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Infection and Immunity, September 2003, p. 5371-5375, Vol. 71, No. 9
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.9.5371-5375.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Accumulation of Clostridium perfringens Epsilon-Toxin in the Mouse Kidney and Its Possible Biological Significance
Eiji Tamai,1 Tetsuya Ishida,2 Shigeru Miyata,1 Osamu Matsushita,1 Hirofumi Suda,3 Shoji Kobayashi,4 Hiroshi Sonobe,5 and Akinobu Okabe1*
Department of Microbiology,1
Department of Anatomy,2
Radioisotope Research Center,3
Department of Diagnostic Pathology, Faculty of Medicine, Kagawa Medical University, 1750-1 Ikenobe, Kita-gun, Kagawa 761-0793,4
Department of Laboratory Medicine and Pathology, National Fukuyama Hospital, Fukuyama City 720-8520, Japan5
Received 10 March 2003/
Returned for modification 23 April 2003/
Accepted 6 June 2003

ABSTRACT
In this paper we show that
Clostridium perfringens epsilon-toxin
accumulates predominantly in the mouse kidney, where it is distributed
mainly in glomeruli, capillaries, and collecting ducts. Although
some pycnotic and exfoliated epithelial cells were observed
in distal tubuli and collecting ducts, there were no findings
indicative of severe renal injury. Bilateral nephrectomy increased
the mouse lethality of the toxin, suggesting that the kidney
contributes to the host defense against the lethal toxicity
of epsilon-toxin.

TEXT
Epsilon-toxin produced by
Clostridium perfringens types B and
D is a potent toxin that is responsible for rapidly fatal enterotoxemia
in livestock (
17,
22). The toxin has been well defined in terms
of the proteolytic activation of epsilon-protoxin (
7,
10), its
pore-forming ability in Madin-Darby canine kidney (MDCK) cell
membranes (
12,
18) and artificial lipid bilayers (
19), and its
heptamerization in detergent-insoluble glycosphingolipid-enriched
microdomains (
10,
11). However, the pathogenic mechanisms involved
in the lethality of epsilon-toxin remain largely unknown, except
that it exhibits toxicity towards neuronal cells (
2,
8,
9) and
blood vessels in the brains of mice and rats (
1,
3,
4). Besides
massive necrosis in the brain, pulpy kidneys are noticeable
in animals that have died due to enterotoxemia (
22). Epsilon-toxin
exhibits cytotoxicity towards MDCK cells derived from dog renal
distal tubules or collecting ducts but not towards any other
cell lines (
17,
21,
24). Moreover, epsilon-toxin was reported
to be most abundant in the kidneys when intravenously (i.v.)
administered to mice (
13). However, nothing is known about its
nephrotoxicity.
The aim of this study was to determine the distribution of epsilon-toxin in the mouse kidney and also its nephrotoxicity. Male ddY mice (4 weeks old; SLC Japan, Shizuoka, Japan) were used. The epsilon-protoxin and epsilon-toxin used in this study were recombinant toxins, which were purified, activated, and labeled with [
-35S]ATP as described previously (11). The intravenous 50% lethal dose of epsilon-toxin was determined to be approximately 20 ng per mouse. The time to death after challenge estimated for mice susceptible to 15 ng of epsilon-toxin was 8.3 ± 0.4 h (mean ± standard error [SE], n = 5), and that for mice receiving 500 ng of epsilon-toxin was 0.35 ± 0.01 h (mean ± SE, n = 5).
First, we examined, by whole-body autoradiography (WBA), whether epsilon-toxin is accumulated predominantly in the mouse kidney in accordance with the results obtained by others using a different methodology (13). One hundred nanograms of 35S-epsilon-toxin or 35S-epsilon-protoxin was i.v. injected into a mouse through a tail vein. The mouse given 35S-epsilon-toxin was frozen in dry ice-acetone at approximately 1 h postinjection (p.i.) immediately after death. The mouse given 35S-epsilon-protoxin was sacrificed by cervical dislocation at 1 h p.i., followed by freezing in dry ice-acetone. Frozen sections (50 µm each) were prepared with an autocryotome (NA-500F; Nakagawa Seisakusho, Tokyo, Japan), dried at -20°C, and then exposed to an imaging plate (Fuji Photo Film, Kanagawa, Japan) for 2 weeks. WBA involving epsilon-toxin and epsilon-protoxin showed the same distribution profile, indicating that their putative receptor(s) is the same (Fig. 1). The toxins were detected most abundantly in the kidneys and fairly abundantly in the brain and spinal cord. In the kidneys the toxins were distributed in the outer and inner regions but not in the intermediate one. They were also detected in other organs, such as the spleen and eyes. Surprisingly, the toxins were deposited densely in the nasal turbinates, suggesting that they are enriched in an epsilon-toxin receptor.
