Am. J. Trop. Med. Hyg., 67(2), 2002, pp. 217–222
Copyright © 2002 by The American Society of Tropical Medicine and Hygiene
BOTHROPS MOOJENI SNAKE VENOM-INDUCED RENAL GLOMERULI
CHANGES IN RAT
PATRÍCIA ALINE BOER-LIMA, JOSÉ ANTONIO ROCHA GONTIJO, AND MARIA ALICE DA CRUZ-HÖFLING
Departamento de Biologia Celular, Instituto de Biologia, Universidade Estadual de Campinas (Unicamp), Campinas, S.P., Brazil;
Núcleo de Medicina e Cirurgia Experimental, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (Unicamp),
Campinas, S.P., Brazil; Departamento de Histologia e Embriologia, Instituto de Biologia, C.P. 6109, Universidade Estadual de
Campinas (Unicamp), Campinas, S.P., CEP 13083-970, Brazil
Abstract. The venom of Bothrops moojeni has potent proteolytic and phospholipase A2 activities. In previous work,
we showed that intravenous injection of this venom in rats decreased creatinine clearance and caused tubular dysfunction and histopathological changes with no alterations in blood pressure. The current study used scanning and transmission electron microscopy to assess the ultrastructural changes caused by B. moojeni venom (0.4 mg/kg i.v.) in rat renal
glomeruli and correlated these alterations with the severity of proteinuria 5 hours, 16 hours, and 48 hours after venom
injection. The changes included mesangiolysis, glomerular microaneurysms, and glomerular basement membrane
(GBM) abnormalities. In addition, there was a reduction in the number and width of podocyte pedicels, which caused
a reduction in the number of filtration slits. Electron-dense amorphous material, which may be proteinaceous in origin,
was found in the pedicels. The severity of the ultrastructural abnormalities correlated with the level of proteinuria. These
morphophysiological changes were attributed to biochemical and physiological disturbances in the components of the
GBM and mesangial matrix as well as in cytoskeleton-associated proteins of podocytic processes, and could account for
the breakdown of optimal glomerular filtration barrier functioning. These results, together with the absence of appreciable glomerular fibrin deposits, support the hypothesis of a direct activity of B. moojeni venom on rat kidneys.
Proteolytic activity of the venom on renal glomeruli could then contribute to the onset of acute renal failure, and would
explain the clinical manifestations of renal injury after bites by this and other Bothrops species.
and received an intravenous injection of 0.4 mg/kg B. moojeni
venom. The dose of 0.4 mg/kg was chosen because it had been
used by Burdmann et al8 in their experimental model of
venom-induced ARF. The venom was dissolved in 0.15 M
NaCl immediately before use. One group of rats was injected
with venom (V), and the control group received the vehicle,
0.15 M NaCl alone (S).
Measurement of proteinuria. The total urine protein excretion of the control group (n ⳱ 5) and of rats injected with B.
moojeni venom was evaluated 5 hours (V, n ⳱ 5), 16 hours
(V, n ⳱ 5), and 48 hours (V, n ⳱ 5) postinjection. At 8:00
a.m., each rat received a tap water load by gavage (5% of
body weight) followed by a second load of equal volume 1
hour later. Twenty minutes after the second load, the rats
were housed individually in metabolic cages, and spontaneously voided urine was collected over a 2 h period. Urine
samples were diluted in 1% sulfasaliycylic acid, and the protein concentrations were determined spectrophotometrically
at a wave length of 550 nm and compared with known albumin standards. Statistical analysis of the data was performed
using nonparametric Kruskal-Wallis and Mann-Whitney
tests. The significance level was set at P ⱕ 0.01.
