C Pharmacology & Toxicology 2003, 92, 14–20.
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Renal Effects of Supernatant from Macrophages
Activated by Crotalus durissus cascavellaVenom:
The Role of Phospholipase A2and Cyclooxygenase
Alice M. C. Martins1, Aldo A. M. Lima2, Marcos H. Toyama3, Sergio Marangoni4, Manasse ´s C. Fonteles5and
Helena S. A. Monteiro2
1Health Science Center, University of Fortaleza-UNIFOR, Fortaleza, Ceara,2Department of Physiology and
Pharmacology, Institute of Biomedicine and Clinical Research Unit, UFC/UECE, Fortaleza, Ceara,3Department
of Physiology and Biophysic- Biological Institute, UNICAMP, Sa ˜o Paulo,4Department of Biochemistry, Biological
Institute, UNICAMP, Sa ˜o Paulo, and5Ceara State University, Fortaleza, Ceara, Brazil
(Received March 4, 2002; Accepted July 1, 2002)
Abstract: In Brazil, the genus Crotalus is responsible for approximately 1500 cases of snakebite annually. The most
common complication in the lethal cases is acute renal failure, although the mechanisms of the damaging effects are not
totally understood. In this work, we have examined the renal effects caused by a supernatant of macrophages stimulated
by Crotalus durissus cascavella venom as well as the potential role of phospholipase A2and cyclo-oxygenase. Rat peritoneal
macrophages were collected and placed in a RPMI medium and stimulated by crude Crotalus durissus cascavella venom
(1, 3 or 10 mg/ml) for 1 hr. They were then washed and kept in a culture for 2 hr. The supernatant (1 ml) was tested in
an isolated perfused rat kidney. The first 30 min. of each experiment were used as an internal control, and the supernatant
was added to the system after this period. All experiments lasted 120 min. A study of toxic effect on perfusion pressure,
glomerular filtration rate, urinary flow, percent of sodium tubular transport and percent of proximal tubular sodium
transport was made. The lowest concentration of venom (1 mg/ml) was not statistically different from the control values.
The most intense effects were seen at 10 mg/ml for all renal parameters. The infusion of the supernatant of macrophages
stimulated with crude venom (3 or 10 mg/ml) increased the perfusion pressure, glomerular filtration rate and urinary flow,
decreased the percent of sodium tubular transport and percent of proximal tubular sodium transport. Dexamethasone
(10 mM) and quinacrine (10 mM) provided protection against the effect of the venom on glomerular filtration rate, urinary
flow, percent of sodium tubular transport, percent of proximal tubular sodium transport and perfusion pressure. Indo-
methacin (10 mM) and nordiidroguaretic acid (1 mM) reversed almost all functional changes, except those of the perfusion
pressure. These results suggest that macrophages stimulated with Crotalus durissus cascavella venom release mediators
capable of promoting nephrotoxicity in vitro. Moreover, phospholipase A2and cyclooxygenase products are involved in
these biologic effects.
Venomous animals are a significant cause of morbidity and
mortality around the world. The poisonous snakes found
around the globe produce a variety of highly effective toxins
and have developed numerous methods of delivery (White
In Brazil, the genus Crotalus contains several species and
subspecies of snakes responsible for approximately 1500
cases of snakebite annually (Santoro et al. 1999). Crotalus
durissus cascavella (C.d. cascavella) is usually found in
scrublands of the Brazilian Northeast (Barraviera 1989).
The bite of this snake is characterized by neurotoxicity, sys-
temic myotoxicity, oedematogenic activity, platelet aggre-
gation and acute renal failure. The pathogenesis of acute
renal failure after snakebites appears to be multifactorial
(Nancy et al.1991; Martins et al. 1998), with the most com-
mon complication in lethal cases being acute renal failure
(Ribeiro et al. 1998). The symptoms are due to the additive
Author for correspondence: Helena Serra Azul Monteiro, Depart-
ment of Physiology and Pharmacology, Faculty of Medicine, Feder-
al University of Ceara ´, CP 3229 Fortaleza – Ce, Brasil (fax π55
085 2815212, e-mail serrazul/truenet-ce.com.br).
or synergistic effect of the different toxins and enzymes
present in the venoms (Gutierrez & Lomonte 1989; Ferreira
et al. 1992). The effect of these toxins on humans is not
limited to poisoning, since these substances are proving in-
valuable as research tools and diagnostic agents, and may
even have a future as precursors of therapeutic agents
(White 2000). The analgesic activity of crotamine, a neuro-
toxin obtained from Crotalus durissus terrificus, for example
was demonstrated by Mancin et al. (1998).
It has also been demonstrated by Martins et al. (1998),
that C.d. cascavella venom produces damaging effects in
isolated rat kidneys, although the mechanisms are not to-
tally understood. The aim of the present investigation was
to examine the renal effect of supernatants from rat macro-
phages stimulated with C.d. cascavella venom to investigate
whether phospholipase A2and cyclooxygenase are involved
in the process.
Materials and Methods
Macrophage cultures. Macrophage culture methods were described
by Rocha et al. (1998). Briefly, rat peritoneal macrophages were
ALICE M. C. MARTINS ET AL.
ferent mechanism is involved in the vascular effect. Indo-
methacin showed a reduction in prostaglandin synthesis in
all segments of the rabbit kidney (Fonteles & Forti 1993).
Most of the observations may be due to prostaglandin
production at the glomeruli, since a great deal of prosta-
glandin is found in the cells of the renal medulla (Azar et
al. 1971). Although, Schlondorff et al. (1980) demonstrated
that prostaglandin can be produced in all kidney zones.
The fact that HPLC analysis shows that the supernatant
of macrophages is free of venom, however strongly suggests
that the venom itself is not acting directly, but rather that
the macrophages are releasing mediators, which are capable
of promoting nephrotoxicity. Moreover, phospholipase and
cyclooxygenase products seem to be involved in the renal
Acknowledgements are made to Domingos Barreto and
Maria Silvia Freire Franc ¸a for their technical assistance. We
also acknowledge the financial support of CNPq, Brazil.
Alice M. C. Martins was supported by a fellowship from
CAPES, Brası ´lia, Brazil.
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