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Toxicon 48 (2006) 44–54

Mechanisms of bee venom-induced acute renal failure
Luciana S.D. Grisotto
a
, Glo´ ria E. Mendes
a
, Isac Castro
b
, Maria A.S.F. Baptista
a
,
Venancio A. Alves
c
, Luis Yu
b
, Emmanuel A. Burdmann
a,Ã
a
Division of Nephrology, Sa˜o Jose´ do Rio Preto Medical School, Av. Brigadeiro Faria Lima, 5416 Sa˜o Jose´ do Rio Preto,
SP 15090-000, Brazil
b
Division of Nephrology, University of Sa˜o Paulo Medical School, Brazil
c
Division of Pathology, University of Sa˜o Paulo Medical School, Brazil
Received 14 February 2006; received in revised form 24 April 2006; accepted 24 April 2006
Available online 6 May 2006
Abstract
The spread of Africanized bees in the American continent has increased the number of severe envenomation after swarm
attacks. Acute renal failure (ARF) is one of the major hazards in surviving patients.
To assess the mechanisms of bee venom-induced ARF, rats were evaluated before, up to 70 min and 24 h after 0.5 mg/kg
of venom injection. Control rats received saline. Bee venom caused an early and significant reduction in glomerular
filtration rate (GFR, inulin clearance, 0.8470.05 to 0.4070.08 ml/min/100 g, po0:0001) and renal blood flow (RBF, laser
Doppler flowmetry), which was more severe in the cortical (À72%) than in the medullary area (À48%), without systemic
blood pressure decrease. Creatine phosphokinase, lactic dehydrogenase (LDH) and serum glutamic oxaloacetic
transaminase increased significantly, pointing to rhabdomyolysis, whereas serum glutamic pyruvic transaminase and
hematocrit remained stable. Twenty-four hours after venom, RBF recovered but GFR remained significantly impaired.
Renal histology showed acute tubular injury and a massive tubular deposition of myoglobin. Venom was added to isolated
rat proximal tubules (PT) suspension subjected to normoxia and hypoxia/reoxygenation (H/R) for direct nephrotoxicity
evaluation. After 60 min of incubation, 0.1, 2 and 10 mg of venom induced significant increases in LDH release: 47%, 64%
and 86%, respectively, vs. 21% in control PT while 2 mg of venom enhanced H/R injury (85% vs. 55%, po0.01).
These results indicate that vasoconstriction, direct nephrotoxicity and rhabdomyolysis are important mechanisms in the
installation of bee venom-induced ARF that may occur even without hemolysis or hypotension.
r 2006 Elsevier Ltd. All rights reserved.
Keywords: Bee; Venom; Nephrotoxicity; Acute renal failure; Acute tubular necrosis; Rhabdomyolysis; Renal blood flow;
Vasoconstriction; Rats; Proximal tubule toxicity
1. Introduction
Bees cause human accidents in two distinct
patterns. One or few stings induce allergic reactions;
sometimes they are severe or fatal. On other hand,
massive attacks with hundreds to thousand stings
cause severe systemic injury affecting many different
organs with high mortality (Vetter et al., 1999;
Schumacher and Egen, 1995).
Bee venom is a unique weapon in the animal
kingdom. It is an efficient and complex mixture of
substances designed to protect bees against a broad
diversity of predators, from other arthropods to
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doi:10.1016/j.toxicon.2006.04.016
Ã
Corresponding author. Tel.: +55 17 3201 5712.
E-mail address: burdmann@famerp.br (E.A. Burdmann).
vertebrates (Winston, 1994). Its main components
are melittin, apamin, peptide 401 (mast cell degra-
nulating peptide), phospholipase A
2
, hyaluronidase,
histamine, dopamine and norepinephrine (Schumacher
and Egen, 1995; Habermann, 1972; Han et al.,
2000). Among then, melittin has been shown to be
the main lethal component in venom (Schmidt,
1995).
Massive inoculation of bee venom may induce
acute renal failure (ARF), adult respiratory distress
syndrome, liver injury, cardiac damage, pancreati-
tis, skin necrosis, shock, hypertension, bleeding,
thrombocytopenia, hemolysis and rhabdomyolysis
(Mejia et al., 1986; Tumwine and Nkrumah, 1990;
Mendes et al., 1990; Munoz-Arizpe et al.,
1992a; Sert et al., 1993; Franca et al., 1994; Daisley,
1998; Diaz-Sanchez et al., 1998; Kolecki, 1999;
Bresolin et al., 2002; Daher et al., 2003; Gabriel
et al., 2004).
