Tokyo 182-0034, Japan
c
Department of Preventive Medicine and Public Health, National Defense Medical College, Saitama 359-8513, Japan
d
Department of Pediatrics, National Hospital Organization, Nishisaitama-Chuo National Hospital, Saitama 359-1151, Japan
e
Department of Nephrology, Showa University School of Medicine, Tokyo 142-8666, Japan
f
Department of Pediatrics, Yamanashi Medical University, Yamanashi 409-3898, Japan
g
Department of Pediatrics, Juntendo University Nerima Hospital, Tokyo 177-8521, Japan
h
Department of Nephrology, Sendai Shakaihoken Hospital, Miyagi 981-8501, Japan

Received 7 September 2009; revised 16 February 2010; accepted 17 February 2010

Keywords: Summary The nephritis-associated plasmin receptor is a recently identified nephritogenic antigen
Acute poststreptococcal
associated with acute poststreptococcal glomerulonephritis and proposed to play a pathogenic role,
glomerulonephritis;
but its precise glomerular localization in acute poststreptococcal glomerulonephritis has not been
Nephritis-associated
elucidated. We therefore analyzed renal biopsy sections from 10 acute poststreptococcal
plasmin receptor;
glomerulonephritis patients by using immunofluorescence staining with antinephritis-associated
Neutrophil;
plasmin receptor antibody and various markers of glomerular components. Nephritis-associated
Plasmin;
plasmin receptor was detected in the glomeruli of all patients, and double staining for nephritis-
Streptococcal pyrogenic
associated plasmin receptor and collagen IV showed nephritis-associated plasmin receptor to be
exotoxin B
predominantly on the inner side of the glomerular tufts. Nephritis-associated plasmin receptor
positive areas within glomerular tufts were further characterized with markers for neutrophils,
mesangial cells, endothelial cells, and macrophages. In 6 of the patients, nephritis-associated plasmin
receptor staining was seen mainly in neutrophils and to a lesser degree in mesangial and endothelial
cells. In the other 4 patients, nephritis-associated plasmin receptor staining was seen mainly in
mesangial cells and to a lesser degree in neutrophils and endothelial cells. In all patients,
macrophages showed little staining. Elevated plasmin activity in glomerular neutrophils was
identified by combining in situ zymography staining for plasmin activity and immunofluorescence
staining for neutrophils. The glomerular localizations of nephritis-associated plasmin receptor and
another nephritogenic antigen, streptococcal pyrogenic exotoxin B, were compared by double
immunofluorescence staining and found to be similar. These findings indicate the nephritogenic

Corresponding author. Department of Internal Medicine, National Defense Medical College, Saitama 359-8513, Japan.
E-mail address: takashio@ndmc.ac.jp (T. Oda).