For immunostaining of epsilon-toxin, anti-epsilon-protoxin antibodies
were generated with formalinized epsilon-protoxin in a female
New Zealand White rabbit (SLC). Anti-epsilon-protoxin immunoglobulin
G (IgG) was purified by means of epsilon-toxin immobilized on
Sepharose 4B and protein A-Sepharose (Amersham Biosciences,
Little Chalfont, Buckinghamshire, United Kingdom). The purified
IgG (0.7 mg/ml) was checked for specificity and sensitivity
by enzyme-linked immunosorbent assay and the Western blot procedure.
Nonspecific control IgG (0.6 mg/ml) was purified from rabbit
preimmune serum by means of protein A-Sepharose. Mice given
i.v. 500 ng of epsilon-toxin or the vehicle (1% Bacto Peptone-0.25%
NaCl solution) were anesthetized with ether at 20 min p.i.,
followed by perfusion with 5 ml of phosphate-buffered saline
(PBS) and then 20 ml of fixative consisting of 4% paraformaldehyde,
2.5% polyvinylpyrrolidone, 2% sucrose, and 0.1 M sodium phosphate
buffer (pH 7.4). The cryostat sections of 5-µm thickness
were prepared as described by Pavelka and Ellinger (
16) except
that treatment with ammonium chloride was omitted. To amplify
the immunohistochemical signal, two signal amplification systems,
a dextran polymer visualization system (EnVision+; Dako, Carpinteria,
Calif.) and a tyramide signal amplification system (NEN Life
Science Products, Boston, Mass.), were used in combination with
some modifications of the suppliers' instructions. Endogenous
peroxidase activity was inhibited by treatment with 3% H
2O
2 in TBST (50 mM Tris-HCl [pH 7.4], 300 mM NaCl, 0.1% Tween 20),
and nonspecific binding of primary antibodies was blocked with
blocking buffer (5% goat serum, 1% Triton X-100 in antibody
dilution buffer [Dako]). After incubation with primary antibodies
(rabbit anti-epsilon-protoxin IgG or nonspecific control IgG,
each diluted to 2.3 µg/ml in blocking buffer), the sections
were subjected to amplification with EnVision+ (goat anti-rabbit
IgG and horseradish peroxidase-immobilized dextran polymer;
Dako). The immunohistochemical signal was further amplified
with a TSA fluorescence system (fluorescein; NEN Life Science
Products). The sections were mounted on glass slides using a
ProLong antifade kit (Molecular Probes, Eugene, Oreg.) and then
viewed under a laser scanning confocal microscope (Olympus Optical,
Nagano, Japan).
Strong labeling was found in the kidney sections from epsilon-toxin-injected mice but not in those from vehicle-injected ones when stained with epsilon-protoxin-specific IgG (Fig. 2A and B). Nonspecific control IgG gave weak signal intensity to the collecting ducts in both sections (Fig. 2C and D). However, the immunoreactivity of nonspecific control IgG was restricted to the apical side, differing from that of epsilon-protoxin-specific IgG, which gave strong signal intensity to the basolateral side in epsilon-toxin-injected mice (Fig. 2A). More importantly, collecting ducts per se were not reactive with epsilon-protoxin-specific IgG (Fig. 2B). Thus, we concluded that the immunostaining involving epsilon-protoxin-specific IgG allows the specific detection of epsilon-toxin distributed in the kidneys. At low magnification, epsilon-toxin was detected in the whole kidney except for the outer medulla (data not shown), this being consistent with the doubly stained WBA profile of the kidney. Epsilon-toxin was detected most prominently in the glomeruli, capillaries, and collecting ducts (Fig. 2A). It was also detected to a large extent in the epithelial cells of the distal tubules and to a lesser extent but significantly in those of the proximal tubules. In the epithelial cells of the proximal tubules epsilon-toxin was mainly detectable on the luminal side, while in the epithelial cells of the distal tubules and collecting ducts it was on the basolateral side (Fig. 2A).