Bee venom-induced ARF after multiple stinging
has been sporadically reported in Europe, Africa
and Asia. In 1957, swarms of African bees escaped
from a research facility in Brazil and hybridized at
random with the local European bees, forming the
so-called Africanized bees (Schumacher and Egen,
1995; Winston, 1994; Tunget and Clark, 1993). This
accident dramatically changed the incidence and
pattern of massive bee attacks in the American
continent. The Africanized and the European bees
have nearly indistinguishable appearance and the
venom composition is practically identical (Schu-
macher and Egen, 1995; Vetter and Visscher, 1998).
However, Africanized bees are peculiarly aggressive,
attacking in swarms with high tenacity after
minimal provocation (Winston, 1994; Tunget and
Clark, 1993). They also have a high reproductive
rate and migratory behavior. Currently, they have
spread throughout the American continent up to the
USA (Texas, New Mexico, Arizona and California),
where they are known as ‘‘killer bees’’ (Vetter et al.,
1999; Schumacher and Egen, 1995; Tunget and
Clark, 1993; Kim and Oguro, 1999). Not surpris-
ingly, the reporting of massive accidents has risen
significantly in affected countries (Franca et al.,
1994; Daisley, 1998; Ariue, 1992; Munoz-Arizpe
et al., 1992b; Taylor, 1986). Correspondingly, the
number of cases of bee venom-induced ARF in the
American continent is more than 6 times higher
than that reported in other continents. The patho-
genesis of bee venom-induced renal injury is not
totally understood. Shock, rhabdomyolysis, hemo-
lysis and direct tubular nephrotoxicity have been
pointed as possible factors for kidney insult (Franca
et al., 1994; dos Reis et al., 1997, 1998).
The aim of this study is to assess the mechanisms
of bee venom-induced ARF in a reproducible and
clinically relevant experimental model.
2. Material and methods
2.1. Animals
Adult male Wistar rats weighing 200–350 g were
housed in a temperature- and light-controlled
environment. They received standard diet and were
allowed free access to tap water. Experiments were
done according to the Brazilian law of protection of
animals.
2.2. Venom
Lyophilized bee venom (Sigma Chemical Co., St.
Louis, MO, USA), 0.5 mg/kg, was diluted in saline
and infused in the jugular vein at the rate of
0.05 ml/min (infusion pump, model 975, Harvard,
USA). Control animals received the same volume of
saline.
2.3. Acute studies
2.3.1. Glomerular filtration rate (GFR)
After anesthesia (intraperitoneal thiopental,
50 mg/kg), tracheostomy was performed and poly-
ethylene tubes (PE-50) were placed in a carotid
artery, in both jugular veins and in the bladder (PE-
90). Animals were maintained in a stable tempera-
ture (36.571 1C) by a thermostatically controlled
warming table (Braile Biome´ dica, Sa˜ o Jose´ do Rio
Preto, Brazil). After surgery, a dose of 1 ml of inulin
solution (25 mg/ml, Sigma Chemical Co., St. Louis,
MO, USA) was given, followed by an infusion of
the same solution at a rate of 0.05 ml/min (Harvard
infusion pump model 975, USA). After a 60-min
equilibration time, urine was collected over two
periods of 20 min each. A blood sample (0.3 ml) was
drawn at the midpoint of each urine collection and
replaced with an equal volume of saline. After these
periods, designed as baseline, venom (venom, n ¼ 8)
or saline (control, n ¼ 8) were infused. After a 30-
min equilibration time, two additional periods of
urine and blood collection were done as already
described. Inulin clearance values, expressed as
ml/min/100 g body wt, represent the means of the
two clearance periods (baseline, venom or saline).
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Diuresis (ml/min) was assessed by the weight
difference in the collection vials before and after
urine collection.
