Effect of Epsilon ToxinGFP on MDCK Cells and Renal Tubules In Vivo
Departament de Biologia Cel·lular i Anatomia Patològica, Campus de Bellvitge, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain (AS-J,JB,IGdA,PN,MM-S), and CNR Anaérobies, Institut Pasteur, Paris, France (MG,MRP)
Correspondence to: Dr. Mireia Martín-Satué, Dpt. Biologia Cel·lular i Anatomia Patològica, Lab 4145, 4a Planta, Pavelló de Govern, Facultat de Medicina Campus de Bellvitge, C/Feixa Llarga s/n, E-08907 L'Hospitalet de Llobregat, Barcelona, Spain. E-mail: martin{at}medicina.ub.es
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Summary |
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Key Words: clostridial toxins epsilon toxin MDCK cells renal tubules pulpy kidney disease
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Introduction |
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C. perfringens can be found in the intestines of most animals. Bacterial numbers remain small because of peristalsis, and clinical disease does not occur unless the microbial balance in the gut is disrupted. Therefore, small amounts of -toxin in the gut of healthy animals are considered innocuous. However, when the intestine is altered by sudden changes in diet or other factors, bacteria proliferate rapidly and produce large amounts of this toxin. Intestinal mucosal permeability is increased by
-toxin, thereby facilitating its absorption into the circulation (reviewed by Finnie 2004
). Disease is principally manifested as severe and often fatal neurological disturbance; edema of lungs, heart, kidneys, and intestine is also a clinical sign related to microvascular damage (Songer 1996
). This pathology is also known as pulpy kidney and overeating disease.
Toxin is encoded by the etx gene (Hunter et al. 1992
) and is synthesized and secreted as an inactive prototoxin of 311 amino acids (32.7 kD) that is converted to the fully active toxin by proteolytic removal of a basic N-terminal peptide of 14 residues and of 23 C-terminal residues (Bhown and Habeerb 1977
; Minami et al. 1997
). This proteolytic activation usually occurs in the gut of infected animals by the actions of trypsin and chymotrypsin, but it can also be achieved in vitro by controlled enzyme digestion.
The MadinDarby canine kidney (MDCK) cell line, of epithelial origin from the distal convoluted tubule, is susceptible to -toxin (Payne et al. 1994
). The cytotoxic effects are very rapid and are enhanced by EDTA (Lindsay 1996
). Cells undergo both cell cycle alterations (Borrmann et al. 2001
) and morphological changes, including swelling and large bleb formation, in a process that is independent of the actin cytoskeleton and endocytosis (Petit et al. 1997
; Borrmann et al. 2001
). The intoxication process correlates with the formation of a membrane complex of
155 kD and the efflux of intracellular K+ and the influx of Na+, Cl, and Ca2+ without entry of the toxin into the cytosol (Petit et al. 1997
,2001
; Borrmann et al. 2001
). The toxin decreases the trans-epithelial resistance of polarized MDCK cells without affecting intercellular junctions (Petit et al. 2003
). The membrane complex is also formed in synaptosomes and corresponds to toxin heptamerization (Miyata et al. 2001
). In artificial lipid membranes,
-toxin induces large pore formation, which appears to constitute its main cytotoxic mechanism (Petit et al. 2001
). Furthermore, heptameric pore formation has recently been described within the detergent-insoluble microdomains of MDCK cells and rat synaptosomes (Miyata et al. 2002
).
Animal models have also been used to study -toxin effects. Vasogenic cerebral edema has been described in mice (Gardner 1973
; Morgan et al. 1975
; Finnie 1984
), rats (Finnie et al. 1999
; Ghabriel et al. 2000
; Zhu et al. 2001
), and calves (Uzal et al. 2002
) injected with the toxin, and pulmonary and cardiovascular lesions have also been described elsewhere (Sakurai et al. 1983
; Uzal et al. 2002
). Specific receptor sites have been identified in the cerebral microvasculature (Buxton 1978
; Nagahama and Sakurai 1991
) as well as disruption of the bloodbrain barrier, which causes a rapid and substantial increase in vascular permeability and leads to severe, diffuse vasogenic edema (reviewed by Finnie 2004
). Moreover, morphological kidney alterations were described on infected domestic animals (pulpy kidney) as well as in IV-injected mice (Buxton 1978
; Tamai et al. 2003
).
