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
RENAL EFFECTS OF CROTALUS VENOM
collected with RPMI medium 4 days after the intraperitoneal injec-
tion of 10 ml thioglycolate (3%) and placed in plastic tissue culture
dishes, as previously described (Ribeiro et al. 1991). After incuba-
tion at 37æ in a 5% CO2atmosphere for 1.5 hr, the non-adherent
cells were removed by washing the dishes three times with RPMI
medium. The cellular pattern was based on cellular morphology
analyzed by optical microscopy. The percentage of macrophages
was calculated from the total number of cells present in the culture.
The total cells, (95% macrophages), were incubated at 37æ in a 5%
CO2atmosphere for 1 hr in a fresh medium (control), in a medium
containing C.d. cascavella venom (1, 3 or 10 mg/ml), or in a medium
containing (10 mg/ml) C.d. cascavella venom plus various pharma-
cological inhibitors. The supernatant was discarded and after ad-
ditional washing, the cells were incubated for another hour with
1ml RPMI medium without venom or drugs. Cell free incubation
medium was obtained by centrifugation (5 min.) and 1 ml of super-
natant was adjusted to 1.3¿107cells /ml by using a Neubauer
Fig. 1. Chromatograms of Crotalus durissus cascavella venom (crot-crotamine; crt-crotapotin; PLA2- phospholipase A2; cvx- convulxin; gyr-
gyroxin) (a) and the venom-stimulated supernatants of macrophages (1, 3 and 10 mg/ml; b, c and d, respectively).
chamber tested in a isolated rat kidney as described below. Macro-
phage viability was determined by trypan blue exclusion as de-
scribed elsewhere (Korzeniewsky & Callewaert 1983; Marshall et al.
1996). Macrophage viability ranged from 89 to 97% in the different
Analysis by HPLC of the venom and supernatant of stimulated
macrophages. Dried venom and the lyophilized supernatant of
macrophages activated by C.d. cascavella venom at doses of 1, 3
and 10 mg/ml were evaluated by analytical (0.39 cm¿30 cm) reverse
phase high pressure liquid chouromatography (HPLC) on m-Bonda-
pack C18 (5 mm) columns (Waters Corp.). Initially, the analytical
reverse phase column was equilibrated with buffer A (0.1% trifluo-
roacetic acid (TFA, in water) and the samples eluted with a linear
gradient of buffer B (0.025% TFA in 66% acetonitrile). The column
was eluted at a flow rate of 1.0 ml/min. and the absorbance meas-
ured at 214 nm. To improve the chromatographic separation of the
ALICE M. C. MARTINS ET AL.
toxins and other fractions, the analytical m-Bondapack C18 column
(0.39¿30 cm) was again equilibrated with a buffer A (0.1% TFA)
and eluted with a discontinuous linear gradient of buffer B (0.025%
of TFA in 66% acetonitrile). The samples were dissolved in 0.1%
TFA in water and centrifuged at 10,000 rmp for 2 min. Before
sample injection, the column was equilibrated for 30 min. with buf-
fer A at a constant flow rate of 1.0 ml/hr and aliquots of samples
were clarified by filtration on the 0.45 mm Millipore filter unit. The
aliquots of 100 ml were injected into the reverse phase m-Bondapack
C18 column (0.39 cm¿30 cm) coupled to an analytical photo diode
array (PDA) HPLC system (Shimadzu LC10AD) (Shimadzu, Inc.,
Japan). The samples were eluted using a linear gradient, which was
modified to non-linear gradient to improve the separation of com-
pounds. The buffer used to separate the compounds was of solution
B (TFA 0.025% in 66% acetonitrile). The chromatographic run was
monitored at 214 nm of absorbance at a column flow rate of 1 ml/
min. The method used for this assays has of high sensibility for
detection of pmoles to nmoles. The AUFS was adjusted for 0.1
AUFS to experimental conditions. The detector was adjusted to
A214 nm ∂A280 nm on the PDA with range of A200 to A300 nm.
Isolated rat kidney. The perfusion method was described by Fontel-
es et al. (1983). Briefly, adult Wistar rats of both sexes (240–280 g)
were fasted with free access to water 24 hr before each experiment.