2.3.2. Cortical and medullary renal blood flow
(RBF)
The rats were anesthetized, placed in the warming
table and polyethylene tubes were placed in trachea,
carotid and jugular veins, as described above. The
abdominal wall was incised, the left kidney exposed,
decapsulated and placed in a kidney micropuncture
cup. For measurement of RBF, needle-type laser-
Doppler flow probes (HL-N 1196 and HL-N 1188,
Transonic Systems Inc., Ithaca. NY, EUA) con-
nected to a Laser Doppler flowmeter (Transonic
Systems Inc., Ithaca. NY, EUA) were used. The
laser Doppler was previously used and validated as
a reliable index of blood flow changes in the
different regions of the kidney (Franchini et al.,
1997). The probes were inserted by a micromani-
pulator approximately 1.5–2 mm depth (cortical
RBF) and 5–6 mm depth (medullary RBF). After
60 min equilibration period, cortical and medullary
RBF and mean arterial pressure were recorded
every 20 min for an hour (baseline period). Then
venom (venom, n ¼ 6) or saline (control, n ¼ 6)
were infused as already described. After a 30-min
equilibration period, RBF and mean arterial pres-
sure were recorded every 20 min for an additional
hour. RBF values, expressed in tissue perfusion
units, represent the mean of all the values recorded.
At the end of the experiment, the kidney was
sectioned to check if the probe was correctly
located.
2.3.3. Arterial blood pressure
The mean carotid arterial blood pressure was
continuously measured throughout the experiments
by an electronic transducer (P23 Db, Statham
Instruments Division, Could Inc., Hato Rey,
USA) connected to a digital polygraph (Blood
Pressure Display Unit, Stoelting, USA).
2.3.4. Hemolysis and rhabdomyolysis evaluation
Anesthesia, surgical preparation and venom
(venom, n ¼ 8) or saline (control, n ¼ 8) infusion
were carried out as previously described. After
60 min, blood was collected for measurement of
hematocrit (Hct), creatine phosphokinase (CPK),
serum glutamic oxaloacetic transaminase (AST),
serum glutamic pyruvic transaminase (ALT) and
lactic dehydrogenase (LDH).
2.3.5. Renal histology
Renal tissue was collected 60 min after venom
(venom, n ¼ 12) or saline (control, n ¼ 11) injec-
tion, fixed in 10% buffered formalin and embedded
in paraffin. Horizontal sections 2–3 mm thick were
stained with periodic acid-Schiff’s reagent (PAS),
hematoxylin–eosin, periodic acid methenamine sil-
ver and Masson Trichrome. The tissue was eval-
uated by light microscopy by an observer masked to
treatment.
2.4. Twenty-four hours after venom studies
Animals received venom or saline as previously
described. After recovery from anesthesia, they were
housed into metabolic cages (Nalgene Co., Roche-
ster, NY, USA) for 24 h for urinary volume
collection.
Twenty-four hours after injection, the animals
were anesthetized and GFR was measured as
previously described. Next, a ventral midline inci-
sion was made, and a suitable probe (R series,
1.5 mm, Transonic Systems, Ithaca, NY, USA) was
placed around the left renal artery. After a 40-min
equilibration time, ultrasonic RBF measurements
were performed (T 106, Transonic Systems Inc,
Ithaca, NY, USA) for 30 min. Arterial pressure was
continuously monitored as previously described. At
the end of the experiment, renal tissue was collected
for renal histology and for assessment of myoglobin
immunohistochemistry.
2.4.1. Immunohistochemical analysis
Paraffin-embedded renal sections (3 mm thick)
were mounted on siliconized slides (S3003, Dako-
cytomation, Carpinteria, CA, USA), dried over-
night at 58 1C in an oven, deparaffinized in xylene
and rehydrated through graded ethanol and deio-
nized water. Sections were then immersed in a 1:10
diluted DAKO
s
Target Retrieval Solution (S1699,
Dakocytomation, Carpinteria, CA, USA), and
heated in a pressure cooker for 3 min. After that,
the sections were washed-out in distilled water and
3% hydrogen peroxide was applied for 10 min to
block endogenous peroxidase. Sections were incu-
bated with 1:10,000 primary nonspecific polyclonal
antibody anti-myoglobin (A324, Dakocytomation,
Carpinteria, CA, USA) diluted with DAKO
s
Antibody Diluent (S3022, Dakocytomation, Car-
pinteria, CA, USA) overnight (4 1C, humid cham-
ber). The amplification system used was Envision
s
System (K1395, Dakocytomation, Carpinteria, CA,
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L.S.D. Grisotto et al. / Toxicon 48 (2006) 44–54 46
USA). Signal amplification was achieved with
dextran-polymer coated with peroxide and color
was developed by incubation with the 3
0
3 diamino-
benzidine tetrachloride (DAB) and hydrogen per-
oxide. Counterstaining was done with Harris
hematoxylin. Slides were cover-slipped with resin.