Here we used recombinant techniques to develop green fluorescence protein-fused -toxin and
-prototoxin that could be directly visualized by fluorescence microscopy. We demonstrate that both proteins bind to the MDCK cell membrane and that only the toxin is cytotoxic, thus retaining the binding properties and cytotoxicity of the non-fluorescent proteins. We analyzed the organ distribution, tissue localization, and histopathology of these proteins in IV-injected mice. Because the kidneys are, in addition to brain, critical target organs for
-toxin intoxication (pulpy kidney disease), we further analyzed these organs by ligandreceptor binding assays on cryostat slices.
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Materials and Methods |
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To improve the efficiency of protein purification, these constructs were used to reamplify and clone -toxinGFP and
-prototoxinGFP into the pGEX-4T-1 expression plasmid (Amersham Biosciences; Freiburg, Germany) by another PCR with new primers.
-ToxinGFP and
-prototoxinGFP were cloned using EcoRI and NotI restriction enzymes. Thus, two new fusion proteins between glutathione-S-transferase (GST) and amplified products were obtained: GST
-toxinGFP and GST
prototoxin-GFP. These plasmids were transformed in Rosetta E. coli host strain (Novagen; Madison, WI).
Protein Expression and Purification
The expression of recombinant proteins was induced overnight at room temperature (RT) in 250 ml LB medium cultures with 0.4 mM isopropyl ß-D-thiogalactopyranoside (IPTG).
Cells were pelleted and resuspended in ice cold buffer containing PBS, 1% Triton X-100, 0.1 mg/ml phenylmethylsulfonylfluoride (PMSF), 10 µg/ml aprotinin, and 10 µg/ml leupeptin, and were sonicated and centrifuged at 15,000 x g for 20 min. The resultant supernatant was incubated with 0.5 ml of previously PBS-equilibrated glutathione Sepharose 4B (Amersham Pharmacia Biotech) beads for 1 hr at 4C. Finally, recombinant proteins were eluted by thrombin elution in 10 mM Tris-HCl, pH 8.0, with 150 mM NaCl and 2.5 mM CaCl2, according to the manufacturer's instructions. We routinely performed a further purification step and isolated -prototoxinGFP by anion exchange chromatography.
For MTT assay, -prototoxinGFP was activated by trypsin proteolysis with trypsin beads (SigmaAldrich; Madrid, Spain) according to the manufacturer's instructions.
Native -toxin was purified by ion exchange chromatography as previously described (Petit et al. 1997
).
Protein Analyses
Samples of -toxinGFP and
-prototoxinGFP were loaded in a 12% acrylamidebisacrylamide gel. After SDS-PAGE, the proteins were transferred onto nitrocellulose membranes (Bio-Rad; Hercules, CA) with a semi-dry unit (SigmaAldrich). After blocking using 5% non-fat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween-20, membranes were incubated with 1:1000 concentration of anti-
-toxin rabbit polyclonal antibody, prepared as previously described (Popoff 1987
), for 1 hr at RT. After three washes in the same buffer, the membrane was incubated with anti-rabbit immunoglobulins conjugated to horseradish peroxidase (DAKO; Glostrup, Denmark). Finally, after five washes the blot was developed using the enhanced chemiluminescence method (ECL).
Cell Cultures
MDCK (MadinDarby canine kidney) cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing glucose (4.5 mg/ml), sodium pyruvate (110 mg/liter), 2 mM glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and supplemented with 10% fetal bovine serum (FBS), and were maintained at 37C in a humidified atmosphere containing 5% CO2.
