Acta Scientiae Veterinariae

ISSN: 1678-0345

ActaSciVet@ufrgs.br

Universidade Federal do Rio Grande do Sul

Brasil

Vargas, Alba; Finol, Héctor; Girón, María; Scannone, Héctor; Fernández, Irma; Rodriguez-Acosta,

Alexis

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

Porto Alegre, Brasil

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EEEEEffffffffffececececects of Lansbts of Lansbts of Lansbts of Lansbts of Lansberererererggggg’’’’’s Hs Hs Hs Hs Hooooognose Pgnose Pgnose Pgnose Pgnose Pit it it it it VVVVVipipipipipers (ers (ers (ers (ers (PPPPPorororororthidium lansbthidium lansbthidium lansbthidium lansbthidium lansbererererergii hutmannigii hutmannigii hutmannigii hutmannigii hutmanni) ) ) ) ) VVVVVenom onenom onenom onenom onenom onRenal Ultrastructure in Experimental Mice*Renal Ultrastructure in Experimental Mice*Renal Ultrastructure in Experimental Mice*Renal Ultrastructure in Experimental Mice*Renal Ultrastructure in Experimental Mice*

Alba VargasAlba VargasAlba VargasAlba VargasAlba Vargas11111, Héctor Finol, Héctor Finol, Héctor Finol, Héctor Finol, Héctor Finol22222, María Girón, María Girón, María Girón, María Girón, María Girón33333, Héctor Scannone, Héctor Scannone, Héctor Scannone, Héctor Scannone, Héctor Scannone11111,,,,,Irma FernándezIrma FernándezIrma FernándezIrma FernándezIrma Fernández11111 & Alexis Rodriguez-Acosta & Alexis Rodriguez-Acosta & Alexis Rodriguez-Acosta & Alexis Rodriguez-Acosta & Alexis Rodriguez-Acosta33333

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. lansbergiilansbergii live in the mountains surrounding the Cordillera de La Costa in mainland Venezuela. The principal damage and themain complication in fatal cases of Viperidae snakebites in Venezuela is acute renal failure (ARF) secondary to acute tubularnecrosis. Kidney alterations in Porthidium snakebite human victims have concerned inconspicuous considerations. There isnot literature description of Porthidium venom activity on the renal structure. The purpose of this study was to determine howintraperitoneal 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 electrophoresiswere carried out. Observing renal tissue under the electron microscope 3 h after CvPlh injection, proximal convoluted tubuleshowed 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 basalregions of distal convoluted tubule showed swollen mitochondria cristae; Golgi apparatus swollen elements and differentwideness of interdigitations were seen. High enlarging of the basal membrane, alteration of the interdigitations with theformation of myelin-like figures were observed. An altered capillary with loss of the wall integrity was visualized. Swollenmitochondria 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 differentsizes. Basal membrane widened in some areas; vacuoles in the endothelial cells were also seen. CvPlh SDS-PAGE profile undernative 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 alterationswere typically restricted to proximal tubules. Mitochondria modifications were intense. The renal damage caused by CvPlhappeared to be due to both systemic effects (mainly renal ischemia) and direct tubulotoxic effects of the venom. It was alsofound 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 structurescan be involved. Tubular necrosis is the important pathological counterpart of acute renal failure. As far as we known this is thefirst time that after intramuscular administration of P. l .hutmanni venom glomerular and tubular kidney changes have beenobserved. It seems likely that mesangiolysis, mitochondria and microvascular damages are a consequence of the high proteolyticand PLA2 activities of this venom. Studies are being designed to identify the fraction (s) venom responsible (s) for suchultrastructural changes.

Keywords: envenoming, glomeruli, kidney, Porthidium lansbergii hutmanni, proximal convoluted tubule, snakebite,venom.

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

ORIGINAL ARTICLEP u b . 9 4 1P u b . 9 4 1P u b . 9 4 1P u b . 9 4 1P u b . 9 4 1

ISSN 1679-9216 (Online)

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

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

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INTRODUCTION

Porthidium lansbergii hutmanni species occursat low and moderate elevations down 800 meters altitudein Margarita Island, Venezuela. It appears to be restrictedto this island. Two different species; Porthidiumlansbergii rozei and P. lansbergii lansbergii live in themountains surrounding the Cordillera de La Costa inmainland Venezuela [4], located in the extremenorthwestern and northeastern portion of the country. Theepidemiological importance at Margarita Island isunknown because the accidents are reported inconjunction with other viper snakes envenomations. Themainly systemic consequence and the most frequentcomplication in fatal cases of snakebites in Venezuela isacute renal failure (ARF) secondary to acute tubularnecrosis [5, 14]. In general, glomerular changes in humanvictims of snakebite envenoming have concerned modestconsideration, and it has not had methodical study of thisproblem. There is not literature description of Porthidiumvenom activity on the renal structure. Otero et al. (2002)in a report of Viperidae envenomed patients described 2cases of envenoming by Porthidium nasutum that didnot have clinically renal damage. The purpose of this studywas to determine how Porthidium lansbergii hutmannivenom injection into mice could lead to severe renal injury.