To examine toxin-induced morphological changes in the mouse
kidney, mice were i.v. given 20 to 500 ng of epsilon-toxin,
followed by transcardial perfusion with 10 ml of PBS and then
20 ml of 4% paraformaldehyde in PBS. The kidneys were cut into
small pieces, which were placed in the fixative for 2 days.
After routine dehydration and embedding, 2-µm cut sections
were stained with hematoxylin-eosin. A histological comparison
between mice in the same dose group showed that renal pathological
changes became more evident as fixation time was delayed. To
detect maximal but not postmortem changes, only mice surviving
at 6.5 to 7.5 h after challenge with 20 to 50 ng of epsilon-toxin
were fixed prior to death. Figure
3 shows morphological changes
detected in both the cortex and the medulla of the kidneys from
a mouse given 50 ng of toxin (Fig.
3). In the cortex, glomeruli
showed apparent shrinkage resulting in dilatation of Bowman's
space. Proximal tubules were almost intact, while distal tubules
and collecting ducts exhibited degenerative changes: a decrease
in the height of epithelial cells with a dilated lumen and cellular
degeneration with karyopycnosis and cellular exfoliation into
the lumen. Since the mouse given 50 ng of the toxin showed the
most prominent renal histological changes among those given
20 to 50 ng of the toxin, the severity and extent of these pathological
changes may also be dependent on the toxin dose. However, they
were less prominent in the kidneys from mice given 100 or 200
ng of epsilon-toxin. Moreover, there was no obvious histological
finding in the kidneys from mice given 500 ng of the toxin,
even in those from a mouse surviving exceptionally long and
fixed at 30 min p.i. (data not shown). Therefore, the changes
described above seem to be the maximal premortem histological
changes, although they are not very severe.
The renal pathological changes and accumulation of epsilon-toxin
may suggest the following possibilities. First, the nephrotoxicity
of epsilon-toxin may be involved in its lethal toxicity not
primarily but still somehow. Second, the kidneys may contribute
to the decrease in the amount of circulating epsilon-toxin and
thereby protect the host from its lethal toxicity. In order
to assess these possibilities, the effect of nephrectomy on
the lethal toxicity of epsilon-toxin was examined. Mice were
anesthetized with ether, and then bilateral nephrectomy was
accomplished by tying a silk suture securely around the hilus
renalis containing the artery, vein, and ureter, followed by
resection of the kidneys. The abdominal incision was closed,
immediately followed by injection with the vehicle alone or
50 or 100 ng of epsilon-toxin through a tail vein. The bilateral
nephrectomy clearly shortened the time required for the toxin
to kill mice (Table
1), indicating that the kidneys protect
the host from the lethal toxicity of epsilon-toxin. The kidneys,
however, could filter any circulating toxins, and the effect
of nephrectomy may reflect such a general feature of the kidneys,
not being specific to epsilon-toxin. To address this issue,
we examined the lethality for nephrectomized mice of two other
toxins,
C. perfringens alpha-toxin, which is similar in molecular
size but not neurotoxicity to epsilon-toxin, and botulinus type
A neurotoxin, which is larger and more neurotoxic than epsilon-toxin.
Nephrectomy did not shorten the time to death in either case
(Table
1). These results exclude the generality of the effect
observed for epsilon-toxin intoxication and suggest that the
accumulation of epsilon-toxin in the kidneys through specific
binding causes the effect.