MTT Cell Death Assay
The assay is based on the capacity of living cells to reduce yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) and thus generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan can be solubilized and quantified by spectrophotometry and is proportional to the number of viable cells.
MDCK cells were grown to 80% confluence. Treated cells were incubated for 30 min at 37C with 5 nM, 50 nM, 100 nM, or 500 nM native -toxin,
-toxinGFP, trypsin-activated
-prototoxinGFP, or
-prototoxinGFP in PBS. Control cells were incubated with PBS. Thereafter, 0.5 mg/ml MTT (SigmaAldrich) was added and maintained for 30 min at 37C. The reaction was stopped by adding an equal volume of isopropanol:1 M HCl (24:1) and the cells were then lysed. Formazan was measured at 550 nm in a 96-microtiter plate. The experiment was done four times for each treatment, and means and standard errors were obtained and compared statistically by Student's t-test.
Binding Assays to Living MDCK Cells
Cells were grown to confluence on 24-well plates. Growing medium was replaced by a buffer containing 5 mM KCl, 140 mM NaCl, 5 mM NaHCO3, 1 mM MgCl2, 1.2 mM Na2PO4, 10 mM glucose, and 20 mM HEPES/NaOH, pH 7.4. Cells were maintained in this buffer for 1 hr, then incubated with 200 nM -prototoxinGFP or
-toxinGFP in the same buffer for 1 hr at 4C and then brought to 37C. After the appropriate periods of time (0, 10, 30, and 60 min), cells were washed three times in PBS, fixed with 4% paraformaldehyde for 20 min, washed twice in PBS, and examined under a LEICA DMIRB/E inverted fluorescence microscope.
Binding Assays to Fixed MDCK Cells
Cells were grown on coverslips to confluence, washed three times in PBS, and fixed with 4% paraformaldehyde. Nonspecific binding was blocked by incubating the cells in PBS containing 0.2% gelatin and 10% FBS for 1 hr. Incubations with 200 nM -toxinGFP or
-prototoxinGFP were performed in the same buffer for 1 hr at 4C or RT. After three washes in PBS, cells were fixed again with 4% paraformaldehyde for 10 min, then washed twice in PBS and mounted on slides using Immuno Floure Mounting Medium (ICN Biomedicals; Costa Mesa, CA), followed by examination under a Leica TCS 4D confocal microscope (Serveis Científico-Tècnics; Universitat de Barcelona, Barcelona, Spain).
To demonstrate the specificity of binding, competition experiments were carried out by co-incubating the cells with native -toxin and
-prototoxinGFP or
-toxinGFP in a 20:1 molar ratio. For detergent experiments, blocking was done at 4C in the presence of 0.2% Triton X-100. The following steps were performed at RT in the absence of detergent.
Electron Microscopy
MDCK cells were grown on Thermanox to 100% confluence, washed three times in PBS, and fixed with 2% paraformaldehyde. Nonspecific binding was blocked by incubating the cells in a buffer containing PBS, 0.2% gelatin, and 10% FBS for 45 min. Incubations with 200 nM -prototoxinGFP were performed in the same buffer for 1 hr at RT. After three washes, another blocking step was done with PBS containing 1% BSA for 20 min. Next, cells were incubated with a 1:100 dilution of anti-
-toxin polyclonal antibody in the same buffer for 1 hr. After three washes, the secondary antibody incubation was performed with 1:25 anti-rabbit immunoglobulins conjugated with 10-nm gold beads in the same buffer for 45 min. After three washes, cells were fixed in 2% paraformaldehyde, 2.5% glutaraldehyde in 100 mM PBS for 2 hr at RT. Primary fixation was followed by postfixation with 1.0% OsO4 in 100 mM PBS for 1 hr. The samples were dehydrated in acetone and embedded in Spurr resin. After sectioning with a ReichertJung Ultracut E ultramicrotome, ultrathin sections were stained with uranyl acetate and lead citrate and examined under a Hitachi H-600 AB transmission electron microscope (Serveis Científico-Tècnics; Universitat de Barcelona, Barcelona, Spain).
