Experimental Evidence for a Direct Cytotoxicity of Loxosceles intermedia (Brown Spider) Venom in Renal Tissue
Department of Cell Biology, Federal University of Paraná (MNL,PHdS,OMC,CRCF,WG,SSV); Health Area, Campos de Andrade University (VLPdS); Department of Medical Pathology, Federal University of Paraná (MFSS); Department of Basic Pathology, Federal University of Paraná (SMZ); and Department of Physiology, Federal University of Paraná (OCM), Curitiba, Paraná, Brazil
Correspondence to: Silvio Sanches Veiga, Dept. of Cell Biology, Federal University of Parana, Jardim das Américas, 81531-990 Curitiba, Parana, Brazil. E-mail: veigass{at}ufpr.br
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Summary |
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Key Words: brown spider venom toxins nephrotoxicity
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Introduction |
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A number of enzymes and biologically active molecules that might contribute to the deleterious effects of the venom have been identified and biochemically characterized. A sphingomyelinase D of 3235 kD isolated from brown spider venom can induce dermonecrosis, platelet aggregation, and experimental hemolysis (Futrell 1992). Metalloproteases of 3235 kD and 2028 kD identified in the venom with gelatinolytic, fibronectinolytic, and fibrinogenolytic activities can also play a role in hemorrhage evoked by envenomation, such as hemorrhage into the dermis, injury of blood vessels, imperfect platelet adhesion, and the defective wound healing observed in some cases (Feitosa et al. 1998
; Veiga et al. 2001a
,b
; da Silveira et al. 2002
; Zanetti et al. 2002
). A hyaluronidase with electrophoretic mobilities at regions of 33 kD and 63 kD is likely to contribute to the gravitational spread of dermonecrotic lesions, a hallmark of brown spider bites (Wright et al. 1973
; Futrell 1992
). A number of other molecules and activities have been identified in the venom, including a lipase, alkaline phosphatase, and proteolytic activities on entactin, basement membranes, and the protein core of a heparan sulfate proteoglycan from endothelial cells (Futrell 1992
; Veiga et al. 2000
,2001b
). The mechanism underlying the involvement of these individual venom constituents or activities in the noxious effects of the venom on cell tissue has not been fully determined.
Some reports have indicated the participation of blood cells and molecular components in the noxious effects of the venom. The serum amyloid P component appears to be a target for platelet activation and ischemic effects and is likely to play a role in the necrosis caused by the venom (Gates and Rees 1990). Leukocytes, and especially polymorphonuclear cells (PMNs) such as neutrophils and eosinophils, appear to play a role in the dermonecrotic lesion evoked by the venom, because histopathological findings have revealed a massive infiltration of these cells into the dermis and related structures in the dermonecrotic regions induced by the venom (Elston et al. 2000
; Ospedal et al. 2002
). Depletion of leukocytes in the blood results in reduction of clinical signs in the skin injected with venom (Smith and Micks 1970
). The complement system in the plasma also appears to participate in the deleterious activities of the venom, especially on erythrocytes, evoking a complement-dependent hemolysis (Futrell 1992
).
Although some clinical signs of loxoscelism have been well described, and putative molecules in the venom and physiopathological events involved in cell destruction have been characterized as described above, data about renal disorders evoked by brown spiders have been limited to earlier reports describing clinical data from victims (Futrell 1992; Lung and Mallory 2000
). We report here the effect of L. intermedia venom on kidney structures. Mice were used because these animals do not develop dermonecrotic lesions induced by Loxosceles venom, so that the occurrence of nephrotoxicity secondary to complications of dermonecrotic lesions can be ruled out. We hope to bring some insight into loxoscelism that could be useful to physicians who diagnose and treat the victims.
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Materials and Methods |
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Spider Venom Extraction
The venom was extracted from spiders captured from the wild and kept for a week without any food but with water ad libitum. The venom was extracted from the fangs of spiders by electrostimulation (15 V) applied to the cephalothorax and was collected with a micropipette, dried under vacuum, and frozen at -85C until use. Pools of venom collected from 100 to 200 spiders in different batches were used during all the experiments, involving approximately 1000 spiders (Feitosa et al. 1998). Protein content was determined by the Coomassie Blue method (Bradford 1976
).