SEM studies. Five hours (V, n ⳱ 2; S, n ⳱ 1) and 16 hours
(V, n ⳱ 2; S, n ⳱ 1) after injection, the rats were anesthesized
with sodium pentobarbital (Sagatal, 60 mg/kg i.p.) and perfused as in the TEM studies discussed later. After perfusion,
renal cortical slices were immersed in Karnovsky’s solution
(2% glutaraldehyde, 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) at 4°C overnight. After rinsing in phosphate buffer for 1 hour, the specimens were postfixed in buffered 1% OsO4 at 4°C in the dark for 2 hours and then immersed in a 2.3 M sucrose solution at 4°C overnight. The
specimens were subsequently immersed for 30 min in liquid
nitrogen and then fractured, washed in the same buffer, dehydrated in a graded acetone series, and critical-point dried
(Balzers CPD 030). After identifying the fractured surface,
INTRODUCTION
Snakebite is a serious health problem in Brazil. Of the
20,000 snakebite accidents reported per year, about 90% are
attributed to the genus Bothrops.1 The most serious systemic
effect and the most common complication in lethal cases is
acute renal failure (ARF) secondary to acute tubular necrosis
and occasionally glomerulonephritis.2–5 Glomerular changes
in human victims of snakebite poisoning have attracted little
attention, and there has been no systematic study of this action of snake venoms. Experimental studies of renal changes
after injection of bothropic venom showed degenerative lesions of tubular cells and glomeruli, with mild proliferation of
the mesangial matrix,6 glomerular congestion,7 and massive
glomerular fibrin deposition immediately after venom infusion.8 We observed a significant decrease in the glomerular
filtration rate, an increase in the fractional urinary Na+ and
K+ excretion, and significant histological changes in the tubular and glomerular structure after intravenous injection of
Bothrops moojeni venom.9 The alterations observed by light
microscopy include glomerulonephritis characterized by hypercellularity, glomeruli lobulation with retraction of the capillary tuft, and condensed mesangial matrix-forming nodular
lesions. The mean arterial pressure remained unchanged.
In this study, transmission and scanning electron microscopy (TEM/SEM) were used to characterize the glomerular
alterations caused by B. moojeni venom in rats. The protein
urinary excretion rates were also examined.
MATERIALS AND METHODS
Sample preparation. Male Wistar rats (200–300 g) were obtained from an established colony maintained by the University’s Central Animal House Service. Lyophilized B. moojeni
venom was donated by the Instituto Butantan (São Paulo)
and stored at −20°C. The rats were anesthetized with ether
217
218
BOER-LIMA AND OTHERS
specimens were mounted on stubs, sputtered with gold (Balzers SCD 050) for 120 s, and examined and photographed with
a Zeiss DSM 940A scanning electron microscope operated at
10 kV.
TEM studies. Five hours (V, n ⳱ 4; S, n ⳱ 2) and 16 hours
(V, n ⳱ 4; S, n ⳱ 2) after injection, the rats were anesthesized
with sodium pentobarbital (Sagatal威, 60 mg/kg, i.p.) and perfused in situ via the left ventricle using gravity flow, initially
with 100 mL of saline containing 0.1% heparin followed by
2.5% glutaraldehyde in 0.1 M phosphate buffer for 20 min.
After perfusion, the kidneys were removed, and cortical slices
were cut into small pieces, which were immersed in the same
fixative with 0.1% tannic acid and 5% sucrose for 3 hours at
room temperature. After rinsing in a sugar-saline solution
(0.15 M NaCl, 0.2 M sucrose), the specimens were postfixed
with 1% OsO4 at 4°C in the glucose-saline solution in the
dark for 2 hours and then rinsed again in the glucose-saline
solution. The samples were dehydrated in a graded ethanol
series and embedded in Epon 812 resin at 60°C for 48 h. Thin
sections (60–70 nm) were double-stained with uranyl acetate
and lead citrate and were observed and photographed with
a LEO 906 transmission electron microscope operated at
60 kV.
the nuclear portion and surrounded the capillary loops. An
interdigitating primary process derived from an adjacent
podocyte always occurred between two primary processes of
the same contiguous podocyte. The primary processes
branched, giving rise to secondary and tertiary processes or
pedicels. The terminal processes or pedicels interdigitated
closely with similar processes from adjacent podocytes, leaving narrow filtration slits between them (Figure 2 A and B).
The bulging surface of the podocyte bodies also showed occasional short pedicel-like microprojections (see Figure 2 A
and B). This elaborate epithelium was anchored to the outer
surface of the underlying thick GBM surrounding the glomerular endothelium.
Five hours (Figure 2 C) and 16 hours (Figure 2 D) after a
venom dose of 0.4 mg/kg, the glomerular visceral epithelium
lost its peculiar cohesive appearance, and the tips of the pedicels projected into the Bowman’s space. Upright pedicels
appeared as long, thin, club-ended projections emerging from
RESULTS
After 5 hours and 16 hours, four animals (two of each
group) voided red-colored urine indicative of hematuria and/
or hemoglobinuria. B. moojeni venom induced massive acute
proteinuria, which was significantly different from control
rats after 5 hours, 16 hours, and 48 hours (P ⱕ 0.01). These
changes were short lived because they partially recovered by
48 hours (Figure 1) and were consistent with the SEM and
TEM observations of drastic morphological alterations in the
mesangium, podocytes and glomerular basement membrane
(GBM).
