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Alba Vargas, Héctor Finol, María Girón, Héctor Scannone, Irma Fernández, Alexis Rodriguez-Acosta
Effects of Lansberg's Hognose Pit Vipers (Porthidium lansbergii hutmanni) Venom on Renal Ultrastructure in
Experimental Mice
Acta Scientiae Veterinariae, vol. 39, núm. 1, 2011, pp. 1-6,
Universidade Federal do Rio Grande do Sul
Brasil

Available in: http://www.redalyc.org/articulo.oa?id=289022244002

Acta Scientiae Veterinariae,

ISSN (Printed Version): 1678-0345
ActaSciVet@ufrgs.br
Universidade Federal do Rio Grande do Sul
Brasil

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 Acta Scientiae Veterinariae, 2011. 39(1): 941.

ORIGINAL ARTICLE ISSN 1679-9216 (Online)

Pub. 941

Effec
ffec ts of Lansb
ects er
Lansber g’s H
erg Hoognose P it V ip
Pit ers (Por
ipers thidium lansb
orthidium er
lansber gii hutmanni) Venom on
ergii
Renal Ultrastructure in Experimental Mice*

Alba Vargas 1, Héctor Finol 2, María Girón3, Héctor Scannone1,

Irma Fernández1 & Alexis Rodriguez-Acosta 3

ABSTRACT

Background: Porthidium lansbergii hutmanni species occurs at Tropical level (800 meters altitude) in Margarita Island,
Venezuela. It seems to be constrained to this island. Two different species; Porthidium lansbergii rozei and P. lansbergii
lansbergii live in the mountains surrounding the Cordillera de La Costa in mainland Venezuela. The principal damage and the
main complication in fatal cases of Viperidae snakebites in Venezuela is acute renal failure (ARF) secondary to acute tubular
necrosis. Kidney alterations in Porthidium snakebite human victims have concerned inconspicuous considerations. There is
not literature description of Porthidium venom activity on the renal structure. The purpose of this study was to determine how
intraperitoneal Porthidium lansbergii hutmanni venom injection into mice could lead to severe renal injury.
Materials, Methods & Results: Lethal dose fifty (2.5 mg/kg body weight) in mice and polyacrylamide gel electrophoresis
were carried out. Observing renal tissue under the electron microscope 3 h after CvPlh injection, proximal convoluted tubule
showed widening and loss of interdigitations with normal mitochondria. Multiform and pleomorphic mitochondria were seen.
Loss of the cytoarchitecture and empty vacuoles was noticed. After 6 h of CvPlh injection, panoramic view of apical and basal
regions of distal convoluted tubule showed swollen mitochondria cristae; Golgi apparatus swollen elements and different
wideness of interdigitations were seen. High enlarging of the basal membrane, alteration of the interdigitations with the
formation of myelin-like figures were observed. An altered capillary with loss of the wall integrity was visualized. Swollen
mitochondria and swollen rough endoplasmic reticulum were observed. After 24 h of CvPlh injection, in the glomeruli,
rupture of the capillary endothelia, with podocytes of different widened were detected: they were irregulars and of different
sizes. Basal membrane widened in some areas; vacuoles in the endothelial cells were also seen. CvPlh SDS-PAGE profile under
native conditions showed approximately 14 bands distributed from 7.6 to 215.0 kDa.
Discussion: The largest renal lesions observed in the experimental mice were tubular degeneration and necrosis. The alterations
were typically restricted to proximal tubules. Mitochondria modifications were intense. The renal damage caused by CvPlh
appeared to be due to both systemic effects (mainly renal ischemia) and direct tubulotoxic effects of the venom. It was also
found visceral epithelial changes that included podocyte alterations, which could be associated with glomerular dysfunction.
The condition usually causes edema, and nephrotic syndrome and it may progress leading to a renal failure. All renal structures
can be involved. Tubular necrosis is the important pathological counterpart of acute renal failure. As far as we known this is the
first time that after intramuscular administration of P. l .hutmanni venom glomerular and tubular kidney changes have been
observed. It seems likely that mesangiolysis, mitochondria and microvascular damages are a consequence of the high proteolytic
and PLA2 activities of this venom. Studies are being designed to identify the fraction (s) venom responsible (s) for such
ultrastructural changes.
Keywords: envenoming, glomeruli, kidney, Porthidium lansbergii hutmanni, proximal convoluted tubule, snakebite,
venom.