Animals
Adult Swiss mice weighing approximately 25 g from the Central Animal House of the Federal University of Paraná were used for in vivo experiments with the venom. All experimental protocols using animals were performed according to the "Principles of Laboratory Animal Care" (NIH Publication 85-23, revised 1985) and Brazilian federal laws.
Venom Administration
Pooled crude venom and mouse samples of 1 mg of protein/kg were diluted in PBS (pH 7.3). These samples were injected IP in a volume of 100 µl in each mouse. The animals were divided into two groups, a control (C) group and a test (T) group. The control group consisted of five animals receiving only PBS and the test group consisted of five animals receiving L. intermedia venom. During the experimental procedures, the envenomation of animals was repeated at least 10 times, completing a number of 50 animals as controls and 50 animals that received the venom. All animals were kept under the same experimental conditions. All kidney and blood samples were collected from living animals.
Kidney and Blood Sample Collections and Laboratory Analysis
Kidney and blood (directly from the heart) samples were obtained from mice anesthetized with ketamine (Agribands; Paulinia, SP, Brazil) and acepromazin (Univet; São Paulo, SP, Brazil). Blood was anticoagulated with EDTA-K3 and used for red cell, hemoglobin, hematocrit, leukocyte, and platelet counts that were determined with an automated CELL-DYN 1,400 blood counter (Abbott Laboratories; Chicago, IL). Urea and C3 complement were determined in serum. Assays were performed using standardized techniques and reagents as described by Kaplan and Pesce (1996) and Henry (2001)
.
Statistical Analysis
The Student's t-test for unpaired observations was used to detect statistically significant differences between control and test groups for red cell concentration (106 ml/blood), hemoglobin (g/dl blood), hematocrit (%), platelets (103 ml/blood), leukocytes (103 ml/blood), serum urea (mg/dl), and C3 complement (mg/dl). The threshold level for significance was P=0.05. All statistical calculations were done with the GraphPad InStat program version 3.00 for Windows 95. Morphometric analysis of sections stained with hematoxylin and eosin or from electronmicrographs were measured by use of software Jandel Sigma Scan Pro.
Gel Electrophoresis
Lysed renal cells were obtained by treatment of kidneys with lysis buffer (50 mM Tris-HCl, pH 7.3, 1% Triton X-100, 50 mM NaCl, 1 mM CaCl2,1 mM phenylmethanesulfonyl fluoride, and 2 µg/ml aprotinin) for 15 min at 4C. The extract was clarified by centrifugation for 10 min at 13,000 x g. Renal extracts or crude venom (normalized for their protein contents) were submitted to electrophoresis under non-reducing conditions. Linear gradient 320% or 818% SDS-PAGE was performed as described by Laemmli (1970). For protein detection, gels were stained with Coomassie Blue. For immunoblotting, proteins were transferred to nitrocellulose filters overnight as described by Towbin et al. (1979)
. The molecular mass markers used were from Sigma. A two-dimensional gel was run with some modifications as previously described by the manufacturers using Immobiline Dry Strip Gel, pH range 310 (Amersham Biosciences). Crude venom 100 µg collected in water was diluted in rehydration solution (6 M urea, 2 M thiourea, 2% w/v CHAPS, 1% IPG buffer, and a trace of bromophenol blue) and applied to an IEF strip. The second dimension was carried out using 818% linear gradient SDS-PAGE under non-reducing conditions. Gel was stained with the silver method as described by Heukeshoven and Dernick (1986)
.
Histological Methods for Light Microscopy
Kidneys were fixed in modified Carnoy's fixative (5% acetic acid instead of 10% as originally proposed) for 3 hr. After fixation, tissues were processed for histology, embedded in paraffin, and cut into 4-µm sections. The sections were stained with hematoxylin and eosin, acid-Schiff (PAS), and silver and by the method of Rosenfeld (Culling et al. 1985; Beautler et al. 1995
).