0046-8177/$ see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.humpath.2010.02.006
Localization of NAPlr in APSGN
potential of nephritis-associated plasmin receptor and offer valuable information with respect to the
pathogenic mechanism of acute poststreptococcal glomerulonephritis.
2010 Elsevier Inc. All rights reserved.
1. Introduction
Acute poststreptococcal glomerulonephritis (APSGN), a
sequel of streptococcus infection and the prototypic acute
nephritic syndrome [1-3], is recently rare in the industrial-
ized world but is often found in rural and Aboriginal
communities [4]. Because of the characteristic manifesta-
tions of this disease (onset of symptoms 1-3 weeks after
streptococcal infection, decrement in serum complement
titer, and glomerular deposition of C3 and/or immunoglob-
ulin [Ig] G in the early phase), its initiation is widely
thought to involve the immunologic response to strepto-
coccal antigens. The most popular theory of the pathogenic
mechanism of APSGN has been the immune complex
theory, which involves the glomerular deposition of so-
called nephritogenic streptococcal antigen and subsequent
formation of immune complexes in situ and/or the
deposition of circulating antigen-antibody complexes
[1-4]. Glomerular immunoglobulin deposition, however,
is often not prominent in this disease. Furthermore, the
reason for the difference between the site of glomerular cell
infiltration and the site of immune complex deposition is
unclear: the major site of inflammation is the inner side of
the glomerular tufts (glomerular endocapillary proliferation
is the histologic hallmark of this disease), whereas in the
early phase of this disease, the immune complexes locate
mainly on the outer (subepithelial) side of the glomerular
tufts [3,5]. Another type of human glomerulonephritis with
subepithelial immune complex deposition, membranous
nephropathy, is rarely accompanied by endocapillary cell
infiltration. Thus, the mechanism of the prominent glomer-
ular endocapillary proliferation occurring in APSGN is
unknown [2,3]; and the identity of the causative antigen(s)
remains a matter of debate [6-11]. We recently isolated and
characterized a nephritogenic antigen from group A
streptococcus [12-15] that we call the nephritis-associated
plasmin receptor (NAPlr) and is homologous to the group A
streptococcus plasmin receptor also known as streptococcal
glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
which has been shown in vitro to bind plasmin and maintain
its proteolytic activity by protecting it from physiologic
inhibitors like 2-antiplasmin [16]. Glomerular deposits of
NAPlr are present in essentially all patients with early-phase
APSGN [14]. The localization of NAPlr differs from that of
C3 or IgG [15] but is almost identical to that of plasmin
activity [17], suggesting that deposited NAPlr induces
glomerular damage by keeping active plasmin trapped in
the glomeruli. The precise intraglomerular localization of
NAPlr in APSGN patients, however, has not been determined.
1277
Furthermore, Batsford and coworkers [11] recently ana-
lyzed a series of APSGN patients and found that
immunohistochemical glomerular positivity for NAPlr
was far less frequent than that for streptococcal pyrogenic
exotoxin B (SPEB). This is critically inconsistent with our
previous findings [14,15,18]. To confirm the glomerular
deposition of NAPlr and identify its precise localization,
we performed single and double immunofluorescence
staining with various glomerular cell markers and evaluated
staining specificity by using antigen absorption tests. We
also compared the localizations of SPEB and NAPlr by
double immunofluorescence staining. The present results
confirm the glomerular deposition of NAPlr in APSGN
patients and show that it is found principally in neutrophils,
mesangial cells, and endothelial cells. We show here that
the distributions of NAPlr and SPEB in the glomeruli of
APSGN patients are similar, and we discuss possible
reasons for this similarity.
2. Materials and methods
2.1. Patients
Renal biopsy tissues were collected from 10 APSGN
patients whose characteristics are listed in Table 1. All
showed overt symptoms of APSGN such as facial edema,
hypertension, and hematuria. Percutaneous needle biopsies
were performed to rule out progressive renal diseases that
can cause acute nephritic syndrome (eg, IgA nephropathy,
membranoproliferative glomerulonephritis, lupus nephri-
tis, rapidly progressive glomerulonephritis). The diagnosis
of APSGN was based on serologic and bacteriologic
evidence of acute streptococcal infection before the onset
of nephritis and on characteristic histologic features of the
biopsy tissue (representative glomerular feature of
APSGN in hematoxylin and eosinstained tissue section
is shown in Fig. 1). The major light microscopic finding
in all of these patients was glomerular endocapillary
proliferation, which was accompanied by mesangial
proliferation or crescent formation in some patients as
shown in Table 1. By routine immunofluorescence
microscopy, all patients showed positive staining for C3,
staining pattern of which was also shown in Table 1. Five
patients with rapidly progressive glomerulonephritis
(defined as the presence of crescents in N60% of
glomeruli) served as disease control. Informed consent
was obtained from each patient in accordance with the
principles of the Declaration of Helsinki.
1278 T. Oda et al.