The visceral epithelium of glomeruli from control rats injected with 0.15 M NaCl solution had a normal ultrastructure
as seen by SEM. The podocytes of the epithelium closely
covered all of the outer surface of the glomerular capillary
loops. The primary processes of the podocytes emerged from
FIGURE 1. Time course of the proteinuria after the injection of B.
moojeni snake venom (0.4 mg/kg i.v.) compared with saline-treated
(S) rats. The columns represent the mean ± SEM. *P ⱕ 0.01 compared with S.
FIGURE 2. Scanning electron micrographs of glomeruli. A. Threedimensional organization of the outer surface of podocytes (p) surrounding capillaries of a renal glomerulus in a control rat (× 4,800,
bar ⳱ 2 ␮m). B. Detail of the entwined processes of the podocytes of
a control glomerulus. Note the primary (1) and secondary (2) processes and the pedicels (arrowhead) among which filtration slits can
be seen (arrow) (× 9,900, bar ⳱ 1 ␮m). C. A damaged glomerulus 5
hours after venom injection in which the swollen podocytes exhibit
villous transformation involving highly thin and branched filamentous pedicels with a chaotic appearance and no cohesive arrangement
(× 4,800, bar ⳱ 2 ␮m). D. View 16 hours after envenomation: note
the numerous wavy microprojections representing noninterdigitating
pedicels and the loss of filtration slits. Note also the club-shaped distal
ends of the pedicels. (× 17,600, bar ⳱ 0.5 ␮m.)
219
GLOMERULAR EFFECTS OF B. MOOJENI VENOM
the podocytes. Consequently, the filtration slits disappeared,
and there was obvious impairment of the filtration barrier. In
light microscopy, this feature appeared as glomerular lobulation. Another change observed was the increased number of
microprojections arising from the nuclear portion of
podocytes in venom-treated rats. This increase in microprojections is characteristic of a disorder known as villous
glomerular transformation.
TEM showed that in saline-treated rats the filtration barrier retained its normal organization of interdigitating pedicels bridged by the filtration slit membrane, the GBM, and
the thin endothelium with fenestrations (Figure 3 A and B).
However, this regular arrangement of the podocyte foot processes disappeared almost completely 5 hours (Figure 3 C and
D) and 16 hours (Figure 3 E and F) after venom injection. In
the presence of venom, the podocytes showed intense simplification, with loss of the filtration slits and the appearance of
wide, irregular profiles at the terminal ends of the pedicels.
There was a reduction in the complexity of cell–cell connections as a result of podocyte simplification or foot process
effacement, which ranged from partial retraction of the foot
processes to total disappearance of the usual interdigitated
pattern. In addition, the cytoplasm of pedicels showed electrondense irregular masses (Figures 3 D–F and 4 C) and the
number of endothelial fenestrations decreased (Figures 3 C
and D and 4 A). The regular width and homogeneous appearance of the GBM was also lost, with this membrane now
FIGURE 3. Transmission electron micrographs showing the filtration barrier in renal corpuscles. A and B. Controls showing all the
structural components of the filtration barrier, including the fenestrations of the glomerular endothelium (arrowheads), the GBM (b),
the podocyte pedicels (pp), and the filtration slits crossed by tenuous
diaphragm membranes (arrows). After 5 hours (C and D) and 16
hours (E and F), there was a drastic decrease in the number of pedicels and slit diaphragms. Electrondense irregular masses occur in the
pedicels (*). Note in C and D the decrease in endothelial fenestrations and in F the rupture of the pedicel apical membrane. (A, C, and
E, × 66,720, bar ⳱ 0.2 ␮m; B, D, and F, × 30,060, bar ⳱ 0.37 ␮m.)
showing discontinuities (Figure 3 D) or becoming narrower
and denser (Figure 3 F) than in the controls.
Additionally, the podocytic processes showed several alterations, including accumulation of absorption droplets in the
cell’s lysosomal system (Figure 4 A), irregular amorphous
masses of granular material (Figure 4 C), pseudocyst formations (Figure 4 A–C), an increasing polyribosome population
(Figure 4 E and F), and a peculiar arrangement of protein
electrondense deposits within greatly enlarged rough endoplasmic reticulum cisternae (Figure 4 E and F) that were not
found in the controls. Degenerative changes were also seen in
the intraglomerular mesangium. The lesions began with local
dissolution of the mesangial matrix or mesangiolysis as well as
local protrusion of the mesangial cells into glomerular capillaries, which, together with expanded glomerular endothelial
cell bodies, could cause occlusion of the capillaries (Figure 5).