In Vivo Studies in a Murine Model
Male OF1 Swiss mice weighing 20 g were anesthetized with sodium pentothal (35 mg/kg). -ToxinGFP and
-prototoxinGFP were prepared in PBS with 1% BSA and injected IV. Tested doses of
-toxinGFP were 7 ng/g, 14 ng/g, 70 ng/g, 750 ng/g, and 2500 ng/g per mouse. Animals injected with
-prototoxinGFP and GFP alone (2.5 µg/g each) were sacrificed after 15 min. Organs (kidneys, brain, heart, lung, liver, spleen, testes, gut) were extracted and fixed by immersion in 4% paraformaldehyde for 12 hr. One half of each organ was embedded in paraffin, cut, and stained with hematoxylin and eosin (HE). The other half was immersed in 30% sucrose, frozen in isobutanol, cut in a cryostat to 6-µm slices, immediately mounted on slides using Immuno Floure Mounting Medium, and observed under a Leica TCS 4D confocal microscope (Serveis Científico-Tècnics).
Control experiments were also performed by injecting 1.65 µg/g native -prototoxin simultaneously with 2.5 µg/g
-prototoxinGFP (10:1 molar ratio). Organs were processed as described above.
Experiments were performed in accordance with the European Communities Council Directive of November 1986 (86/609/EEC) and were approved by the Institutional Review Board at the University of Barcelona.
-ToxinGFP and
-PrototoxinGFP Incubations on Kidney Slices
Kidneys from OF1 Swiss mice were processed to obtain cryostat slices as described above. Nonspecific binding was blocked by incubating the slices in PBS containing 0.2% gelatin and 10% FBS for 1 hr. The incubation with -toxinGFP or
-prototoxinGFP was performed in the same buffer at a 200 nM concentration for 1 hr at RT. After three PBS washes, the samples were fixed in 4% paraformaldehyde for 10 min, mounted with Immuno Floure Mounting Medium, and examined under a Nikon Eclipse E800 fluorescence microscope.
In some experiments, blocking steps were performed in the presence of 0.2% Triton X-100.
For competition experiments, slices were co-incubated with native -toxin and
-toxinGFP or
-prototoxinGFP in a 20:1 molar ratio.
The same incubation experiments were done with kidneys from rat, sheep, cow, and human. Sheep and cow samples were obtained from Mercabarna slaughterhouse in Barcelona. Slices from human healthy kidney were provided by the Servei d'Anatomia Patològica from Hospital Princeps d'Espanya, (L'Hospitalet de Llobregat, Spain). Samples were fixed by immersion in 4% paraformaldehyde for 12 hr and processed as described for mice to obtain cryostat slices and perform overlay experiments.
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Results |
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Binding of -ToxinGFP and
-PrototoxinGFP to Living MDCK Cells
To characterize the cell binding of GFP fusion proteins on living cells, we performed time-course experiments on MDCK cells. After incubation for 1 hr at 4C with -toxinGFP or
-prototoxinGFP and without previous washing, cells were brought to 37C and maintained at that temperature for various time periods (0 to 60 min) before washing and fixation with paraformaldehyde (Figure 2) . Figure 2A shows results for
-toxinGFP and Figure 2B for
-prototoxinGFP.
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On the other hand, cells incubated with -toxinGFP underwent the morphological alterations described for MDCK cells treated with
-toxin (Hambrook et al. 1995
; Petit et al. 1997
; Borrmann et al. 2001
): nuclear condensation, vacuolation, and progressive swelling, leading to cell death (Figures 2A2, 2A4, 2A6, and 2A8). Dead cells finally detach from the cell culture substrate (Figure 2A8). Fluorescence images revealed time-dependent binding as for
-prototoxinGFP (Figures 2A1, 2A3, 2A5, and 2A7).