The animals were anaesthetized with sodium pentobarbital (50 mg/
kg body weight). The perfusion fluid was a modified Krebs-Hensele-
it solution with the following composition in mmoles/l: Naπ147,
Kπ5, Caππ2.5, Mgππ2, Clª110, HCO3ª2.5, SO4ª1, PO4ª1.
Bovine serum albumin (BSA 6 g%; fraction V), urea (0.075 g), inu-
lin (0.075 g) and glucose (0.15 g) were added to the solution, giving
a final perfusate volume of 100 ml. All BSA was previously dialyzed
for 48 hr at 4æ in 1.5 liters of Krebs solution, which was changed
in the solution every 24 hr (Hanson & Ballard 1968; Greg et al.
1978; Fonteles et al. 1983; Lima et al. 1992). The pH was adjusted
to 7.4 and the perfusion system, based on Bowman’s technique
(Bowman 1970), was modified (Hamilton et al. 1974; Fonteles et al.
1998) by the addition of an artificial lung to improve oxygenation
(Balhlmann et al. 1967) and a 1.2 mm Millipore filter (Pegg 1971).
Flow calibration and the resistance of the system were determined
before each experiment. Perfusion pressure was determined at the
tip of the stainless steel cannulae. The organ was isolated according
to Balhlmann et al (1967), Nishiitsutji-Uwo et al. (1967) and Ross
(1978), as modified by Fonteles et al. (1983), to allow perfusion
without interruption of kidney flow thourough a cannulation of the
mesenteric artery reaching the right renal artery. After an equili-
bration period of 15 to 20 min., the experiments were carried out
for 120 min. with the supernatants of macrophages stimulated by
Fig. 2. Effect of supernatants (SUP) from macrophages (MjS) stimulated of Crotalus durissus cascavella venom (1, 3 and 10 mg/ml) on the
glomerular filtration rate. The supernatants were tested in isolated rat kidneys (Values represent mean∫S.E.M. for four periods of 30 min.
C.d. cascavella venom with or without pharmacological inhibitors
added after 30 min. of the perfusion. Perfusion pressure was meas-
ured at 5 min. intervals. Samples of the urine and perfusate were
collected every 10 min. for the determination of sodium, potassium,
inulin levels and osmolality. Sodium and potassium concentrations
were determined by flame photometry (flame photometer Model
445) and inulin levels were also determined (Bowman 1970; Martins
et al. 1998). The osmolality of the samples was measured in an
advanced instrument osmometer (WESCOR 5100c vapor pressure).
The experiments follows the methodology recommended by the
international ethical standards of the scientific committee of our
university (Comite ˆ de E´tica e Pesquisa do Complexo Hospitalar da
Universidade Federal do Ceara ´, COMEPE).
Reagents. Crotalus durissus cascavella venom was obtained from the
regional ophiology laboratory of Fortaleza (LAROF-CE). Dexa-
methasone was obtained from Merck Sharp & Dohme (Sa ˜o Paulo,
Brazil). Quinacrine, indomethacin, nordiidroguaretic acid RPMI
medium, albumin, inulin and glucose were purchased from Sigma
Chemical Co. (St.Louis, MO, USA). Thioglycolate was obtained
from Difco Laboratories, Detroit, USA.
Statistical analysis. The data were analyzed using analysis of vari-
ance (ANOVA), and results were expressed as mean∫S.E.M. (level
of significance of P?0.05) for the six experiments of each experi-
Analysis by HPLC of the venom and supernatant of stimu-
The chromatographic profile of 250 mg whole C.d. cascavel-
la venom, showed the presence of 10 major fractions. Frac-
tions 1 and 2 were characterized by two crotamine isoforms,
which 3 and 4 were identified as crotapotins, fraction 5 was
PLA2, while 6 to 10 were components of gyroxin, convuxin
The fractionation of the supernatants of macrophages
activated by C.d. cascavella revealed similar chromato-
graphic profiles, with the first peaks being identified as salt
and several small peaks of non proteic components were
also revealed (fig.. 1b, 1c and 1d).
RENAL EFFECTS OF CROTALUS VENOM
Functional data from control kidneys perfused with supernatant of macrophages without Crotalus durissus cascavella venom in Krebs-
Henseleit solution containing 6g% of bovine serum albumin.