Other prominent features in glomeruli 5 hours and 16 hours
after venom injection were capillary ballooning and the eventual formation of microaneurysms (Figure 6).
DISCUSSION
The results of this study show that in rats B. moojeni venom
causes proteinuria and ultrastructural changes in the visceral
epithelium and glomerular capillaries tufts compatible with
FIGURE 4. Details of the changes in podocytes 5 hours (A) and 16
hours (B, C, D, and F) after venom injection. A. The cytoplasm of the
pedicels contains an increased number of lysosomes (ly) or protein
globules. A and D. Several pinocytotic invaginations (arrows) occur
along the surface of the epithelial cell membrane facing the urinary
spaces (us); small profiles of upright pedicel can also be seen. B and
D. Note pseudocyst formations (夝) and long and extremely thin
projections (arrowheads). C. Dense masses in the cytoplasm of a
broaded pedicel (*); filtration slits are absent. E and F. The enlarged
cisternae of podocyte rough endoplasmic reticulum containing bizarre masses of electrondense material are shown. (A, × 9,187, bar ⳱
1 ␮m; B, × 5,755, bar ⳱ 2 ␮m; C, × 58,380, bar ⳱ 0.2 ␮m; D, × 8,992,
bar ⳱ 1 ␮m; E, × 26,720, bar ⳱ 0.5 ␮m; F, × 16,275, bar ⳱ 0.5 ␮m.)
220
BOER-LIMA AND OTHERS
FIGURE 5. Partial view of renal glomeruli 16 hours after venom
injection. A and inset a are electron micrographs showing the cell
body of a mesangial cell and components of the mesangial matrix,
both occluding the glomerular capillary. A, left side. A capillary with
several bulbous projections (夝), which are less electrondense than
endothelium, can be seen. B. The glomerular capillaries occluded by
the cell bodies of endothelial (e) and mesangial (m) cell. Some
cytoplasmic projections from mesangial cells imbricate with the endothelium. Note the hypertrophied Golgi apparatus (arrowheads)
of the mesangial cell. C. Diapedesis of a mesangial cell through
the constricted lumen of a capillary. (A, × 6,681, bar ⳱ 1.5 ␮m; inset
a, × 9,000, bar ⳱ 1 ␮m; B, × 11,150, bar ⳱ 1 ␮m; C, × 14,400, bar ⳱
1 ␮m.)
the renal dysfunction described elsewhere.9 Significant proteinuria occurred 5 hours, 16 hours, and 48 hours after venom.
However, the level of proteinuria had recovered considerably
after 48 hours, indicating that the process was reversible. Proteinuria has been associated invariably with effacement of the
podocyte foot processes of glomerular epithelia10 and lysosomal accumulation.11 In agreement with this, we found visceral epithelial changes that included podocyte simplification
as well as lysosomal clusters accumulated in podocyte endings. In addition, the appearance of electron-dense clumps of
amorphous bodies in the cytoplasm of the remaining processes, the formation of pseudocysts, and the loss of normal
slit-diaphragm integrity and orientation were associated with
glomerular dysfunction. These features were seen in both
SEM and TEM. Arakawa and Tokunaga12 showed that the
loss of foot processes is caused by their withdrawal into the
cell body and not by the fusion of adjacent processes to form
a syncytium. Foot process effacement or simplification would
represent a reduction in the complexity of the usual interdigitating pattern of cell–cell connections and an obvious diminution of filtration capacity.
FIGURE 6. Two partial views of glomeruli 5 hours (B) and 16
hours (A) after venom injection. A. The capillary (c1) forms a complete ring (microaneurysm) with fragments of mesangium (arrows);
in the urinary space (us), there are numerous microprojections and/or
segments of wavy pedicels. Inset a. The decreased density of the
mesangial matrix. B. The abnormal location of a mesangial cell (m1)
beneath the endothelium (e); another mesangial cell (m2) is in its
usual location between the capillary loops. Note that capillary represents a microaneurysm, and its lumen contains endothelial and mesangial cell fragments. c, capillary; b, basal lamina; co, collagen bundle.