A slide from a well-known myoglobin staining was
run in parallel as an additional control. It was
submitted to each step of the reaction except for the
incubation with the primary antibody, which was
omitted.
2.4.2. Proximal tubule (PT) direct nephrotoxicity
2.4.2.1. Isolation of PT. Male Wistar rats were
anesthetized with sodium thiopental (40 mg/kg
ip.). Kidneys were flushed in vivo with 60 ml
oxygenated solution (solution A), pH 7.2, 4 1C,
containing (in mM) NaCl 112; NaHCO
3
25; KCl 5;
NaH
2
PO
4
2; CaCl
2
1.6; MgSO
4
1.2; HEPES 2.5;
mannitol 10; glutamine 1; butyric acid 1; lactic acid
1 and heparin (4000 UI). Subsequently, perfusion
was continued with solution A (without heparin)
containing 15 mg of collagenase type V (Sigma
Chemical Co., USA) and 15 mg of hyaluronidase
type III (Sigma Chemical Co., USA). After perfu-
sion, kidneys were decapsulated, removed and
transferred to ice-cold and oxygenated solution A.
Renal cortices were removed and minced on a cold
Petri dish. After 3 washes with solution A, tissue
was incubated in 60 ml of 37 1C oxygenated solution
A containing 40 mg collagenase and 10 mg hyalur-
onidase. Digestion was performed for 25 min in
oxygenated shaking water bath. After digestion was
completed, tissue was washed and placed for 10 min
in 30 ml of ice-cold solution A containing 1 g of
fatty-acid-free albumin (Sigma Chemical Co.,
USA). After filtering the tissue through a tea
strainer, 3 washes were carried out to remove
albumin. Tubules were separated using 45% Percoll
gradient. After centrifugation for 10 min (11000 G),
PTs were recovered from the lowest band. This
band contained no glomeruli and was composed
primarily (95%) of PTs. The tubules suspension was
washed 3 times to remove Percoll, and was
subsequently incubated in pH 7.2, 4 1C oxygenated
solution C containing (in mM) NaCl 106; NaHCO
3
18; KCl 5; CaCl
2
5; NaHOP
4
2; MgSO
4
1, glucose 5;
HEPES 2.5; glutamine 2; butyric acid 10 and lactic
acid 4. Aliquots of 6 ml (containing 1–1.5 mg of
protein/ml) were placed in siliconized Erlenmeyer
flasks and gassed on ice for 5 min with 95% O
2
/5%
CO
2
, followed by a 10-min period at room
temperature. At this moment, tubules were ready
for the experiment (Gesek et al., 1987).
2.4.2.2. PT toxicity protocol. Bee venom concen-
trations of 0.1, 2.0 and 10 mg/ml were added at
baseline to oxygenated PT suspensions.
2.4.2.3. Hypoxia/reoxygenation (H/R) protocol. -
After equilibration, tubules were divided into an
experimental and a time control group. The pO
2
in
the control groups was kept throughout the experi-
ment in the 200–300 mmHg range. The experimental
groups were rendered hypoxic (pO
2
averaged
20–40 mmHg) by gassing with 95% N
2
/5% CO
2
for 5 min. The duration of the hypoxic period was
15 min. After hypoxia, tubules were reoxygenated
by gassing with 95% O
2
/5% CO
2
for 5 min. The pO
2
after reoxygenation returned to the 200–300 mmHg
range. The flasks were then resealed and tubules
were kept reoxygenated for 45 min. Bee venom at
concentrations of 0.1 and 2 mg/ml were added at
baseline to PT.
2.4.2.4. Cell injury. Cell injury was assessed by
LDH release (Bergmeyer, 1974). LDH was mea-
sured after removal of 1 ml sample from tubules
suspension (venom and time control groups) at
baseline, after hypoxia (15 min) and after reoxy-
genation (60 min). LDH release was calculated by
dividing supernatant LDH by total LDH (super-
natant+pellet) and expressed as a percentage
(n ¼ 5).
2.4.3. Analytical methods
Inulin was determined by chemical anthrone
method. CPK, AST, ALT and LDH were assessed
by colorimetry (Hitachi auto-analyzer model 917,
Japan). Hct was assessed by a microhematocrit
method. Sodium concentrations were determined by
electrolyte analyzer (9180 Electrolyte Analyzer,
AVL Scientific Co., Rosweel, GA, USA) and
creatinine by colorimetry (Jaffe, Spectrophotometer
ByoSystems BTS 310, Barcelona, Spain).