2.2. Immunofluorescence staining for NAPlr and

End

Abbreviations: UP, urinary protein; U-RBC, urinary red blood cells; HPF, high-power field; CH50, 50% hemolytic unit of complement; ASO, anti-streptolysin O; Mes, mesangial; Cres, crescent; Cap, capillary;
+

+
+
+
+
+
+



antigen absorption tests
Mes

+++

+++
Unfixed cryostat sections of APSGN and rapidly

++

++
+

+
+
+

+
progressive glomerulonephritis patients were stained with
NAPlr localization d

fluorescein isothiocyanate (FITC)conjugated polyclonal

M

rabbit anti-NAPlr antibody by using procedures described

previously [14], and selected sections were also stained with
FITC-conjugated monoclonal mouse anti-NAPlr antibody
+++

+++

+++
Neu

(1F10) (Abcam Inc, Cambridge, MA). The specificity of the

++
++

++
+

1F10 has been confirmed by Western blotting [19].

Antibody absorption tests were used to rule out cross-
Mes

reactivity between the polyclonal anti-NAPlr antibody and

++

++

++
++

++
++

+

+
+
IF for C3 c

human GAPDH. Blue A-Sepharose (Amersham Bios-

ciences, Piscataway, NJ) was used to isolate human
+++
Cap

GAPDH from the red blood cells of a healthy volunteer as

++

++
++

++
+
+

+
+
+

described by Thompson et al [20]. Polyclonal anti-NAPlr

antibody was incubated with a 5-fold excess concentration
The existence or absence of glomerular mesangial proliferation and crescent formation under light microscopy was identified as + or .

Intraglomerular NAPlr-positive portion was identified by double staining as neutrophil, macrophage, mesangial cell, or endothelial cell.
Cres

of human GAPDH for 2 hours. Unless otherwise stated, all

+
+

procedures were performed at 27C. On some sections,

microscopy b

direct immunofluorescence staining with absorbed anti-

The level of immunofluorescence (IF) staining for C3 along capillary or on mesangial area was graded as , +, ++, or +++.

NAPlr antibody was performed, which was compared with

Light

Mes

that with nonabsorbed anti-NAPlr antibody.

+
+
+
+
+

+
Clinical and histologic profiles of patients with APSGN and the summary of NAPlr localization

2.3. Double immunofluorescence staining for NAPlr

(U/mL)

and glomerular markers

691
226
1373
254
1530
536
539
214
601
166
ASO

Double staining for NAPlr and collagen IV was used to

determine the localization of NAPlr relative to the
(U/mL)

glomerular basement membrane. After being washed in

CH50

15.2
5.1
16.9
6.7
23.9
9.4
12.5
12.0
19.2
4.7

phosphate-buffered saline (PBS), sections were incubated in

Accurate date could not be obtained because the onset date was not recorded exactly.

a mixture of FITC-conjugated rat anti-human collagen IV 3

chain antibody (a gift from Dr Yoshikazu Sado, Shigei
Creatinine

Medical Research Institute, Okayama, Japan) and Alexa

(mg/dL)

Fluor 594conjugated rabbit anti-NAPlr antibody for 30

1.7

0.9
0.8

1.0
2.2
1.0
1.3
0.6
0.9
1.7

minutes, washed in PBS, and mounted. The rabbit anti-

NAPlr antibody was labeled with Alexa Fluor 594 by using a
protein labeling kit (Molecular Probes, Inc, Eugene, OR) as
(cells/HPF)

described elsewhere [15].

U-RBC

50-99

10-19
30-49

30-49
30-49

20-29

To identify intraglomerular NAPlr staining, we used 4

N100

N100
5-9
5-9

Neu, neutrophil; M, macrophage; End, endothelial cell.

murine monoclonal antibodies from Dako Cytomation

Duration between disease onset and renal biopsy.