View this table:
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TABLE 1. Lethality of epsilon-toxin, alpha-toxin, and botulinus toxin for nephrectomized and unnephrectomized mice
|
Epsilon-toxin is accumulated predominantly in the kidneys, which
attenuates its lethality. This apparently paradoxical finding
implies the biological significance of the epsilon-toxin accumulation
in the kidneys. We propose that the kidneys contribute to the
host defense against epsilon-toxin by trapping the toxin and
thereby protecting more susceptible organs, e.g., the brain,
from its lethal toxicity. Our hypothesis is of interest in the
context of the host defense against bacterial infection. Another
example of specialized host defense in kidneys is the protection
from urinary tract infection by Tamm-Horsfall glycoprotein secreted
from the surface of cells of the Henles loop, which binds
to type 1 fimbriated
Escherichia coli, preventing infection
by the organism (
14,
15,
20). Interestingly, both
C. perfringens and
E. coli are intestinal commensal bacteria. Mammals might
have developed defense mechanisms specific to individual bacteria
or toxins during their interaction with commensal bacteria.

ACKNOWLEDGMENTS
We thank K. Oguma (Department of Bacteriology, Okayama University
Graduate School of Medicine and Dentistry, Okayama, Japan) for
providing purified botulinus neurotoxin and J. Wada (Department
of Medicine and Clinical Science, Okayama University Graduate
School of Medicine and Dentistry) for advice and help on nephrectomy.
This work was supported by a grant-in-aid from the Japan Society for the Promotion of Science and also by the Sasagawa Scientific Research Grant from Japan Science Society.

FOOTNOTES
* Corresponding author. Mailing address: Department of Microbiology, Faculty of Medicine, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan. Phone and fax: 81-87-891-2129. E-mail:
microbio{at}kms.ac.jp.

Editor: A. D. O'Brien

REFERENCES
1 - Finnie, J. W. 1984. Ultrastructural changes in the brain of mice given Clostridium perfringens type D epsilon toxin. J. Comp. Pathol. 94:445-452.[CrossRef][Medline]
2 - Finnie, J. W., P. C. Blumbergs, and J. Manavis. 1999. Neuronal damage produced in rat brains by Clostridium perfringens type D epsilon toxin. J. Comp. Pathol. 120:415-420.[CrossRef][Medline]
3 - Finnie, J. W., P. C. Blumbergs, J. Manavis, T. D. Utteridge, V. Gebski, J. G. Swift, B. Vernon-Roberts, and T. R. Kuchel. 2001. Effect of global system for mobile communication (gsm)-like radiofrequency fields on vascular permeability in mouse brain. Pathology 33:338-340.[Medline]
4 - Ghabriel, M. N., C. Zhu, P. L. Reilly, P. C. Blumbergs, J. Manavis, and J. W. Finnie. 2000. Toxin-induced vasogenic cerebral oedema in a rat model. Acta Neurochir. Suppl. 76:231-236.[Medline]
5 - Inoue, K., Y. Fujinaga, T. Watanabe, T. Ohyama, K. Takeshi, K. Moriishi, H. Nakajima, K. Inoue, and K. Oguma. 1996. Molecular composition of Clostridium botulinum type A progenitor toxins. Infect. Immun. 64:1589-1594.[Abstract]
6 - Katayama, S.-I., O. Matsushita, J. Minami, S. Mizobuchi, and A. Okabe. 1993. Comparison of the alpha-toxin genes of Clostridium perfringens type A and C strains: evidence for extragenic regulation of transcription. Infect. Immun. 61:457-463.[Abstract/Free Full Text]
7 - Minami, J., S. Katayama, O. Matsushita, C. Matsushita, and A. Okabe. 1997. Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N- and C-terminal peptides. Microbiol. Immunol. 41:527-535.[Medline]
8 - Miyamoto, O., J. Minami, T. Toyoshima, T. Nakamura, T. Masada, S. Nagao, T. Negi, T. Itano, and A. Okabe. 1998. Neurotoxicity of Clostridium perfringens epsilon-toxin for the rat hippocampus via the glutamatergic system. Infect. Immun. 66:2501-2508.[Abstract/Free Full Text]