GFR and fractional excretion of sodium (FeNa)
were determinate by the usual formulas.
2.4.4. Statistical analysis
Results are presented as mean7standard error of
mean (SEM). Comparisons were done by two-tail
paired, unpaired Student’s t-test or one-way ANO-
VA with Tukey multiple comparisons test, as
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L.S.D. Grisotto et al. / Toxicon 48 (2006) 44–54 47
appropriate. p values o0.05 were considered
significant.
3. Results
3.1. Renal function and hemodynamics
There was a significant decrease in GRF and
urinary volume after venom infusion. In the same
way, venom caused a sharp and immediate decrease
in RBF, which was more pronounced in the cortex
(72% vs. 48% of decrease in medulla). BP remained
stable. In the control group, saline infusion did not
change GFR, RBF, urinary volume or blood
pressure (Table 1).
After 24 h of injection, diuresis and RBF were
similar in the two groups, but GFR remained
significantly depressed in venom-infused animals.
The FeNa and urinary sodium were higher in the
venom group; however, this difference did not reach
statistical significance. BP was similar and remained
within normal limits in both groups (Table 2).
3.2. Direct tubular toxicity
Bee venom caused cellular injury to normoxic PT
in all concentrations utilized (Fig. 1). At 15 min of
hypoxia, bee venom had no additional effect on
hypoxia-induced PT injury. LDH was 3674% in
hypoxic PT (po0:01 vs. control PT) compared with
hypoxic PT plus 0.1 mg of venom (4276%, NS vs.
hypoxia alone) and 2.0 mg of venom (48710%, NS
vs. hypoxia alone). However, 2.0 mg of venom
enhanced the H/R injury observed after reoxygena-
tion (LDH 8573% in H/R plus venom vs. 5573%
in H/R alone, po0.01).
3.3. Enzymes and Hct
There was a significant increase in serum LDH,
CPK and AST in the venom group as compared to
control animals, whereas ALT and Hct did not
change significantly (Table 3).
3.4. Histology
3.4.1. Acute study
In the venom group, the lesion ranged from mild
and moderate glomerular tuft retraction to mild
acute tubular cell degeneration and tubular dilata-
tion. In the control group, renal histological was
normal or presented mild glomerular tuft retraction.
3.4.2. Twenty-four-hour study
In the venom group, rats presented flattened
tubular epithelium, acute degeneration of tubular
cells, presence of detached epithelial cells in tubular
lumen and foci of tubulorrhexis (Fig. 2). In the
control group, animals showed normal renal histol-
ogy or mild tubular dilatation.
Immunohistochemical analysis revealed massive
presence of myoglobin in lumen and cells of renal
tubules of the venom group (Fig. 3).
4. Discussion
Very few studies have addressed experimentally
the effects of bee venom on renal function. dos Reis
ARTICLE IN PRESS
Table 1
Comparison of renal function, renal hemodynamics and mean
arterial blood pressure levels before (baseline) and immediately
after the injection of bee venom (venom) or saline (control) to
male Wistar rats
Baseline Venom Baseline Control
GFR (ml/min/
100 g)
0.8470.05 0.4070.08
a
0.9670.05 0.9070.05
Diuresis (ml/
min)
13.072.2 6.671.3
b
13.470.6 17.972.3
Cortical RBF
(TPU)
34.974.9 9.970.9
c
28.175.8 30.178.1
Medullary
RBF (TPU)
7.771.2 4.070.8
d
7.470.4 7.770.6
BP (mmHg) 12175 12177 12874 12774
Mean7SE; GFR: glomerular filtration rate; RBF: renal blood
flow; BP: blood pressure.
a
po0.0001 vs. baseline.
b
p ¼ 0:002 vs. baseline.
c
p ¼ 0:004 vs. baseline.
d
p ¼ 0:024 vs. baseline.