Received: August 2010 www.ufrgs.br/actavet Accepted: November 2010

*
This work was supported by Grants: FONACIT G- 2002000447 and FONACIT G-2005000400.1Laboratorio de Investigaciones Farmacéuticas
de la Facultad de Farmacia de la Universidad Central de Venezuela. 2Centro de Microscopía Electrónica de la Facultad de Ciencias de la
Universidad Central de Venezuela. 3Sección de Inmunoquímica del Instituto de Medicina Tropical/Laboratorio de Inmunoquímica y Ultraestructura
del Instituto Anatómico de la Universidad Central de Venezuela. Apartado 47423, Caracas 1041, Venezuela. CORRESPONDENCE: A. Rodriguez-
Acosta [alexis.rodriguez@ucv.ve].

1
A.V ar
A.Var gas
gas,, H.F
argas inol, M.G
H.Finol, irón, et al . 2011. Effects of Lansberg’s Hognose Pit Vipers ( Porthidium lansbergii hutmannii )
M.Girón,
Venom on Renal Ultrastructure in Experimental Mice.
INTRODUCTION
Porthidium lansbergii hutmanni species occurs
at low and moderate elevations down 800 meters altitude
in Margarita Island, Venezuela. It appears to be restricted
to this island. Two different species; Porthidium
lansbergii rozei and P. lansbergii lansbergii live in the
mountains surrounding the Cordillera de La Costa in
mainland Venezuela [4], located in the extreme
northwestern and northeastern portion of the country. The
epidemiological importance at Margarita Island is
unknown because the accidents are reported in
conjunction with other viper snakes envenomations. The
mainly systemic consequence and the most frequent
complication in fatal cases of snakebites in Venezuela is
acute renal failure (ARF) secondary to acute tubular
necrosis [5, 14]. In general, glomerular changes in human
victims of snakebite envenoming have concerned modest
consideration, and it has not had methodical study of this
problem. There is not literature description of Porthidium
venom activity on the renal structure. Otero et al. (2002)
in a report of Viperidae envenomed patients described 2
cases of envenoming by Porthidium nasutum that did
not have clinically renal damage. The purpose of this study
was to determine how Porthidium lansbergii hutmanni
venom injection into mice could lead to severe renal injury.
MATERIALS AND METHODS
Porthidium lansbergii hutmanni venom
Crude venom from Porthidium lansbergii
hutmanni (CVPlh) was obtained milking every 30 days
twelve mature snakes, captured in Juan Griego and
Porlamar, Margarita Island (Venezuela) and
maintained in captivity in the Serpentarium of the
Laboratory of Investigations, Pharmacy Faculty of the
Universidad Central de Venezuela. The extracted
venom was dehydrated in vacuum desiccators,
maintained with calcium chloride1 at 4ºC until its total
crystallization.
Mice
Fifteen male mice INH strain weighing 18-22
g from the Instituto Nacional de Higiene “Rafael
Rangel”, Caracas, Venezuela were used. The mice
boxes were kept in a colony room maintained at 23
ºC on a 12/12-h lumen/dark cycle.
Envenomed mice
Fifteen mice (18-22 g) were intramuscularly
injected with 0.1 mL of a sub-lethal dose (2.0 mg/kg body
2
Acta Scientiae Veterinariae 39(1): 941.
weight) of CVPlh and sacrificed at 3, 6, and 24 h later.
Two mice injected with saline were use as normal controls.
Ethical statement
Expert personnel prepared all the experimental
procedures relating to the use of live animals. The
Venezuelan pertinent regulations as well as
institutional guidelines, according to protocols
approved by the Institute of Anatomy of the
Universidad Central de Venezuela and the norms
obtained from the guidelines for the care and use of
laboratory animals, published by the US National
Institute of Health [1] were followed.
Determination of protein concentration
Protein concentrations of all tested venoms were
measured by the Lowry method [9], using bovine
serum albumin1 as standard protein.
Lethality assay
The Spearman-Karber [20] method was used
to calculate the P.l. hutmanni crude venom lethal dose
fifty (LD50). Six groups of five mice (18-22 g) were
intraperitoneally injected with 0.1 mL of several
concentrations of venom and the number of mice that
died was counted after 48 h.
Transmission electron microscopy
Kidney biopsies were obtained at 3, 6, and 24
h after venom injection from envenomed mice under
anesthesia2. Samples were immediately fixed in situ
with 3% glutaraldehyde3 and 1% OsO4 (both fixatives
diluted in pH 7.4, 320 mM phosphate buffer saline),
dehydrated in ethanol and embedded in LX-112
resin4. Ultrathin sections were stained with uranyl
acetate3 and lead citrate3 and observed in a Hitachi
H-500 transmission electron microscope with an
accelerating voltage of 100 KV.
SDS-PAGE
Electrophoresis using a MINI-PROTEAN II
(BioRad, USA) chamber was performed. Sodium
dodecyl sulphate-polyacrylamide gel electrophoresis
was carried out according to the Laemmli method [8]
using 12.5% gels under reducing conditions 2 .
Molecular weight markers5 were run in parallel and
gels were stained with Comassie Blue R-2501.
CVPlh samples were dissolved in 1:1 proportion
in the solubilizer solution 0.5 M tris-HCl buffer (pH 6.8)
with 10% (v/v) SDS1, 10% (v/v) b-mercaptoethanol1,
A.V ar
A.Var gas
gas,, H.F
argas inol, M.G
H.Finol, irón, et al . 2011. Effects of Lansberg’s Hognose Pit Vipers ( Porthidium lansbergii hutmannii )
M.Girón,
Venom on Renal Ultrastructure in Experimental Mice. Acta Scientiae Veterinariae 39(1): 941