9 - Miyamoto, O., K. Sumitani, T. Nakamura, S.-I. Yamagami, S. Miyata, T. Itano, T. Negi, and A. Okabe. 2000. Clostridium perfringens epsilon-toxin causes excessive release of glutamate in the mouse hippocampus. FEMS Microbiol. Lett. 189:109-113.[CrossRef][Medline]
10 - Miyata, S., O. Matsushita, J. Minami, S. Katayama, S. Shimamoto, and A. Okabe. 2001. Cleavage of a C-terminal peptide is essential for heptamerization of Clostridium perfringens epsilon-toxin in the synaptosomal membrane. J. Biol. Chem. 276:13778-13783.[Abstract/Free Full Text]
11 - Miyata, S., J. Minami, E. Tamai, O. Matsushita, S. Shimamoto, and A. Okabe. 2002. Clostridium perfringens epsilon-toxin forms a heptameric pore within the detergent-insoluble microdomains of MDCK cells and rat synaptosomes. J. Biol. Chem. 277:39463-39468.[Abstract/Free Full Text]
12 - Nagahama, M., S. Ochi, and J. Sakurai. 1998. Assembly of Clostridium perfringens epsilon-toxin on MDCK cell membrane. J. Nat. Toxins 7:291-302.[Medline]
13 - Nagahama, M., and J. Sakurai. 1991. Distribution of labeled Clostridium perfringens epsilon toxin in mice. Toxicon 29:211-217.[Medline]
14 - Pak, J., Y. Pu, Z. T. Zhang, D. L. Hasty, and X. R. Wu. 2001. Tamm-Horsfall protein binds to type 1 fimbriated Escherichia coli and prevents E. coli from binding to uroplakin Ia and Ib receptors. J. Biol. Chem. 276:9924-9930.[Abstract/Free Full Text]
15 - Parkkinen, J., R. Virkola, and T. K. Korhonen. 1988. Identification of factors in human urine that inhibit the binding of Escherichia coli adhesins. Infect. Immun. 56:2623-2630.[Abstract/Free Full Text]
16 - Pavelka, M., and A. Ellinger. 1989. Pre-embedding labeling techniques applicable to intracellular binding site, p. 199-214. In H. Plattner (ed.), Electron microscopy of subcellular dynamics. CRC Press, Inc., Boca Raton, Fla.
17 - Payne, D., and E. Oyston. 1997. The Clostridium perfringens
-toxin, p. 439-447. In J. I. Rood, B. A. McClane, J. G. Songer, and R. W. Titball (ed.), The clostridia: molecular biology and pathogenesis. Academic Press, London, United Kingdom.
18 - Petit, L., M. Gibert, D. Gillet, C. Laurent-Winter, P. Boquet, and M. R. Popoff. 1997. Clostridium perfringens epsilon-toxin acts on MDCK cells by forming a large membrane complex. J. Bacteriol. 179:6480-6487.[Abstract/Free Full Text]
19 - Petit, L., E. Maier, M. Gibert, M. R. Popoff, and R. Benz. 2001. Clostridium perfringens epsilon toxin induces a rapid change of cell membrane permeability to ions and forms channels in artificial lipid bilayers. J. Biol. Chem. 276:15736-15740.[Abstract/Free Full Text]
20 - Reinhart, H. H., N. Obedeanu, and J. D. Sobel. 1990. Quantitation of Tamm-Horsfall protein binding to uropathogenic Escherichia coli and lectins. J. Infect. Dis. 162:1335-1340.[Medline]
21 - Shortt, S. J., R. W. Titball, and C. D. Lindsay. 2000. An assessment of the in vitro toxicology of Clostridium perfringens type D epsilon-toxin in human and animal cells. Hum. Exp. Toxicol. 19:108-116.[Abstract/Free Full Text]
22 - Songer, J. G. 1996. Clostridial enteric diseases of domestic animals. Clin. Microbiol. Rev. 9:216-234.[Medline]
23 - Tsutsui, K., J. Minami, O. Matsushita, S. Katayama, Y. Taniguchi, S. Nakamura, M. Nishioka, and A. Okabe. 1995. Phylogenetic analysis of phospholipase C genes from Clostridium perfringens types A to E and Clostridium novyi. J. Bacteriol. 177:7164-7170.[Abstract/Free Full Text]
24 - Uzal, F. A., B. E. Rolfe, N. J. Smith, A. C. Thomas, and W. R. Kelly. 1999. Resistance of ovine, caprine and bovine endothelial cells to Clostridium perfringens type D epsilon toxin in vitro. Vet. Res. Commun. 23:275-284.[CrossRef][Medline]
Infection and Immunity, September 2003, p. 5371-5375, Vol. 71, No. 9
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.9.5371-5375.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
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