(A, × 4,008, bar ⳱ 3 ␮m; a, × 7,515, bar ⳱ 1 ␮m; B, × 5,344, bar ⳱
2 ␮m.)
Club-ended pedicels (by SEM) and enlarged processes (by
TEM) were seen resting over the GBM and contained clumps
of electrondense material. An explanation for such changes
described elsewhere13 includes podocyte fixation to the GBM
via broad, sheet-like processes that contained a highly organized network of cytoskeletal proteins formed by microfilaments regularly cross-linked to dense bodies, with prominent
expression of ␣-actinin. These findings have been interpreted
as indicative of an adaptative change in the cell in which the
rearrangement and hypertrophy of the contractile apparatus
would reinforce podocyte adherence to the GBM.13 Similar
alterations have been found in human glomerulonephritis associated with nephrotic syndrome14,15 and in nephrosis induced by puromycin aminonucleosides, which are toxic to
visceral epithelial cells.11,16,17 Changes in the state of cytoskeletal aggregation are associated with decreased fibrillar
attachment of the epithelium to the GBM and with the onset
of massive proteinuria.16,18,19 Our results reproduced these
abnormal features and provided evidence for a toxic effect of
B. moojeni venom on visceral epithelial cells.
To understand how B. moojeni venom may induce such
podocytic alterations, it must be recalled that most of the
glomerular F actin is concentrated in the foot process of
GLOMERULAR EFFECTS OF B. MOOJENI VENOM
podocytes, and that these densely-packed cytoskeletal filaments are associated with the ability to maintain and/or generate significant changes in podocytes shape as well as to
provide support for the foot processes.20,21 Consequently, alterations in the state of F actin filament polymerization responsible for anchoring the pedicels to the GBM would alter
the function of the filtration barrier.
Andrews21 reported that the treatment of glomeruli in vitro
with cytochalasin B (a drug that inhibits the contraction of
actin filaments) changed the shape of podocyte foot processes
and thereby altered the number of open filtration rifts. These
alterations may have been involved in the changes in the
ultrafiltration coefficient and, consequently, in the rate of glomerular filtration. Because the glomerular and renal tubule
epithelial cells are strategically interposed between the extraand intramilieu, they are potential targets for numerous nephrotoxic agents, and the glomeruli are the first structure of the
nephron to come in contact with circulating venom.
The proteolytic and phospholipase A2 activities of Bothrops venoms have important cytotoxic effects in many cell
types,22–24 and could contribute to the nephrotoxicity seen in
the current study. A hypothetical cytotoxin-like mechanism
of action has been proposed for bothropic myotoxins acting
on cultured cells, liposomes, and skeletal muscle cells in
vivo.23 After binding to an unidentified site on the cell plasma
membrane, the myotoxins would penetrate the bilayer
through hydrophobic interactions, destabilizing the membrane and causing impaired permeability to ions and macromolecules. A prominent calcium influx would probably be the
most important consequence of membrane disturbance and
would be responsible for the onset of a variety of destructive
mechanisms such as cytoskeletal alterations, mitochondrial
damage, and the activation of calcium-dependent proteases
and endogenous phospholipases, which, in turn, would account for the cellular damage.
The ultrastructural alterations induced by the snake venom
in the trilaminar structure of the GBM suggest the involvement of physicoelectrostatic barrier sites. Podocytes and endothelial cells retain heparin sulfate25 in the lamina rara externa and interna of the GBM, and the feet processes are
coated with sialoproteins that contribute to the anionic
charges of the filtration barrier.26 This ionic barrier of glomerular filtration was shown to be destroyed by Vipera russelli venom in isolated rat kidneys.27
Several proteins, including type IV collagen, laminin, entactin, and heparin sulfate proteoglycan are common components of basement membranes, although there are important
tissue-to-tissue differences in the relative proportion.28,29 The
strong attachment of podocytes to the GBM is based on ␣3-␤1
integrin-fibronectin–laminin interactions.30,31 Baramova et
al32 demonstrated the ability of crotalic hemorrhagin to proteolytically degrade the major components of the basal
lamina and extracellular matrix, such as laminin, fibronectin,
type IV collagen, entactin/nidogen, and gelatins. Ohsaka et
al33 studied the action of Trimeresurus flavoviridis snake
venom on isolated GBM in vitro and demonstrated that proteins (or peptides) and carbohydrates were released from the
GBM by venom fractions. Because the GBM is formed by the
union of the basal lamina of the capillary endothelium and the
visceral epithelium, and the endothelium of these capillaries
is fenestrated, thereby facilitating contact between venom
hemorrhagins and the components of the filtration lamina, B.