Moreover, no cytotoxic effect was detected when cells treated with -toxinGFP were washed after the 4C incubation period and then brought to 37C, confirming the absence of binding at 4C (data not shown).
Binding of -ToxinGFP and
-PrototoxinGFP to Paraformaldehyde-fixed MDCK Cells
To further characterize the binding ability of GFP fusion proteins, we performed binding assays with -toxinGFP or
-prototoxinGFP on paraformaldehyde-fixed MDCK cells. Both proteins bound to the MDCK cell membrane, as revealed by direct fluorescence detection on confocal microscopy (Figure 3A
for
-prototoxinGFP and Figure 3B for
-toxinGFP).
-Prototoxin immunodetection by electron microscopy showed the binding to the external face of plasma membrane, in particular, to the apical side of polarized cells (a detail of this area is shown in Figure 3I). Specific binding was demonstrated by competition experiments in which fluorescence labeling was abolished when non-labeled
-toxin was co-incubated with GFP fusion proteins (20:1 molar ratio) (Figures 3C and 3D). Moreover, no fluorescence was detected when incubations were performed with GFP alone (data not shown).
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The presence of 0.2% Triton X-100 in the blocking step diminished fluorescence and changed the uniform membrane labeling to a discontinuous spotted pattern (Figures 3G and 3H).
In Vivo Studies in a Murine Model
To determine in vivo cytotoxic effects and organ distribution of -toxinGFP and
-prototoxinGFP, we injected mice IV with these recombinant proteins.
Mice injected with -toxinGFP died after various periods of time in a dose-dependent manner (from 4 min for 2500 ng/g to 134 min for 7 ng/g). Animals injected with either
-prototoxinGFP or GFP alone were sacrificed after 15 min.
After postmortem observation, organs were processed for both histological studies and fluorescence distribution. HE examination of toxin-injected kidneys revealed more congested and hemorrhagic medullae (Figure 4B)
than in animals injected with either -prototoxinGFP or GFP alone (Figures 4E and 4H). Toxin-injected animals also showed severe alterations on renal distal tubules, including pyknotic nuclei and desquamated epithelia (Figure 4A). These alterations were observed in all animals injected with toxin but were more pronounced in those that received the highest doses. Morphological alterations were not found in animals injected with either
-prototoxinGFP or GFP alone (Figures 4D and 4G). We did not find any significant morphological alterations in other organs analyzed, although an edematogenous process, especially in brain, is well documented after
-toxin intoxication (reviewed by Finnie 2004
). We sporadically detected perivascular edema in cerebral microvasculature (data not shown), although the routine histopathological techniques used here are not the most appropriate to visualize such changes.
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Moreover, immunohistochemistry with anti--toxin and anti-GFP antibodies revealed complete co-localization of both molecules (data not shown).
Fluorescence also accumulated at the luminal surface of proximal tubules but not at the distal tubules in GFP-injected animals (Figure 4I), nor was fluorescence detected on vascular endothelia in these control animals (data not shown).
In summary, the three recombinant proteins crossed the glomerular barrier and bound to the proximal tubules but only -prototoxinGFP and
-toxinGFP bound to distal tubules and only
-toxinGFP specifically degenerated them. Finally, both
-prototoxinGFP and
-toxinGFP, but not GFP alone, bound to endothelial cells.
To show the specificity of toxin binding, displacement experiments were performed in which excess amounts of native -prototoxin were injected simultaneously with
-prototoxinGFP. Fluorescence label in cerebral endothelia diminished drastically (Figures 5C and 5D) and was abolished in distal renal tubules (Figures 5E and 5F), confirming specificity of binding in both structures. Vascular endothelia from other tissues were also displaced but to a lesser extent, which probably indicates that this binding has a nonspecific component (not shown). Interestingly, under these experimental conditions we detected increased label in the proximal tubules (Figure 5F), revealing that
-prototoxinGFP was displaced from its specific binding sites and accumulated unspecifically.