(Glostrup, Denmark) as markers of intraglomerular cell

components: anti-human -smooth muscle actin antibody
+++
+++

+++
+++

+++
UP

++

1A4 to mark mesangial cells, anti-human CD31 antibody

+

+
+

JC70A to mark endothelial cells, anti-human neutrophil

elastase antibody NP57 to mark neutrophils, and anti-human
Onset a

CD68 antibody EBM11 to mark macrophages. After the

20 e
(d)

8
8
8
8
12
14
20
20

32

sections were stained with FITC-conjugated rabbit anti-NAPlr

antibody, they were fixed in 4% paraformaldehyde for 5
Age/sex

minutes, blocked with 7% nonfat skim milk in PBS for 5

21/M

48/M
16/M
16/M
10/M

45/M
44M
37/F

44/F

minutes, and incubated with one of the monoclonal antibodies

9/M

for 1 hour. Bound antibodies were visualized by incubating

the sections in Alexa Fluor 594conjugated goat anti-mouse
Table 1
Patient

IgG antibody (Molecular Probes) for 30 minutes. Digital

b

d
a

images of the stained sections were obtained with a confocal

no.

10
1
2
3
4
5
6
7
8
9

microscope (Zeiss LSM 510; Carl Zeiss, Gttingen,

Localization of NAPlr in APSGN
Fig. 1 A representative photomicrograph of light microscopic
image of hematoxylin and eosin staining in a patient (patient 5) with
APSGN (original magnification 260).
Germany). Double staining was assessed as follows: for no
double staining, for less than 10% of the NAPlr-positive area
being double positive, + for 10% to 30% of the NAPlr-positive
area being double positive, ++ for 31% to 50% of the NAPlr-
positive area being double positive, and +++ for greater than
50% of the NAPlr-positive area being double positive.
2.4. Double staining for neutrophils and
plasmin activity
Plasmin activity in glomerular neutrophils was evaluated
by performing in situ zymography for plasmin activity [17]
and indirect immunofluorescence staining for neutrophil
elastase sequentially in the cryostat sections of APSGN and
rapidly progressive glomerulonephritis patients. After sec-
tions were fixed in 4% paraformaldehyde for 5 minutes, they
were incubated for 30 minutes with a reaction mixture
containing 0.1% Fast Violet B and 0.5 mmol/L p-
toluenesulfonyl-L-lysine -naphthyl ester (Torii Pharmaceu-
tical Co, Ltd, Tokyo, Japan) in 67 mmol/L phosphate buffer.
Indirect immunofluorescence staining for neutrophil elastase
was then performed in the same manner as described above
except that Alexa Fluor 488conjugated goat anti-mouse
IgG antibody (Molecular Probes) was used as the secondary
antibody. Light microscope images of plasmin activity and
fluorescence microscope images of neutrophil elastase
staining were acquired, digitized, and merged.
2.5. Double immunofluorescence staining for NAPlr
and SPEB
Because of the limited availability of renal biopsy sections
for the present series of patients, localization profiles of
1279
NAPlr and SPEB were compared by double immunofluo-
rescence staining in renal biopsy sections from only 6 of the
APSGN patients (patients 1, 4, 5, 7, 9, and 10). Cryostat
sections were fixed for 5 minutes in 4C acetone, incubated
for 60 minutes in a mixture of Alexa Fluor 594conjugated
rabbit anti-NAPlr antibody and FITC-conjugated rabbit anti-
zymogen/SPEB antibody (a gift from Dr Stephen Batsford,
Department of Immunology, Institute of Medical Microbi-
ology, Freiburg, Germany), washed in PBS, and mounted.
Digital images were obtained with a confocal microscope
(Zeiss LSM 510, Carl Zeiss). To rule out an influence of
antibody mixing on the staining results, several consecutive
sections were also stained for either NAPlr or SPEB.
3. Results
3.1. Single staining for NAPlr and evaluation of
staining specificity
Staining with polyclonal anti-NAPlr antibody (Fig. 2A)
showed glomerular positivity in all APSGN patients
analyzed in the present study, although the staining pattern
and intensity varied, and diffuse (ie, glomerular as well as in
tubulointerstitial) nuclear background staining was ob-
served in several sections. Similar results were observed
with monoclonal anti-NAPlr antibody (Fig. 2B), but both
the specific staining and background staining were
relatively weak. On the other hand, glomerular NAPlr
staining was not found in any biopsies of rapidly
progressive glomerulonephritis patients stained with either
polyclonal anti-NAPlr antibody or monoclonal anti-NAPlr
antibody (data not shown). We previously reported that
preabsorption of polyclonal anti-NAPlr antibody with
recombinant plasmin receptor or pretreatment of tissue
sections with unlabeled anti-NAPlr antibody diminished
glomerular NAPlr immunofluorescence staining [14,15].
Because NAPlr is the same molecule as streptococcal
GAPDH, the nonspecific positivity due to the cross reaction
with human GAPDH is a matter of concern. In the present
study, however, absorption of the polyclonal anti-NAPlr
antibody with human GAPDH did not affect the glomerular
NAPlr staining (Fig. 2C), indicating that the anti-NAPlr
antibody does not react with human GAPDH and stains
NAPlr (ie, streptococcal GAPDH) specifically.
3.2. Double staining for NAPlr and
glomerular markers
Double staining for NAPlr and collagen IV showed that
glomerular NAPlr positivity was localized predominantly on
the inner side of glomerular tufts (Fig. 3A-C) and to a lesser
degree on the parietal epithelial cells lining Bowman capsule.
NAPlr staining was rarely detected on the outer side of
glomerular tufts (subepithelial sites or podocytes). The
1280 T. Oda et al.