Table 2
Renal function, renal blood flow and mean arterial blood
pressure levels 24 h after the injection of bee venom (venom) or
saline (control) to male Wistar rats
Venom (n ¼ 7) Control (n ¼ 6) p
GFR (ml/min/100 g) 0.6770.10 1.0870.06 0.0077
RBF (ml/min) 5.470.7* 5.270.7 0.8998
FeNa (%) 0.9570.18 0.6570.05 0.1655
UNa (mEq/L) 189736 113710 0.0868
Diuresis (ml/24 h) 10.170.8 9.471.4 0.6534
BP (mmHg) 11476 13075 0.0608
Mean 7 SE; GFR: glomerular filtration rate; RBF: renal blood
flow; FeNa: fractional excretion of sodium; UNa: urinary
sodium; BP: blood pressure; *n ¼ 6 for RBF in venom.
L.S.D. Grisotto et al. / Toxicon 48 (2006) 44–54 48
et al. observed GFR decrease, FeNa increase, acute
tubular necrosis and presence of myoglobin in renal
tubules 3–8 h after an intravenous injection of
Africanized bee venom in rats. Twenty to 30 h after
venom injection, the functional and histological
changes were still present (dos Reis et al., 1997,
1998). The current paper discloses new aspects of
the mechanisms of bee venom-induced renal injury.
Accidents with 500 or more bee stings are
potentially lethal to humans due to the massive
amount of venom injected (Vetter et al., 1999;
Schumacher and Egen, 1995; Vetter and Visscher,
1998). Although the composition and lethality of
venom are similar between European and Africa-
nized bees (Schmidt, 1995; Vetter and Visscher,
1998; Schumacher et al., 1990), European bees have
around 40% more of venom per venom reservoir
(Schumacher et al., 1990, 1992; Funari et al., 2001).
On the other hand, African bees required signifi-
cantly smaller electro stimulus to promote stinging
and liberated larger percentages of reservoir venom
when stimulated (Funari et al., 2001). Indeed, the
main reason for the serious systemic reactions after
Africanized bee attacks is the high number of stings
rather than differences in the potency or amount of
venom per bee. A sac of venom of an Africanized
bee contains around 100 mg of venom (Schumacher
et al., 1990, 1992; Funari et al., 2001), so an accident
with 500–1000 stings would deliver approximately
0.7–1.4 mg/kg of venom in an individual of 70 kg.
Thus, the amount of venom used in the present
study (0.5 mg/kg) is clinically relevant.
The experimental injection of bee venom caused a
picture alike to the observed in patients with bee
venom-induced ARF, i.e. an important and early
GFR and diuresis decrease. In fact, there are clinical
reports of oliguria and serum creatinine increase as
soon as 2 h after the attack (Franca et al., 1994).
Early histology disclosed retraction of the glomer-
ular tuft and mild acute tubular injury. Glomerular
retraction is usually associated to glomerular
ischemia, what is consistent with the striking RBF
decrease observed after venom infusion. After 24 h,
the lesion evolved to frank tubular injury, which is
the lesion found in renal biopsies or autopsies of
patients with bee venom-induced ARF (Mendes
et al., 1990; Franca et al., 1994; Beccari et al., 1992;
Bourgain et al., 1998; Hommel et al., 1998).
Although GFR decrease was previously demon-
strated after bee venom injection in rats (dos Reis
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Control 0.1 µg 2 µg 10 µg Control 0.1 µg 2 µg 10 µg
0
10
20
30
40
50
60
70
venom
*
**
***
L
D
H

(
%
)
0
25
50
75
100
venom
**
***
***
L
D
H

(
%
)
* p<0.05 vs control
** p<0.01 vs control
*** p<0.001 vs control
15 minutes of incubation 60 minutes of incubation
Fig. 1. LDH release from control proximal tubules and from proximal tubules incubated with bee venom concentrations of 0.1, 2 and
10 mg/ml for 15 and 60 min. Note that even 0.1 mg/ml of venom caused significant tubular injury.
Table 3
Enzymes and hematocrit immediately after the injection of bee
venom (venom) or saline (control) to male Wistar rats
Venom (n ¼ 8) Control (n ¼ 8) p
CPK (IU/L) 8217141 277754 0.0057
LDH (IU/L) 12127210 357761 0.0045
ALT (IU/L) 146711 8778 0.0006
AST (IU/L) 7474 7077 0.6275
Hematocrit (%) 4873 4477 0.6076
Mean7SE; CPK: creatine phosphokinase; LDH: lactic dehy-
drogenase; ALT: serum glutamic pyruvic transaminase; AST:
serum glutamic oxaloacetic transaminase.