Incubation Experiments of -ToxinGFP and
-PrototoxinGFP on Cryostat Kidney Slices
We developed another approach, already used in ligandreceptor interaction studies, to analyze the specificity of -toxinGFP and
-prototoxinGFP binding on kidneys, consisting of incubations of cryostat kidney slices of mice with these recombinant proteins. Fluorescence images revealed that both
-toxinGFP and
-prototoxinGFP bind to the luminal surface of distal tubule cells (Figure 6A
for prototoxin; not shown for toxin). The specificity of this interaction was demonstrated by co-incubation with native
-toxin, which completely abolished fluorescence (Figure 6B).
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We obtained similar results in incubation experiments with slices from rat (Figure 6D), sheep (Figure 6E), cow (Figure 6F), and human kidneys (Figure 6G). Specificity of binding was also demonstrated in these species (Figure 6H for human).
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Discussion |
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Among the cell lines initially examined in search of the lethal effects of -toxinGFP, only MDCK was sensitive, in agreement with previous reports indicating that these cells are the most appropriate cell model in which to study
-toxin toxicity (Lindsay 1996
; Petit et al. 1997
; Miyata et al. 2002
). More recently, Shortt et al. (2000)
identified another cell line (G-402), of human kidney origin, susceptible to effects of
-toxin exposure. It is relevant because of the effect of the toxin on the kidneys in vivo.
Here we show that -toxinGFP fusion protein retains its full toxic activity on MDCK cells, and no differences from the native
-toxin were detected. MTT assays revealed a maximum of
70% cell death, which was not affected by increasing the dose or the incubation period, consistent with a previously reported subpopulation of resistant cells in MDCK cultures (Shortt et al. 2000
). Furthermore, both the time course and morphological changes on incubation with GFP recombinant
-toxin were similar to those also described here for native
-toxin. It has been described that the lethality of
-toxin in MDCK cells is drastically reduced by lowering temperature (Lindsay 1996
; Petit et al. 2003
). We show here that incubation at 4C prevents
-prototoxinGFP and
-toxinGFP binding and, in consequence, cytotoxicity, even at very long incubation periods.
Furthermore, co-incubation of -prototoxinGFP in excess with
-toxinGFP prevented cell death in MDCK cells, thus revealing that both proteins compete for the same binding sites but that only
-toxinGFP kills the cells (data not shown). Direct fluorescence studies confirmed that both
-toxinGFP and
-prototoxinGFP have similar binding properties to paraformaldehyde-fixed MDCK cells. The labeling showed a peripheral cell pattern, as reported elsewhere using indirect immunofluorescence (Petit et al. 1997
). Moreover, according to our electron microscopy observations, most of the toxin was found in the apical cell membrane of polarized MDCK cells, where binding has a stronger cytotoxic effect (Petit et al. 1997
).
Interestingly, pretreatment of fixed cells with detergent at 4C clearly changed the fluorescence distribution on the cell membrane to a punctate pattern. These observations represent the visual evidence of detergent-resistant membrane domains (DRMs) recently described on MDCK cells in association with toxin heptamerization (Miyata et al. 2002).
At this point we confirm that -prototoxinGFP is a convenient tool for studies of toxin binding mechanisms, since it binds to the same sites as active toxin but has a less damaging effect.
We injected mice IV with the above reported recombinant GFP proteins and analyzed their organ distribution and histopathology. Macroscopically, we found that kidneys of mice injected with -toxinGFP were more hematic, especially in the medullary area, than those injected with
-prototoxinGFP or GFP alone. Coincident with these morphological observations, histological examination revealed hemorrhagic medullae. Moreover, we show images of selective degeneration found on distal tubules, mainly located in the cortical area, pyknotic nuclei and disorganized epithelia being the main observations. These results may help to understand the soft, friable, and very congested kidneys of infected livestock (pulpy kidney disease). On the other hand, no histopathological alterations were observed in other organs at the
-toxin doses administered here.