Fig. 2 Glomerular deposition of NAPlr in a patient (patient 5) with APSGN. Representative photomicrographs of immunofluorescence
staining for NAPlr (A) with polyclonal rabbit anti-NAPlr antibody, (B) with monoclonal mouse anti-NAPlr antibody (1F10), or (C) with
polyclonal rabbit anti-NAPlr antibody that had been preabsorbed with isolated human GAPDH (original magnification 260).

Fig. 3 NAPlr localization relative to the glomerular basement membrane. Confocal microscopy images of double immunofluorescence
staining for collagen IV (A, FITC) and NAPlr (B, Alexa Fluor 594) with a merged image (C) in an APSGN patient (patient 9). NAPlr
immunofluorescence was mostly seen on the inner side of glomerular tufts and rarely seen on the outer side of glomerular tufts (subepithelial
site or podocytes) (scale bar = 10 m).

intratuft NAPlr positivity was therefore further investigated
by double staining with markers of glomerular intratuft cell
components. Representative microphotographs of double
staining are shown in Fig. 4A-L, and the results are
summarized in the Table 1. In 6 of the 10 patients (1, 3, 5,
6, 9, and 10), NAPlr staining was localized mainly in
neutrophils (Fig. 4A-C) and to a lesser degree in mesangial
and endothelial cells (Fig. 4G-I). In the other 4 patients
(patients 2, 4, 7, and 8), NAPlr staining was localized mainly
in mesangial cells (Fig. 4J-l) and to a lesser degree in
neutrophils and endothelial cells. In all patients, some
macrophages were positive for NAPlr staining (Fig. 4D-F);
but this staining was minor. As summarized in Table 1, no
significant correlation was found between glomerular NAPlr
distributions with certain clinical or histologic features of
APSGN patients.
3.3. Plasmin activity of glomerular neutrophils
Because NAPlr staining was found to be localized mainly
in neutrophils and NAPlr is known to bind to plasmin and
maintain its proteolytic activity in vitro [16], we examined

Fig. 4 Precise intraglomerular localization of NAPlr by double staining. Representative confocal microscopy images of double
immunofluorescence staining for NAPlr (A, D, G, J; FITC) and various markers of intratuft glomerular cell components (B, neutrophils:
neutrophil elastase; E, macrophages: CD68; H, endothelial cells: CD31; K, mesangium: -smooth muscle actin) (Alexa Fluor 594). In most
patients, NAPlr immunofluorescence was localized predominantly in neutrophils (A-C, patient 1). Macrophages showed only minor NAPlr
immunofluorescence in all patients (D-F, double-positive portion is indicated by arrow, patient 1). NAPlr immunofluorescence was also
localized in endothelial cells (G-I, double-positive portions are indicated by arrows, patient 5). In some patients, the mesangium showed the
most NAPlr staining (J-L, patient 8) (scale bar = 20 m).
Localization of NAPlr in APSGN 1281
1282 T. Oda et al.