L.S.D. Grisotto et al. / Toxicon 48 (2006) 44–54 49
et al., 1997, 1998), the effects of the venom on RBF
were never assessed before. The venom caused a
remarkable and immediate RBF decrease, which
was more profound in the cortical area. RBF
returned to normal 24 h after venom infusion. This
disparity in regional RBF impairment has been
described in ARF models and has been related to
severe renal ischemia (Dworkun et al., 2000). The
main component of the venom is melittin, a 26
amino acid highly basic peptide, accounting for
approximately 50% of the venom dry weight
(Schumacher and Egen, 1995; Habermann, 1972;
Vetter and Visscher, 1998). Melittin might be
related to RBF impairment in many ways. It can
damage the vascular endothelium (Schumacher and
Egen, 1995), cause vasoconstriction (Garcia-Sainz
et al., 1990; Koyama et al., 2002; White and
Robertson, 1987), smooth muscle cells contraction
(Franca et al., 1994; Tunget and Clark, 1993),
increased renal rennin secretion (Churchill et al.,
1990), catecholamine (Franca et al., 1994) and
arachidonic acid release (Hassid and Levine, 1977)
and enhanced thromboxane B
2
production (Garcia-
Sainz et al., 1990). Phospholipase A
2
accounts for
around 12% of the venom dry weight (Han et al.,
2000). It has been related to diverse situations of
intrarenal vasoconstriction (Imig and Deichmann,
1997; Kawaguchi et al., 1987; Rossi et al., 1989) and
may induce RBF impairment by the release of
vasoconstrictor eicosanoids and catecholamine. The
venom sac contains about 2 mg of histamine, so 500
stings may deliver 14 mg/kg of exogenous histamine
in a 70 kg adult (Schumacher and Egen, 1995). The
mast cell degranulating peptide present in the
venom may induce more endogenous histamine
release. Since 1 mg/kg of histamine can produce
cardiovascular changes in healthy humans (Schu-
macher and Egen, 1995), it is conceivable that
histamine might play a role in bee venom-induced
RBF decrease. Actually, severe hypotension has
been described after massive bee attacks resulting in
ARF (Mejia et al., 1986; Franca et al., 1994; Diaz-
Sanchez et al., 1998; Humblet et al., 1982). That
does not seem to be the case in the present model,
since BP remained stable after venom injection.
Dopamine and norepinephrine are other venom
constituents potentially related to renal hemody-
namics changes. There are descriptions of hyperten-
sion after massive bee accidents, possibly due to
excess of catecholamine directly injected with the
venom and endogenously released by melittin and
phospholipase A
2
(Mendes et al., 1990; Munoz-
Arizpe et al., 1992a; Sert et al., 1993; Franca et al.,
1994; Bresolin et al., 2002; Bourgain et al., 1998).
Finally, RBF impairment might be related to
venom-induced cardiac changes. Injection of ve-
nom, 0.8 ml/100 g, produced an infarct-like myocar-
dial injury in rats (Ferreira et al., 1995). Clinically,
myocardial infarction has been described after
massive bee venom inoculation (Franca et al.,
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Fig. 2. Acute tubular damage 24 h after venom infusion. Note flattened proximal tubule epithelium, degeneration and loss of proximal
tubule epithelial cells and tubulorrhexis (HE, 400 Â).
L.S.D. Grisotto et al. / Toxicon 48 (2006) 44–54 50
1994). This event is improbable in the current
experiment. The amount of venom infused was
almost half of that used in studies producing
myocardial necrosis and blood pressure remained
stable after venom injection.
Bee venom produced clear dose-dependent PT
toxicity. Additionally, to the best of our knowledge,
for the first time it was demonstrated that this
venom may enhance tubular hypoxia/reperfusion
injury. Franca et al. (1994) found high serum and
urine concentrations of venom in two fatal cases
that developed bee venom-induced ARF. In the
most severe case, concentrations of 1.9 and 3.1mg/ml
of venom were found in urine, respectively 24 and
53 h after the attack. In contrast, in a surviving
patient who did not develop ARF, the maximal
urinary level of venom was 0.19 mg/ml. Indirect
evidences of proximal tubular toxicity were also
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Fig. 3. (a) Immunostaining for myoglobin in a bee venom-injected rat. There is a massive presence of myoglobin (brown) in the tubular
lumen and in the cytoplasm of proximal tubular cells. (b) In frank contrast, there is no significant immunostaining in the proximal tubular
cells nor in the luminal tubular area of control rats.