Transmission Electron Microscopy
Kidneys were fixed with modified Karnovsky's fixative (without calcium chloride and with glutaraldehyde 2.5%) (Karnovsky 1965) for 2 hr, washed in 0.1 M cacodylic acid buffer, pH 7.3, postfixed in 1% OsO4 in 0.1 M cacodylic acid buffer, pH 7.3, for 1 hr, dehydrated with ethanol and propylene oxide, embedded in Epon 812, contrasted with uranyl acetate and lead citrate, and examined with a JEOL-JEM 1200 EX II transmission electron microscope at an accelerating voltage of 80 kV (Peabody, MA).
Immunofluorescence and Fluorescence Cytochemistry
For immunofluorescence microscopy, kidney tissues were fixed with 2% formaldehyde in PBS for 30 min at 4C, incubated with 0.1 M glycine for 3 min, and blocked with PBS containing 1% BSA for 1 hr at room temperature (RT). Histological sections were incubated for 1hr with specific antibodies raised against laminin (0.33 µg/ml), type IV collagen (1:40), and venom toxins (2.0 µg/ml) as described above. The sections were washed three times with PBS, blocked with PBS containing 1% BSA for 30 min at RT, and incubated with fluorescein- or rhodamine-conjugated anti-IgG secondary antibodies (Chemicon) at RT for 40 min. After washing with PBS, samples were observed under a confocal fluorescence microscope (Confocal Radiance 2,100; BioRad, Hercules, CA) coupled to a NikonEclipse E800 with Plan-Apochromatic objectives (Sciences and Technologies Group Instruments Division; Melville, NY). For nuclear fluorescence cytochemistry, samples of renal tissue were incubated with DAPI (O.5 µg/mL diluted in PBS) for 5 min (Molecular Probes). The samples were washed and observed under a confocal microscope as above. For antigen competition assay, the immunofluorescence protocol was the same as described above except that the hyperimmune serum to venom toxins was incubated previously for 1 hr with 50 µg/ml of crude venom diluted in PBS. Then the mixture was incubated with renal biopsies identically as above.
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Results |
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Discussion |
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To obtain additional information about the extent of renal damage and the mechanisms of the disorders induced by L. intermedia venom, we presented here laboratory and biochemical data and morphological data from examinations of kidney structures from mice exposed to the venom. Histological studies showed that envenomation induced a complex pattern of nephrotoxicity. A general view of renal tissue revealed alterations at the level of glomerular and tubule structures, supporting the nephrotoxic activity of venom toxins. The glomerular damage becomes apparent by the presence of extravascular red blood cells around the glomerular capillaries in Bowman's space. A protein-rich exudate inside Bowman's space confirms some damage at the glomerular level, suggesting loss of vascular integrity. In addition, tubule injuries are supported by the presence of proteinaceous material in their lumen in addition to tubule edema, causing the tubules to be susceptible to ischemic damage caused by occlusion. Ultrastructural features of nephrotoxicity were further supported by glomerular and tubule disorders. The azotemia detected by the increase in serum urea strengthened this evidence.
The mechanism by which Loxosceles venom induces nephrotoxic lesions has remained elusive and is currently unknown. In contrast to cutaneous lesions evoked by the venom, in which leukocytes (neutrophils) play an essential role in pathogenesis (Smith and Micks 1970; Futrell 1992
; Patel et al. 1994
; Ospedal et al. 2002
), in the model studied here there was no leukocyte infiltration in the presence of the renal injuries, which were not associated with inflammatory disease.
On the other hand, several case reports have described intravascular hemolysis associated with brown spider envenomation (Murray and Seger 1994; Williams et al. 1995
), and many studies have indicated a direct hemolytic activity of Loxosceles venom on erythrocytes (Futrell 1992
). Such hematological disturbances could be related to renal injuries as secondary agents (Futrell 1992
; Williams et al. 1995
; Lung and Mallory 2000
). We found that, in the model and under the conditions used, despite the renal injuries caused by the venom there was neither a direct hemolytic effect in vivo nor hemoglobin deposition in the kidney structures. These results agree with those described by Futrell (1992)
, who pointed out the susceptibility of some animal species to venom-induced hemolysis. Human and swine erythrocytes are more susceptible to hemolysis than rabbit and guinea pig erythrocytes. We cannot rule out the involvement of venom hemolysis in the human renal failure evoked by the venom. Nevertheless, because the venom did not induce dermonecrosis (data not shown) or hemolysis in mice and was extremely active against renal integrity, we can speculate about a direct and primary activity of the venom on kidney structures.