Fig. 5 Glomerular infiltrating neutrophils and plasmin activity in APSGN and rapidly progressive glomerulonephritis. Representative
photomicrographs of double staining for neutrophil elastase (A, D, indirect immunofluorescence staining) and plasmin activity (B, E, in situ
zymography) from a patient with APSGN (A-C, patient 5) and with rapidly progressive glomerulonephritis (D-F). The same fields were
observed under fluorescence microscopy (A, D) and light microscopy (B, E) and were merged (C, F). The merged image (C) shows up-
regulated plasmin activity in a large portion of glomerular neutrophils in APSGN patients but not in rapidly progressive glomerulonephritis
patients (F) (original magnification 260).

the plasmin activity of glomerular neutrophils and found that
many were positive for plasmin activity in renal tissues from
APSGN patients (Fig. 5A-C). On the other hand, glomerular
neutrophils were not positive for plasmin activity in renal
tissues from rapidly progressive glomerulonephritis patients
(Fig. 5D-F), which suggests disease specificity of the
relationship between plasmin activity and neutrophils.
Adding 0.1 U/L aprotinin (plasmin inhibitor) to the reaction
mixture eliminated this activity, providing further evidence
for the specificity of the in situ zymographic staining (data
not shown).
3.4. Relative distributions of NAPlr and SPEB
Like NAPlr staining, SPEB staining was detected in the
glomeruli of all 6 patients whose renal biopsy sections
were double stained for NAPlr and SPEB. The glomerular
distributions of NAPlr and SPEB were similar but not
identical. The NAPlr staining generally predominated, and
most of the SPEB staining was within NAPlr-positive
areas (Fig. 6A-C). The nonspecific background staining,
however, was also more strongly evident in NAPlr
staining than in SPEB staining. This staining pattern was
confirmed by staining serial sections for either SPEB or
NAPlr (data not shown).
4. Discussion
Identifying the streptococcal antigen(s) causing APSGN
is an essential part of elucidating the mechanism of APSGN
pathogenesis [6-11,21], and the most likely are NAPlr and
SPEB. NAPlr is a 43-kd antigen recently isolated from group
A streptococcus by our laboratory and has been shown to
have plasmin(ogen)-binding capacity [14]. Our proposal that
NAPlr-bound plasmin plays a pathogenic role in the
development of APSGN was based on the identical
glomerular distributions of NAPlr and 2-antiplasmin
resistant plasmin activity [17]. We had also found APSGN
patients to have elevated urinary plasmin activity that is
resistant to 2-antiplasmin [22]. Plasmin might damage
renal tissue directly by degrading extracellular matrix
Localization of NAPlr in APSGN
Fig. 6 Similar localization of NAPlr and SPEB in APSGN. Representative confocal microscopy images of double immunofluorescence
staining for SPEB (A, FITC) and NAPlr (B, Alexa Fluor 594) in an APSGN patient (patient 5). The merged image (C) shows that the
distributions of NAPlr and SPEB staining are similar but not identical. Arrows indicate a representative intraglomerular region positive for
SPEB but negative for NAPlr (scale bar = 20 m).
proteins such as fibronectin or laminin and might also affect
almost all extracellular matrix proteins indirectly by
activating promatrix metalloproteases [23,24]. Plasmin
can also mediate inflammation by accumulating and
activating monocytes and neutrophils in situ [25,26]. Thus,
we think that glomerular damage may initially be induced by
immunodetectable free NAPlr, which can bind plasmin and
maintain its proteolytic activity, rather than by subepithelial
immune complexes. In this respect, the present finding that
immunodetectable NAPlr is localized on the inner side of
glomerular tufts (endocapillary) is consistent with the
predominantly endocapillary glomerular inflammation in
APSGN. In other words, endocapillary localization of
immunodetectable NAPlr might account for the different
sites of glomerular inflammation and immunocomplex
deposition in APSGN.
SPEB is a cationic cysteine protease secreted as a 42-kd
zymogen and subsequently cleaved to a 28-kd active