MATERIALS AND METHODS

Porthidium lansbergii hutmanni venomCrude venom from Porthidium lansbergii

hutmanni (CVPlh) was obtained milking every 30 daystwelve mature snakes, captured in Juan Griego andPorlamar, Margarita Island (Venezuela) andmaintained in captivity in the Serpentarium of theLaboratory of Investigations, Pharmacy Faculty of theUniversidad Central de Venezuela. The extractedvenom was dehydrated in vacuum desiccators,maintained with calcium chloride1 at 4ºC until its totalcrystallization.

MiceFifteen male mice INH strain weighing 18-22

g from the Instituto Nacional de Higiene “RafaelRangel”, Caracas, Venezuela were used. The miceboxes were kept in a colony room maintained at 23ºC on a 12/12-h lumen/dark cycle.

Envenomed miceFifteen mice (18-22 g) were intramuscularly

injected with 0.1 mL of a sub-lethal dose (2.0 mg/kg body

weight) of CVPlh and sacrificed at 3, 6, and 24 h later.Two mice injected with saline were use as normal controls.

Ethical statementExpert personnel prepared all the experimental

procedures relating to the use of live animals. TheVenezuelan pertinent regulations as well asinstitutional guidelines, according to protocolsapproved by the Institute of Anatomy of theUniversidad Central de Venezuela and the normsobtained from the guidelines for the care and use oflaboratory animals, published by the US NationalInstitute of Health [1] were followed.

Determination of protein concentrationProtein concentrations of all tested venoms were

measured by the Lowry method [9], using bovineserum albumin1 as standard protein.

Lethality assayThe Spearman-Karber [20] method was used

to calculate the P.l. hutmanni crude venom lethal dosefifty (LD50). Six groups of five mice (18-22 g) wereintraperitoneally injected with 0.1 mL of severalconcentrations of venom and the number of mice thatdied was counted after 48 h.

Transmission electron microscopyKidney biopsies were obtained at 3, 6, and 24

h after venom injection from envenomed mice underanesthesia2. Samples were immediately fixed in situwith 3% glutaraldehyde3 and 1% OsO4 (both fixativesdiluted in pH 7.4, 320 mM phosphate buffer saline),dehydrated in ethanol and embedded in LX-112resin4. Ultrathin sections were stained with uranylacetate3 and lead citrate3 and observed in a HitachiH-500 transmission electron microscope with anaccelerating voltage of 100 KV.

SDS-PAGEElectrophoresis using a MINI-PROTEAN II

(BioRad, USA) chamber was performed. Sodiumdodecyl sulphate-polyacrylamide gel electrophoresiswas carried out according to the Laemmli method [8]using 12.5% gels under reducing conditions2.Molecular weight markers5 were run in parallel andgels were stained with Comassie Blue R-2501.

CVPlh samples were dissolved in 1:1 proportionin the solubilizer solution 0.5 M tris-HCl buffer (pH 6.8)with 10% (v/v) SDS1, 10% (v/v) b-mercaptoethanol1,

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10% (v/v) glycerol1 and 0.05% (w/v) bromophenol blue1,and heated at 100 ºC for 10 min.

RESULTS

Determination of lethalityThe LD50 for crude venom was 2.5 mg/kg body weight.

Transmission electron microscopyElectron-micrographs of mice kidney injected

with saline presented a normal tissue aspect asdescribed everywhere (data not shown).

Observing renal tissue under the electronmicroscope 3 h after CvPlh injection, in a proximalconvoluted tubule section is showed the basal regionwith loss of interdigitations, being the epithelial cellcytoplasm occupied by pleomorphic mitochondria.Lysosomal structures were noticed. In the extracellularspace membranous debris, probably from endotheliaorigin were seen (Figure 1).

Electron-micrographs of mice kidney after 3 h CvPlh

injection, in a proximal convoluted section showed theendothelium capillary cytoplasm very edematous (Figure 2).

Electron-micrographs of mice kidney after 6 hCvPlh injection, a panoramic view of the proximalconvoluted tubule section showed pleomorphicmitochondria with calcic deposits and anautophagolysosome (rhombus) with myelinic figuresinside (Figure 3).

In electron-micrographs of mice kidney after 6hCvPlh injection, a section of the region formerly occupiedby the interdigitations was observed; vacuoles andvesicles, in addition to swollen mitochondria with variablenumber of cristae in different orientations were noticed.The capillary showed widened fenestrae. In the tubule,at the right position, an area of interdigitations withmembranous debris was observed (Figure 4).