221
moojeni venom34 could digest some of these components. In
addition, proteolytic enzymes in B. moojeni venom could alter the connections between laminin and the integrins of the
pedicels, releasing the latter from their anchorage to the
GBM. Structural and functional disturbances of the GBM in
venom-treated rats could indirectly cause podocyte RE cisternae dilatation with accumulation of proteinaceous material. This, in turn, could result in an anomalous rise in protein
synthesis to replace the GBM and/or podocyte cytoskeletal
constituents.
Another characteristic ultrastructural alteration induced by
B. moojeni venom was a rapid onset of mesangiolysis, capillary ballooning, and formation of microaneurysms. The first
report of mesangiolysis-like lesions after experimental envenomation with snake (Crotalus adamanteus) venom was
published in 1909.35 Agkistrodon halys snake venom also induces mesangiolysis,36 and this damage has been reported
after bites from the habu snake Trimeresurus flavoviridis.37,38
The high proteolytic activity of the latter venom was thought
to cause most of the damage to the glomerular mesangium39,40 and could also account for B. moojeni venom-induced
mesangiolysis.
The formation of glomerular microaneurysms is poorly
understood, although a failure in the supporting system of
the glomerular tuft because of mesangiolysis is usually
the underlying cause.40 In addition to the mesangium, the
podocytes41,42 and GBM43 also provide a structural basis for
capillary tuft support, and all undoubtedly participate in protecting the capillaries from ballooning or microaneurysm formation otherwise seen after mesangiolysis.44
In summary, the current study demonstrates for the first
time that glomerular changes are responsible for proteinuria
and that both contribute to the nephrotoxicity in ARF caused
by the intravenous administration of B. moojeni venom. It
seems likely that mesangiolysis, microaneurysm formation,
and pedicel damage are a consequence of the high proteolytic
and PLA2 activities of this venom. Studies are being designed
to identify the biological phenomena responsible for such ultrastructural changes.
Acknowledgments: We thank Gustavo Henrique da Silva for technical assistance, Dr. Stephen Hyslop (Universidade Estadual de Campinas/UNICAMP) for revising the language of an earlier version of this
article, Dr. Luiz Antonio Ribeiro de Moura (Universidade Federal de
São Paulo/UNIFESP-EPM) and Dr. Fábio Bucaretchi (Universidade
Estadual de Campinas /UNICAMP) for helpful suggestions, and Dr.
Elliot Watanabe Kitajima (ESALQ-USP) for use of the SEM.
Financial support: This work was supported by Coordenação de
Aperfeiçoamento de Pessoal de Nı́vel Superior, Fundação de Amparo à Pesquisa do Estado de São Paulo (Proc. 93/4995-5, 98/00340-4,
98/00341-0), Fundo de Apoio ao Ensino e à Pesquisa (FAEP—
UNICAMP), and Conselho Nacional de Pesquisa e Desenvolvimento
Cientı́fico e Tecnológico (Proc. 522131/95-6).
Authors’ addresses: Patrı́cia Aline Boer-Lima, Departamento de
Biologia Celular, Instituto de Biologia, C.P. 6109, Universidade Estadual de Campinas (UNICAMP), S.P., CEP 13083-970, Brazil. José
Antonio Rocha Gontijo, Núcleo de Medicina e Cirurgia Experimental, Faculdade de Ciências Médicas, Universidade Estadual de
Campinas (UNICAMP), S.P., CEP 13083-970, Brazil. Maria Alice da
Cruz-Höfling, Departamento de Histologia e Embriologia, Instituto
de Biologia, C.P. 6109, Universidade Estadual de Campinas
(UNICAMP), S.P., CEP 13083-970, Brazil.
Reprint requests: Maria Alice da Cruz-Höfling, Ph.D., Departamento
de Histologia e Embriologia, Instituto de Biologia, C.P. 6109, Uni-
222
BOER-LIMA AND OTHERS
versidade Estadual de Campinas (UNICAMP), Campinas, S.P., CEP
13083-970, Brazil, E-mail: hö[email protected]
21.
REFERENCES
1. Ministério da Saúde do Brasil, 1997. Manual de Diagnóstico e
Tratamento de Acidentes por Animais Peçonhentos. Brasilia:
Fundação Nacional da Saúde.
2. Rosenfeld G, 1971. Symptomatology, pathology and treatment of
snake bites in South America. Bücherl W, Buckley EE, eds.