10% (v/v) glycerol1 and 0.05% (w/v) bromophenol blue1, injection, in a proximal convoluted section showed the
and heated at 100 ºC for 10 min. endothelium capillary cytoplasm very edematous (Figure 2).
RESULTS
Electron-micrographs of mice kidney after 6 h
Determination of lethality CvPlh injection, a panoramic view of the proximal
The LD50 for crude venom was 2.5 mg/kg body weight. convoluted tubule section showed pleomorphic
Transmission electron microscopy mitochondria with calcic deposits and an
autophagolysosome (rhombus) with myelinic figures
Electron-micrographs of mice kidney injected
inside (Figure 3).
with saline presented a normal tissue aspect as
In electron-micrographs of mice kidney after 6h
described everywhere (data not shown).
CvPlh injection, a section of the region formerly occupied
Observing renal tissue under the electron
by the interdigitations was observed; vacuoles and
microscope 3 h after CvPlh injection, in a proximal
vesicles, in addition to swollen mitochondria with variable
convoluted tubule section is showed the basal region
number of cristae in different orientations were noticed.
with loss of interdigitations, being the epithelial cell
The capillary showed widened fenestrae. In the tubule,
cytoplasm occupied by pleomorphic mitochondria.
at the right position, an area of interdigitations with
Lysosomal structures were noticed. In the extracellular
membranous debris was observed (Figure 4).
space membranous debris, probably from endothelia
origin were seen (Figure 1).
Electron-micrographs of mice kidney after 3 h CvPlh

Figure 2. Electron-micrographs of mice kidney after 3 h CvPlh

Figure 1. Electron-micrographs of mice kidney after 3 h CvPlh injection, proximal convoluted section showing in the endothelium
injection, proximal convoluted tubule section showing in the basal capillary cytoplasm very edematous (stars) [X 20.000].
region loss of interdigitations, being the epithelial cell cytoplasm
occupied by pleomorphic mitochondria (oval). Lysosomal structures
were noticed (arrows). In the extracellular space membranous debris,
probably from endothelia origin were seen (X 20.000).

Figure 4. Electron-micrographs of mice kidney after 6 h CvPlh

Figure 3. Electron-micrographs of mice kidney after 6 h CvPlh
injection, panoramic view of proximal convoluted tubule section
showing pleomorphic mitochondria with calcic deposits (arrows)
and an autophagolysosome (rhombus) with mylenic figures inside
(X 20.000).
injection, in this section a region formerly occupied by the
interdigitations was observed; vacuoles (V) and vesicles (v), in
addition to swollen mitochondria (stars) with variable number of
cristae in different orientations were noticed. The capillary showed
widened fenestrae (arrows). In the tubule at the right position, an
area of interdigitations with membranous debris (rhombus) was
observed (X 16.000).