We confirmed this possibility by confocal immunofluorescence microscopy using antibodies to venom toxins. We were able to detect toxins as "planted antigens" deposited along the kidney structures of animals exposed to the venom. A competition assay using crude venom toxins in solution blocked the immunofluorescence positivity in the kidney of venom-treated animals, further supporting this evidence and the idea of "planted toxins." The present results agree with several reports indicating the binding of exogenous molecules, such as bacterial products, viral antigens, and drugs, to intrinsic components of renal structures as etiological agents of renal injuries (Kerjaschki and Neale 1996; Cotran et al. 1999
). In the model used here, we did not find a role for immune mechanisms in renal injuries. Because the biopsies were collected just 4 hr after exposure to the venom, there was no alteration in C3 complement levels in the serum of venom-treated animals compared to controls, and an immunofluorescence assay with an anti-mouse IgG was negative (data not shown), excluding the possibility of renal deposits of immunoglobulins. Taken together, the above results support the notion that venom toxins act as direct ligands in renal structures and as primary agents, playing a role in renal disorders.
In addition, our data also showed that venom toxins bind to glomerular and tubule cells and basement membranes but do not bind to kidney cell nuclei. This conclusion was based on double-staining immunofluorescence reactions that demonstrated co-localization of venom toxins with basement membrane constituents such as type IV collagen and laminin, but no co-localization along the chromosomes revealed by DAPI. The basement membranes are specialized extracellular matrices involved in several physiological events, especially those dependent on cell adhesion. In the kidney these structures play a role in glomerular filtration during urine formation in addition to organizing podocyte, endothelial, and epithelial cell adhesion (Courtoy et al. 1982; Rohrbach and Timpl 1993
; Cotran et al. 1999
). The deposition of venom toxins along the renal basement membranes can explain glomerular epithelial cell injury, fenestrated endothelial cell cytotoxicity, hyalinosis, and proteinuria, as well as tubule cell damage. Because some L. intermedia venom toxins are proteases, with the ability to degrade basement membrane constituents (as discussed above), such venom injuries can result from loss of renal basement membrane integrity with consequent cytotoxicity to epithelial and endothelial cells and detachment and loss of the glomerular basement membrane charge barrier.
Using two-dimensional electrophoresis, we observed that L. intermedia venom is enriched in basic proteins with molecular masses ranging from 35 to 20 kD. Immunoblotting analysis using antibodies to venom proteins identified venom toxin(s) at 30 kD as direct ligands of renal structures. Physicochemical properties such as the molecular charge and size of "planted antigens" along the kidney are very important factors that affect the interaction of these antigens along the renal basement membranes, especially with glomerular structures (Cotran et al. 1999). Highly cationic molecules (as is the case for Loxosceles venom toxins) tend to bind to glomerular basement membrane anionic sites (proteoglycans) (Cotran et al. 1999
). In addition, the glomerular barrier function is dependent on the molecular mass of proteins. Molecules with mass larger than 70 kD are less permeable than low molecular mass proteins (as is the case for a large number of Loxosceles venom toxins) (Cotran et al. 1999
). This charge and size properties of venom toxins can account for their binding to glomerular basement membranes in a first step and later to tubule structures accumulated in tubule epithelial cells.
On the basis of the above findings, we have identified a possible cellular and molecular mechanism for the nephrotoxicity that occurs after envenomation by Loxosceles spiders. Although the renal injuries occurring after envenomation can be increased by dermonecrotic products and hemolysis, we conclude that Loxosceles venom toxins are direct and potentially nephrotoxic agents.
We hope that this report will bring some insight into loxoscelism, opening the possibility for a rational basis for therapy after brown spider bites.
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Acknowledgments |
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We acknowledge LIPAPE for spider capture and venom extraction and Prof Marco A. F. Randi (Department of Cell Biology, Federal University of Paraná), who helped us during morphometic analysis.
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Footnotes |
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Literature Cited |
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