L.S.D. Grisotto et al. / Toxicon 48 (2006) 44–54 51
provided by an impressive elevation of the frac-
tional excretion of sodium and mitochondrial
structural changes suggestive of direct venom
toxicity in rats infused with bee venom (dos Reis
et al., 1997, 1998). Han et al. (2000) provided
additional substantiation of direct nephrotoxicity.
They showed that bee venom decreased cell
viability, increased LDH release, impaired apical
transporter activity and increased calcium uptake in
cultured rabbit PT cells. Melittin had identical
effects as the whole venom. Subsequently, the same
group found that bee venom or melittin increased
the formation of lipid peroxide by cultured PT cells.
This lipid peroxide release was blocked by antiox-
idants, phospholipase A
2
inhibitors, lipoxygenase
inhibitors and epoxygenase inhibitors (Han et al.,
2002). Supporting a role for phospholipase A
2
in
bee venom renal toxicity, Zager et al. (1993)
demonstrated that bee venom phospholipase A
2
produced direct toxicity to oxygenated isolated rat
PT and had an additive effect on hypoxia-mediated
injury.
Rhabdomyolysis is a well-known cause of ARF.
Myoglobin toxicity has been related to renal
vasoconstriction, intraluminal cast formation and
direct heme-protein cytotoxicity (Zager, 1996). In
the present experiment, bee venom caused an early
and significant increase in CPK, a reliable marker of
the presence and intensity of muscle injury (Van-
holder et al., 2000), and a massive tubular deposi-
tion of myoglobin. Other intramuscular cell
enzymes like LDH and AST increased as well.
Experimental bee venom-induced rhabdomyolysis
with enzyme elevations was previously described by
Azevedo-Marques et al. (1992). However, these
authors used a much higher amount of intravenous
venom (1.5 ml/100 g), causing apnea 70 s to 13 min
after venom infusion, and did not assessed renal
function. The main factor responsible for rhabdo-
myolysis in bee venom is likely melittin (Ownby et
al., 1997). This substance inserts itself into the
phospholipid bilayer of cell membranes causing
hydrolysis and cell disruption (Schumacher and
Egen, 1995). Phospholipase A
2
may also damage the
cell membrane and in fact bee venom phospholi-
pases have already been implicated in rhabdomyo-
lysis (Ownby et al., 1997). Clinically, many cases of
bee venom-induced ARF presented rhabdomyolysis
evidenced by enzymes increase or myoglobinemia
and myoglobinuria (Mejia et al., 1986; Sert et al.,
1993; Franca et al., 1994; Diaz-Sanchez et al., 1998;
Kolecki, 1999; Bresolin et al., 2002; Bourgain et al.,
1998; Hommel et al., 1998; Humblet et al., 1982).
However, cases of rhabdomyolysis without ARF
(Franca et al., 1994) and ARF without rhabdomyo-
lysis after massive bee inoculation have also been
reported (Franca et al., 1994; Beccari et al., 1992)
suggesting a secondary role for muscle injury in
human bee venom-induced ARF.
In the present experiment, there was no evidence
of hemolysis. Clinically, ARF was described both
with (Munoz-Arizpe et al., 1992a; Franca et al.,
1994; Bresolin et al., 2002; Humblet et al., 1982;
Deshmukh and Borse, 1996) and without concomi-
tant hemolysis (Diaz-Sanchez et al., 1998; Beccari
et al., 1992; Bourgain et al., 1998; Hommel et al.,
1998), suggesting that hemolysis is not obligatory
for ARF development after massive bee venom
inoculation.
In summary, in a clinically relevant model of bee
venom-induced ARF, the mechanisms for renal
injury were found to be severe vasoconstriction,
intense direct tubular toxicity and rhabdomyolysis.
Renal lesion occurred in the absence of hypotension
and hemolysis.
Acknowledgments
Dr. Emmanuel A. Burdmann is partially sup-
ported by a grant from the National Council for
Scientific and Technological Department (CNPq),
Brazil.
Parts of this study were presented at the 33rd
Annual Meeting of the American Society of
Nephrology, Toronto, Canada, and published in
abstract form (J. Am. Soc. Nephrol. 11:129A, 2000).
The authors are grateful to Kleber G. Franchini
for valuable support and advice regarding the laser
renal blood flow measurements. In addition, we
thank Livia C. Burdmann for the excellent grammar
review of the manuscript.
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