Event(ml ¡ gª1¡ min.ª1) (ml ¡ gª1¡ min.ª1) %TNaπ
a) Results are expressed as means∫S.E.M. of control kidneys (nΩ6).
b) UFΩurinary flow, GFRΩglomerular filtration rate, %TNaπΩpercent of sodium tubular transport, %pTNaπΩpercent of proximal tubular
sodium transport, PPΩperfusion pressure.
Rat kidneys perfused with supernatants of macrophages with-
out venom in modified Krebs-Henseleit solution.
Selected functional parameters of renal function during
stable experimental conditions were evaluated in a rat kid-
ney perfused with the supernatant of macrophages (SUP.
MjS) without venom stimulation. The data are presented
as mean∫S.E.M. for the four periods of 30, 60, 90 and 120
min. (table 1). For each experiment, the first 30 min. of
perfusion were considered as an internal control. The phar-
macological antagonists did not modify the functional kid-
ney parameters (data not shown).
Renal effects of supernatant of macrophages stimulated by
Crotalus durissus cascavella venom.
The supernatant from the macrophages stimulated by (1, 3
and 10 mg/ml) C.d. cascavella venom was administrated into
the perfusion system. The lowest concentration of venom (1
mg/ml) resulted in values, which were not statistically differ-
ent from the controls, while the most intense effects on all
parameters were seen with 10 mg/ml.
The infusion of 1 ml of the supernatant stimulated by
C.d. cascavella venom (3 and 10 mg/ml) caused a significant
increase in glomerular filtration rate, with a maximal effect
at 120 min. (fig. 2). The urinary flow was also significantly
increased by the infusion with the supernatant activated by
Fig. 3. Effect of supernatants (SUP) from macrophages (MjS) stimulated of Crotalus durissus cascavella venom (1, 3 and 10 mg/ml) on the
urinary flow. The supernatants were tested in isolated kidneys. (Values represent mean∫S.E.M. for four periods of 30 min. each; nΩ6).
the venom, again with a maximal response at 120 min. (fig.
3). The treated kidneys showed a significant decrease in per-
cent of sodium tubular transport (fig. 4). Regarding percent
of proximal tubular sodium transport, we observed no dif-
ference with percent of sodium tubular transport (fig. 5).
The perfusion pressure increased significantly after adminis-
tration of the supernatant stimulated by the venom at doses
of 3 and 10 mg/ml (fig. 6).
Effects of pharmacological inhibitors on renal functional par-
When we studied the renal effects promoted by venom in
supernatant of macrophages using 1, 3 and 10 mg/ml we
noticed that the highest concentration showed the most in-
tense effect. This concentration was choosen to evaluate the
major pharmacological blockers on this venom renal
Dexamethasone (10 mM) or quinacrine (10 mM) 30 min.
before and during the stimulation of macrophages with C.d.
cascavella venom (10 mg/ml), significantly blocked the in-
crease in isolated kidney glomerular filtration rate and the
increase in urinary flow. The treatment with dexamethasone
and quinacrine also reversed the effects on percent of so-
dium tubular transport and perfusion pressure. Similarly,
indomethacin (10 mM), a cyclo-oxygenase inhibitor, and
nordiidroguaretic acid (1 mM), a dual cyclo- and lipooxy-
ALICE M. C. MARTINS ET AL.
Fig. 4. Effect of supernatants (SUP) from macrophages (MjS) stimulated of Crotalus durissus cascavella venom (1, 3 and 10 mg/ml) on the
percent of sodium tubular transport (%TNaπ). The supernatants were tested in isolated rat kidneys. (Values represent mean∫S.E.M. for
four periods of 30 min. each; nΩ6).
genase inhibitor, also reduced the ability of the supernatant
of macrophages activated by C.d. cascavella venom to cause
increase in glomerular filtration rate and urinary flow and
reversed the decrease of percent of sodium tubular trans-
port. On the other hand, these drugs did not change the
effect observed on perfusion pressure (table 2).
The most common complication seen in lethal cases of
snakebites in Brazil is acute renal failure (Ribeiro et al.