Venomous Animals and Their Venoms. Volume 2. New York:
Academic Press, 345–384.
3. Amaral CFS, Rezende NA, Silva AO, Ribeiro MMF, Magalhães
RA, Reis RJ, Carneiro JG, Castro JRS, 1986. Insuficiência
renal aguda secundária a acidentes ofı́dicos botrópico e crotálico. Análise de 63 casos. Rev Inst Med Trop São Paulo 28:
220–227.
4. Secretaria de Saúde do Estado de São Paulo, 1993. Acidentes por
Animais Peçonhentos. Identificação Diagnóstico e Tratamento.
Manual de Vigilância Epidemiológica. São Paulo, 61–62.
5. Barraviera B, Pereira PCM, 1994. Acidentes por serpentes do
gênero Bothrops. Barraviera B, ed. Venenos Animais: Uma
Visão Integrada. Rio de Janeiro: Publicações Cientı́ficas Ltda,
261–280.
6. Rezende NA, Amaral CFS, Bambirra EA, Lachatt JJ, Coimbra
TM, 1989. Functional and histopathological renal changes induced in rats by Bothrops jararaca venom. Braz J Med Biol Res
22: 407–416.
7. Chaves F, Gutiérrez JM, Lomonte B, Cerdas L, 1989. Histopathological and biochemical alterations induced by intramuscular
injection of Bothrops asper (terciopelo) venom in mice. Toxicon 10: 1085–1093.
8. Burdmann EA, Woronik V, Prado EBA, Abdulkader RC,
Saldanha LB, Barreto OCO, Marcondes M, 1993. Snakebiteinduced acute renal failure: an experimental model. Am J Trop
Med Hyg 48: 82–88.
9. Boer-Lima PA, Gontijo JAR, Cruz-Höfling MA, 1999. Histologic
and functional renal alterations caused by Bothrops moojeni
snake venom in rats. Am J Trop Med Hyg 61: 698–706.
10. Brains R, Furness PN, Critchley DR, 1997. A quantitative immunofluorescence study of glomerular cell adhesion proteins in
proteinuric states. J Pathol 183: 272–280.
11. Caulfield JP, Reid JJ, Farquhar MG, 1976. Alterations of the
glomerular epithelium in acute aminonucleoside nephrosis.
Evidence for formation of occluding junctions and epithelial
cell detachment. Lab Invest 34: 43–59.
12. Arakawa M, Tokunaga J, 1972. A scanning electron microscope
study of the glomerulus. Further consideration of the mechanism of the fusion of podocyte terminal processes in nephrotic
rats. Lab Invest 27: 366–373.
13. Kriz W, Gretz N, Lemley KV, 1998. Progression of glomerular
diseases: is the podocyte the culprit? Kidney Int 54: 687–697.
14. Grishman E, Churg J, 1975. Focal glomerular sclerosis in nephrotic patients: an electron microscopic study of glomerular
podocytes. Kidney Int 7: 111–122.
15. Yoshikawa V, Ito H, Acamatsu R, Hazikano H, Okada S, Matsuo
T, 1986. Glomerular podocyte vacuolization in focal segmental
glomerulosclerosis. Arch Pathol Lab Med 110: 394–398.
16. Whiteside CI, Cameron R, Munk S, Levy J, 1993. Podocytic cytoskeletal disaggregation and basement-membrane detachment in puromycin aminonucleoside nephrosis. Am J Pathol
142: 1641–1653.
17. Ricardo SD, Bertram JF, Ryan GB, 1994. Reactive oxygen species in puromycin aminonucleoside nephrosis: in vitro studies.
Kidney Int 45: 1057–1069.
18. Messina A, Davies DJ, Dillane PC, Ryan GB, 1987. Glomerular
epithelial abnormalities associated with the onset of proteinuria in aminonucleoside nephrosis. Am J Pathol 126: 220–229.
19. Venkatachalam MA, Cotran RS, Karnovsky MJ, 1969. Glomerular studies in experimental nephrosis using horseradish peroxidase as a tracer. J Exp Med 132: 1168–1180.
20. Vasmant D, Maurice M, Feldmann G, 1984. Cytoskeleton ultra-
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
structure of podocytes and glomerular endothelial cells in man
and in rat. Anat Rec 210: 17–24.
Andrews PM, 1981. Investigations of cytoplasmic contractile and
cytoskeletal elements in the kidney glomerulus. Kidney Int 20:
549–562.