Direct fluorescence images revealed that fluorescence accumulated on two structures: vascular endothelia and renal distal tubules. Several organs of animals injected with either -prototoxinGFP or
-toxinGFP showed fluorescence on the luminal surface of blood vessel endothelia, coinciding with the increased vascular permeability associated with this pathology and the edematogenous renal medullae described here. Vascular binding was, however, detected mainly in brain, where receptor sites for the toxin have been already described (Buxton 1978
; Nagahama and Sakurai 1991
). Experiments in which non-labeled
-prototoxin was injected in excess and simultaneously with
-prototoxinGFP further confirmed specificity of toxin binding to brain vascular endothelia because fluorescence label drastically decreased under these experimental conditions.
On kidneys, fluorescence was mainly associated with the luminal surface of proximal tubules of all the mice analyzed, including those injected with -toxinGFP,
-prototoxinGFP, or GFP alone, which indicates that these proteins cross the glomerular filter barrier and accumulate, somehow nonspecifically, on the luminal surface of these tubules. Fluorescence was detected to a lesser extent on the distal tubules of
-toxinGFP- and
-prototoxinGFP-injected mice, but never in the case of GFP alone. The control experiments confirmed our hypothesis about the coexistence of two binding components in renal tubules, one related to binding to proximal tubules, which was not specific and therefore non-displaceable with non-labeled
-prototoxin, and a specific component related to distal tubules, which was displaceable. These findings elucidate a previous report (Nagahama and Sakurai 1991
) in which radioactive
-prototoxin was accumulated mainly in kidneys of injected mice but only a small fraction of radioactivity was displaced by
-prototoxin.
To complete this study of -toxin pathology on kidneys, we incubated cryostat-obtained mice kidney slices with recombinant
-prototoxinGFP,
-toxinGFP, or GFP alone as control. Both forms of toxin, but not GFP alone, bound to distal tubules in a displaceable way, reinforcing our hypothesis about binding specificity. Furthermore, experiments in which samples were pretreated with 0.2% Triton X-100 revealed the presence of an
-toxin receptor mainly associated with detergent-sensitive areas. Further experiments are under way to identify this receptor.
Because pulpy kidney disease mainly affects livestock, we also performed the incubation experiments on cryoslices of sheep and cows and obtained the same distribution pattern as that of mice and rats.
Interestingly, incubations performed using human kidney cryoslices further confirm this distribution and could explain the sensitivity to -toxin of a cell line of human kidney origin recently described (Shortt et al. 2000
). This is the first evidence that
-toxin binds specifically to distal tubules of kidneys of different species, including those such as sheep and cows, in which pulpy kidney disease can be fatal, or those such as humans, in which
-toxin-associated pathology has not been reported.
In conclusion, we have generated useful recombinant toxins to study by direct fluorescence the effects and distribution of -toxin. We have shown for the first time the specific binding on distal renal tubules of different species. In addition, we hypothesize that although several animals including mice, rats, and also humans could be sensitive to the toxin, intoxication occurs only in animals in which Clostridia overgrow, which leads to an excess of toxin production.
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Acknowledgments |
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We are grateful to Serveis Científic Tècnics of the Universitat de Barcelona for confocal and electron microscopy assistance and to the Mercabarna slaughterhouse for providing biological samples. We also thank Prof Dr Marta Carreras (Anatomia Patològica, Hospital Princeps d'Espanya, Bellvitge, Barcelona) for providing human samples. We thank Robin Rycroft for careful reading of the manuscript. A.S.-J. is a recipient of a predoctoral fellowship from Instituto de Salud Carlos III (BEFI N/ref: CPC/CLC).
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Footnotes |
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Literature Cited |
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