proteinase [10,27]. It is an exotoxin produced by pyro-
genic streptococci and has attracted considerable atten-
tion as an important toxin in severe invasive streptococcal
infections [27], and Poon-King et al [28] have suggested
that it is also a nephritogenic antigen that plays a role in
the pathogenesis of APSGN. They reported a previously
identified nephritogenic antigen for APSGN, termed
nephritis strain-associated protein, as the same molecule
as SPEB and possessing plasmin-binding capacity. Vogt
and his colleagues [7] previously reported that the
glomeruli of APSGN patients frequently contained a
cationic extracellular streptococcal antigen later shown
to be the same molecule as SPEB. More recently, Cu et al
[10] analyzed the renal biopsy tissues of APSGN patients
by using polyclonal anti-SPEB antibody and found
SPEB in the glomeruli of 67% of the APSGN patients
they examined.
An evaluation of SPEB and streptococcal GAPDH
(NAPlr) as nephritogenic antigens for APSGN was
1283
recently reported by Batsford et al [11], who found
anti-SPEB antibodies in the sera of all 53 of the APSGN
patients in their sample and found SPEB deposition in
the glomeruli of 14 of the 17 renal biopsies of the
APSGN patients they examined. They also found high
colocalization of SPEB and C3 by double immunofluo-
rescence staining and found SPEB within electron-dense
subepithelial deposits by immunoelectron microscopy.
They found that serum antibody responses and glomerular
depositions were far less common for streptococcal
GAPDH (NAPlr) than for SPEB. However, we previously
reported that anti-NAPlr antibody was detected at high
titers in 92% (46/50) of sera from APSGN patients and
that glomerular deposition of NAPlr was observed in
100% (25/25) of APSGN patients within 2 weeks after
disease onset or in 83.7% (36/43) of APSGN patients
within 30 days after onset [14,15]. Thus, the result
reported by Batsford et al [11] is critically different from
what we had found. This discrepancy may be in part due
to the different antibodies used in each of these studies;
in their study, they used the anti-streptococcal GAPDH
antibody that they generated, whereas in ours, we used
the anti-NAPlr antibody that we generated. The immu-
nostaining results for an antigen not infrequently differ
when different antibodies are used [18].
In the present study, the glomerular localization profiles
of SPEB and NAPlr were compared using the same
antibodies and the same methodology in a well-defined
group of APSGN patients. The similar localizations of
NAPlr and SPEB in glomeruli were unexpected because our
previous study [15] showed different distributions of C3
and NAPlr staining and because Batsford et al [11] reported
a high degree of colocalization of C3 and SPEB staining. In
contrast to NAPlr, SPEB has cationic characteristics; so it is
plausible that it would deposit in the subepithelial space and
colocalize with C3. NAPlr and SPEB are surely different
molecules, although the reported molecular weights are
1284
close: 43 and 42 kd, respectively. NAPlr was isolated from
cytoplasmic fraction of group A streptococcus, whereas
SPEB was isolated from extracellular fraction; amino acid
sequences of both molecules were quite different; and
structural and functional properties of both molecules were
generally different except for several functional properties
such as the capacity for binding plasmin. The present
results are unlikely to be artifactual results or due to cross
reactivity of the 2 antibodies because (1) we confirmed
similar staining patterns of both antigens in serial sections
by single staining, (2) preabsorption of anti-SPEB antibody
with NAPlr did not affect the staining results (data not
shown), (3) preincubation of sections with unlabeled anti-
NAPlr antibody did not affect SPEB staining (data not
shown), and (4) preincubation of sections with anti-SPEB
antibody did not affect NAPlr staining (data not shown).
Thus, we could hardly expect the reason for this
discrepancy. As suggested by Rodriguez-Iturbe and Bats-
ford [1] and by Batsford et al [11], the discrepancy might be