1998). This can happen even after treatment with specific
antivenom and the pathogenesis is not well understood.
Monteiro et al. (2001) have demonstrated the nephrotoxi-
city of Crotalus durissus terrificus venom and crotoxin using
an isolated rat kidney model. A direct cascavella venom ef-
fect in the isolated kidney has also been demonstrated
(Martins et al. 1998), but the mechanisms of this effect are
not totally understood.
Macrophages signal the presence of foreign bodies thour-
ough the elaboration and release of several substances, in-
Fig. 5. Effect of supernatants (SUP) from macrophages (MjS) stimulated of Crotalus durissus cascavella venom (1, 3 and 10 mg/ml) on the
percent of proximal tubular sodium transport (%pTNaπ). The supernatants were tested in isolated rat kidneys. (Values represent
mean∫S.E.M. for four periods of 30 min. each; nΩ6).
cluding cytokines and arachidonic acid metabolites (Ferrei-
ra 1980; Nathan 1987; Laskin & Pendino 1995; Rocha et
To investigate whether macrophages are important in the
renal effect of C.d. cascavella venom, we evaluated the abil-
ity of the supernatant of macrophages stimulated with
venom to cause alterations in renal parameters as glomeru-
lar filtration rate, urine flow, percent of sodium tubular
transport, percent of proximal tubular sodium transport
and perfusion pressure.
The lowest concentration of venom did not significantly
change from the control values. The renal alterations at 10
mg/ml were more intense in all parameters than the effect
observed with 3 mg/ml.
The supernatant stimulated with 10 mg/ml of the venom
caused a significant increase in glomerular filtration rate
and urinary flow, as well as a decrease in sodium transport.
This is in agreement with recent findings that C.d. cascavella
venom is toxic to kidney (Martins et al. 1998). The renal
toxic effect of C.d. cascavella venom on macrophages was
determined and has been shown to correlate with the direct
RENAL EFFECTS OF CROTALUS VENOM
Fig. 6. Effect of supernatants (SUP) from macrophages (MjS) stimulated of Crotalus durissus cascavella venom (1, 3 and 10 mg/ml) on the
perfusion pressure. The supernatants were tested in isolated rat kidneys. (Values represent mean∫S.E.M. for four periods of 30 min. each;
venom renal effect. These data suggest that renal effects
may be caused indirectly by the release of mediators by
The ability of quinacrine and dexamethasone (phos-
pholipase A2inhibitors), to block the toxic effects of the
supernatants on the kidney indicated that arachidonic acid
metabolites are involved in these processes (Barnes & Ad-
cock 1993; Rocha et al. 1998). The hypothesis that arachi-
donate-derived metabolites are involved in the renal effects
Effects of pharmacological inhibitors in renal perfusion with supernatant of macrophages stimulated with venom on functional parameters.
Treatment(ml ¡ gª1¡ min.ª1) (ml ¡ gª1¡ min.ª1) %TNaπ
Results are expressed as means∫S.E.M. Supernatant of macrophages (SUP.MjS) with venom group was compared with the other experimen-
tal groups (nΩ6). The (SUP.MjS) stimulated by venom on the containing C.d. cascavella (10 mg/ml; ANOVA; * P?0.05) and the pharmaco-
logical inhibitors were infusion after 30 min. of the perfusion. The first 30 min. of perfusion were considered as an internal control. vΩ
venom, Dexa Ωdexamethasone (10 mM), QuinacΩquinacrine (10 mM), IndoΩindomethacin (10 mM), NDGAΩnordiidroguaretic acid (1
mM). UFΩurinary flow, GFRΩglomerular filtration rate, %TNaπΩpercent of sodium tubular transport, PPΩperfusion pressure.
induced by the supernatant of venom-stimulated macro-
phages is also supported by the fact that the venoms showed
important phospholipase A2activity.
Indomethacin (a cyclooxygenase inhibitor) and nordiid-
roguaretic acid (a dual cyclo and lipoxygenase inhibitor),
reversed almost all renal alterations induced by super-
natants of macrophages stimulated by C.d. cascavella
venom, although, these inhibitors did not change the effect
observed on perfusion pressure, which suggests that a dif-
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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