3
A.V ar
A.Var gas
gas,, H.F
argas inol, M.G
H.Finol, irón, et al . 2011. Effects of Lansberg’s Hognose Pit Vipers ( Porthidium lansbergii hutmannii )
M.Girón,
Venom on Renal Ultrastructure in Experimental Mice.
After 24 h CvPlh injection, in the glomerule,
fenestrated areas of the capillary endothelia lost and
widened basal membrane in some regions (parallelogram)
were seen. Podocytes irregular form and dimensions was
observed (Figure 5).
Figure 5. Electron-micrographs of mice kidney after 24 h CvPlh
injection, in the glomerule, fenestrated areas of the capillary endothelia
lost (circle) and widened basal membrane in some regions
(parallelogram) were observed. Podocytes irregular form and
dimensions was noticed (X 20.000).
Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE)
CvPlh SDS-PAGE profile is shown in Figure 6.
Under native conditions approximately 14 bands
distributed from 7.6 to 215.0 kDa were observed.
Figure 6. Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis of Porthidium lansbergii hutmanni crude
venom. (CvPlh), MWM: molecular weight markers.
DISCUSSION
Lansberg’s hog-nosed pit viper is a nocturnal
animal. They are not violent snakes (excluding their prey)
but will respond energetically if threatened.
The results of this study show that in mice CvPlh
causes ultrastructural changes in the tubular and
4
Acta Scientiae Veterinariae 39(1): 941.
glomerular structures and capillaries cluster compatible
with the renal dysfunction caused by snakebite described
elsewhere [3]. The predominant renal lesions observed
in the experimental animals were tubular degeneration
and necrosis. The changes were mostly confined to
proximal tubules. Mitochondria changes were intense. The
renal damage caused by CvPlh seemed to be due to both
systemic effects (mainly renal ischemia) and direct
tubulotoxic effects of the venom. It was also found visceral
epithelial changes that included podocyte alterations, which
could be associated with glomerular dysfunction [21].
Arakawa and Tokunaga [2] showed that the loss
of podocyte processes is caused by their extraction into
the cell body and not by the fusion of adjacent processes
to form a syncytium. Podocytes process removing
caused by the venom, which it was observed in the above
experiments, would represent a diminution in the
complexity of the usual interdigitating pattern of cell–cell
connections and an evident diminution of filtration capacity.
Comparable modifications have been found in human
glomerulonephritis linked with nephrotic syndrome[6] and
in nephrosis induced by antibiotics, which are toxic to
visceral epithelial cells [15, 19] as well as in renal lesions
caused by Uracoan rattlesnake venom [5], where
important podocytes alterations were described. Our
results reproduced these anomalous features and offer
evidence for a toxic effect of CvPlh on visceral epithelial
cells.
Investigational studies of renal modifications after
injection of Crotalic venom [5] demonstrated
degenerative lesions of tubular cells and glomerule, with
mild proliferation of the mesangial matrix, glomerular
congestion, and massive glomerular fibrin deposition
immediately after venom infusion. This mesangial lesion
is caused by inflammation of the glomeruli, and specifically
an increase in number (proliferation) of these cells.
Otsuji et al. [12] proposed that Agkistrodon halys
snake venom induces mesangiolysis, and this damage has
been reported after bites from the habu snake
Trimeresurus flavoviridis [10]. The high proteolytic
activity of the last venom was thought to produce most of
the harm to the glomerular mesangium [16] and could
also be responsible for CvPlh-induced mesangiolysis.
The disorder usually causes edema, and nephrotic
syndrome and it may progress to renal failure. It is seen
more commonly in Crotalus envenomed patients who
develop acute renal failure. All renal structures can be
involved. Tubular necrosis is the important pathological
A.V ar
A.Var gas
gas,, H.F
argas inol, M.G
H.Finol, irón, et al . 2011. Effects of Lansberg’s Hognose Pit Vipers ( Porthidium lansbergii hutmannii )
M.Girón,
Venom on Renal Ultrastructure in Experimental Mice. Acta Scientiae Veterinariae 39(1): 941

counterpart of acute renal failure. This is the first time
that these kinds of lesions have been observed in
Porthidium genus.
The proteolytic and phospholipase A2 (PLA2)
activities of Porthidium venoms [18] have significant
cytotoxic consequences in many cell types [13] and
could contribute to the nephrotoxicity seen in the
current study. A theoretical cytotoxin-like activity has
been estimated for members of the same family
(Viperidae) myotoxins acting on skeletal muscle cells in
vivo [7]. Following fixing to an unknown place on the
cell plasma membrane, the myotoxins would go in the
bilayer through hydrophobic interactions, producing
enhanced permeability to ions and macromolecules. An
important calcium influx would almost certainly be the
most significant consequence of membrane disorder and
would be dependable for the beginning of an array of
critical mechanisms such as cytoskeleton modifications,
the activation of calcium-dependent proteases,
mitochondrial injure, and intracellular phospholipases,
which, sequentially, would explain the cellular harm [17].
The electrophoretic profile of CvPlh showed
protein bands with molecular masses between 7.0 and
206 kDa (Fig. 6), corresponding to the range in which the
main components of Viperidae venoms are found and
most likely equivalent to metalloproteinases (40-64 kDa),
serine proteinases (20-40 kDa) and phospholipases A2
(13-16 kDa) [7,13].
CONCLUSIONS
In synopsis, the present study for the first time
demonstrates that intramuscular administration of P.l.
hutmanni venom causes glomerular and tubular kidney
changes and that this contribute to the nephrotoxicity in
ARF. It seems likely that mesangiolysis, mitochondria and
microvascular damages are a consequence of the high
proteolytic and PLA2 activities of this venom. Studies
are being designed to identify the fraction(s)venom
responsible(s) for such ultrastructural changes.
SOURCES AND MANUFACTURERS
1
Sigma Aldrich. St. Louis, MO, USA.
2
Merck. Darmstadt , Germany).
3
Electron Microscopy Sciences. Hatfield, PA, USA.
4
Ladd Research Inc. Williston, VT , USA.
5
Bio-Rad. USA.
Conflict of interest. The authors declare that there are no
conflicts of interest concerned with this work.

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M.Girón,
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