Bruses JL, Capaso J, Katz E, Pilar G, 1993. Specific in vitro
biological activity of snake venom myotoxins. J Neurochem 60:
1030–1042.
Gutiérrez JM, Lomonte B, 1995. Phospholipase A2 myotoxins
from Bothrops snake venoms. Toxicon 33: 1405–1424.
Reichl AP, Serrano SMT, Assakura MT, Mandelbaum F, 1989.
Isolation and properties of a phospholipase A2 from the
venom of the snake B. moojeni (caissaca). Mem Inst Butantan
51: 225–237.
Stow JL, Sawada H, Farquhar MG, 1985. Basement membrane
heparin sulfate proteoglycans are concentrated in the laminae
rarae and in podocytes of the rat renal glomerulus. Proc Natl
Acad Sci USA 82: 3296–3300.
Kerjaschki D, Sharkley, DJ, Farquhar, MG, 1984. Identification
and characterization of podocalyxin—the major sialoglycoprotein of the renal glomerular epithelial cell. J Cell Biol 98: 1591–
1596.
Willinger CC, Thamaree S, Schramek H, Gstraunthaler G,
Pfaller W, 1995. In vitro nephrotoxicity of Russell’s viper
venom. Kidney Int 47: 518–528.
Martin GR, Timpl R, 1987. Laminin and other basement membrane components. Annu Rev Cell Biol 3: 57–85.
Yurchenko P, Schittny J, 1990. Molecular and cellular aspects of
basement membranes. FASEB J 4: 1577–1590.
Kerjaschki D, Ojha PP, Susani M, Horvat R, Binder S, Hovorka
A, Hillemanns P, Pytela R, 1989. A ␤1-integrin receptor for
fibronectin in human kidney glomeruli. Am J Pathol 143: 481–
489.
Korhonen M, Ylänne J, Laitinen L, Virtanen I, 1990. The ␣1-␣6
subunits of integrins are characteristically expressed in distinct
segments of developing and adult human nephron. J Cell Biol
111: 1245–1254.
Baramova EN, Shannon JD, Bjarnason JB, Fox JW, 1989. Degradation of extracellular matrix proteins by hemorrhagic metalloproteinases. Arch Biochem Biophys 275: 63–71.
Ohsaka A, Just M, Habermann E, 1973. Action of snake venom
hemorrhagic principles on isolated glomerular basement membrane. Biochim Biophys Acta 323: 415–428.
Assakura MT, Reichl AP, Asperti MCA, Mandelbaum FR, 1985.
Isolation of the major proteolytic enzyme from the venom of
the snake Bothrops moojeni (caissaca). Toxicon 23: 691–706.
Pearce RM, 1909. An experimental glomerular lesion caused by
venom (Crotalus adamanteus). J Exp Med 11: 532–540.
Otsuji Y, Irie Y, Ueda H, Yotsueda K, Kitahara T, Yokoyama K,
Higashi, 1978. A case of acute renal failure caused by mamushi
(Agkistrodon halys) bite. Med J Kagoshima Univ 30: 129–135.
Morita T, Churg J, 1983. Mesangiolysis. Kidney Int 24: 1–9.
Kitamura H, Sugesaki Y, Yamanaca N, 1995. Endothelial regeneration during the repair process following habu-snake venom
induced glomerular injury. Virchows Arch 427: 195–204.
Sakaguchi H, Kawamura S, 1963. Electron microscopic observations of mesangiolysis: the toxic effects of the “habu snake”
venom on the renal glomerulus. Keio J Med 12: 99–106.
Uiker S, Kriz W, 1995. Structural analysis of the formation of
glomerular microaneurysms in the habu venom model. Virchows Arch 426: 281–293.
Kretzler M, Koeppen-Hagemann I, Kriz W, 1994. Podocyte damage is a critical step in the development of glomerulosclerosis
in uninephrectomized-desoxycorticosterone hypertensive rat.
Virchows Arch 425: 181–193.
Kriz W, Hackenthal E, Nobiling R, Sakai T, Elger M, 1994. A
role for podocytes to counteract capillary wall distension. Kidney Int 45: 369–376.
Sakai T, Kriz W, 1987. The structural relationship between mesangial cells and basement membrane of the renal glomerulus.
Anat Embryol 176: 373–386.
Morita T, Yamamoto T, Churg J, 1998. Mesangiolysis: an update.
Am J Kidney Dis 31: 559–573.