due to the difference in the genetic and demographic
backgrounds of the patients analyzed in the different
studies. Cu et al [10], however, analyzed the renal tissues
of APSGN patients in the United States and Chile and
described the localization of SPEB in mesangial and
endocapillary areas as diffuse and granular, similar to the
patterns of NAPlr and SPEB staining observed in the
present study. Our double-staining results (similar localiza-
tions of 2 distinct antigens in the same glomerulus) were
surprising because many researchers, including us, have
been assuming that APSGN is the result of a single
nephritogenic streptococcal antigen. The present results
indicate that this assumption may be incorrect. We should
consider the possibility that 2 or more antigens interact in
the induction of this disease. Rodriguez-Iturbe and Batsford
[1] indicated in their recent review that multiple mechan-
isms should be responsible for each case of APSGN and
elegantly summarized the potential cooperative roles of
NAPlr and SPEB. It is also interesting that NAPlr and
SPEB share a common function. Both bind plasmin,
thereby protecting it from physiologic inhibitors, and thus
might cause chemotaxis of inflammatory cells and degra-
dation of glomerular basement membranes due to the
activity of plasmin [4,17,28,29]. The binding site on
plasmin for these molecules has not been clarified yet.
There have been many studies using single staining to
investigate the glomerular localization of nephritogenic
antigens potentially responsible for APSGN [6,7,10,21].
The study reported here is one of the few using double
staining to identify the precise intraglomerular localization of
nephritogenic antigens. NAPlr may deposit on glomerular
mesangial cells and endothelial cells by adhering to matrix
proteins [14] and may cause histologic and functional change
on those cells by promoting plasmin-catalyzed proteolysis.
With respect to the pathogenic role of NAPlr on neutrophils,
the hyperproteolytic state of NAPlr-positive neutrophils
in the present study indicates a possible role of these
T. Oda et al.
neutrophils in the induction of proteolytic glomerular
damage in situ. Regarding the mechanism of localization
of NAPlr on neutrophils, we suggest 2 possibilities. NAPlr
may bind the urokinase-type plasminogen activator receptor
expressed on neutrophils [30,31], which has recently been
shown to be the receptor for streptococcal GAPDH (NAPlr)
[32]. Alternatively, NAPlr may be phagocytosed by
neutrophils as a foreign bacterial antigen. These mechan-
isms, however, would not explain why NAPlr staining was
rarely found in macrophages. Macrophages, like neutrophils,
express urokinase-type plasminogen activator receptor
[31,33] and engage in phagocytosis; and similar levels of
macrophage and neutrophil infiltration were observed in the
glomeruli of the present series of APSGN patients. The
relative infrequency of NAPlr staining in macrophages might
be due to different levels of urokinase-type plasminogen
activator receptor expression on the neutrophils and macro-
phages of patients with APSGN. Alternatively, it might be
due to the relatively quicker infiltration of neutrophils than
that of macrophages in this disease [34,35]. Most of the
NAPlr deposited in glomeruli might be phagocytosed by
neutrophils accumulated in the early phase of the disease,
leaving little NAPlr free when macrophages eventually
migrate into the glomeruli.
In summary, we have confirmed that NAPlr is found
in the glomeruli of APSGN patients and have found its
principal locations to be in glomerular neutrophils,
mesangial cells, and endothelial cells. We had previously
found the location of glomerular NAPlr staining to
correspond to that of glomerular plasmin activity [17]
and, in the present study, have found the patterns of NAPlr
and SPEB staining to be similar. Finding out how and
when these 3 variables colocalize and interact will require
further studies.
Acknowledgment
We are grateful to our colleague Ms Yasuko Sugimoto for
expert secretarial assistance.
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