Figure 1. Electron-micrographs of mice kidney after 3 h CvPlhinjection, proximal convoluted tubule section showing in the basalregion loss of interdigitations, being the epithelial cell cytoplasmoccupied by pleomorphic mitochondria (oval). Lysosomal structureswere noticed (arrows). In the extracellular space membranous debris,probably from endothelia origin were seen (X 20.000).

Figure 2. Electron-micrographs of mice kidney after 3 h CvPlhinjection, proximal convoluted section showing in the endotheliumcapillary cytoplasm very edematous (stars) [X 20.000].

Figure 3. Electron-micrographs of mice kidney after 6 h CvPlhinjection, panoramic view of proximal convoluted tubule sectionshowing pleomorphic mitochondria with calcic deposits (arrows)and an autophagolysosome (rhombus) with mylenic figures inside(X 20.000).

Figure 4. Electron-micrographs of mice kidney after 6 h CvPlhinjection, in this section a region formerly occupied by theinterdigitations was observed; vacuoles (V) and vesicles (v), inaddition to swollen mitochondria (stars) with variable number ofcristae in different orientations were noticed. The capillary showedwidened fenestrae (arrows). In the tubule at the right position, anarea of interdigitations with membranous debris (rhombus) wasobserved (X 16.000).

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After 24 h CvPlh injection, in the glomerule,fenestrated areas of the capillary endothelia lost andwidened basal membrane in some regions (parallelogram)were seen. Podocytes irregular form and dimensions wasobserved (Figure 5).

Sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE)

CvPlh SDS-PAGE profile is shown in Figure 6.Under native conditions approximately 14 bandsdistributed from 7.6 to 215.0 kDa were observed.

DISCUSSION

Lansberg’s hog-nosed pit viper is a nocturnalanimal. They are not violent snakes (excluding their prey)but will respond energetically if threatened.

The results of this study show that in mice CvPlhcauses ultrastructural changes in the tubular and

glomerular structures and capillaries cluster compatiblewith the renal dysfunction caused by snakebite describedelsewhere [3]. The predominant renal lesions observedin the experimental animals were tubular degenerationand necrosis. The changes were mostly confined toproximal tubules. Mitochondria changes were intense. Therenal damage caused by CvPlh seemed to be due to bothsystemic effects (mainly renal ischemia) and directtubulotoxic effects of the venom. It was also found visceralepithelial changes that included podocyte alterations, whichcould be associated with glomerular dysfunction [21].

Arakawa and Tokunaga [2] showed that the lossof podocyte processes is caused by their extraction intothe cell body and not by the fusion of adjacent processesto form a syncytium. Podocytes process removingcaused by the venom, which it was observed in the aboveexperiments, would represent a diminution in thecomplexity of the usual interdigitating pattern of cell–cellconnections and an evident diminution of filtration capacity.Comparable modifications have been found in humanglomerulonephritis linked with nephrotic syndrome[6] andin nephrosis induced by antibiotics, which are toxic tovisceral epithelial cells [15, 19] as well as in renal lesionscaused by Uracoan rattlesnake venom [5], whereimportant podocytes alterations were described. Ourresults reproduced these anomalous features and offerevidence for a toxic effect of CvPlh on visceral epithelialcells.

Investigational studies of renal modifications afterinjection of Crotalic venom [5] demonstrateddegenerative lesions of tubular cells and glomerule, withmild proliferation of the mesangial matrix, glomerularcongestion, and massive glomerular fibrin depositionimmediately after venom infusion. This mesangial lesionis caused by inflammation of the glomeruli, and specificallyan increase in number (proliferation) of these cells.

Otsuji et al. [12] proposed that Agkistrodon halyssnake venom induces mesangiolysis, and this damage hasbeen reported after bites from the habu snakeTrimeresurus flavoviridis [10]. The high proteolyticactivity of the last venom was thought to produce most ofthe harm to the glomerular mesangium [16] and couldalso be responsible for CvPlh-induced mesangiolysis.

The disorder usually causes edema, and nephroticsyndrome and it may progress to renal failure. It is seenmore commonly in Crotalus envenomed patients whodevelop acute renal failure. All renal structures can beinvolved. Tubular necrosis is the important pathological

Figure 5. Electron-micrographs of mice kidney after 24 h CvPlhinjection, in the glomerule, fenestrated areas of the capillary endothelialost (circle) and widened basal membrane in some regions(parallelogram) were observed. Podocytes irregular form anddimensions was noticed (X 20.000).

Figure 6. Sodium dodecyl sulphate-polyacrylamide gelelectrophoresis of Porthidium lansbergii hutmanni crudevenom. (CvPlh), MWM: molecular weight markers.

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5

counterpart of acute renal failure. This is the first timethat these kinds of lesions have been observed inPorthidium genus.

The proteolytic and phospholipase A2 (PLA2)activities of Porthidium venoms [18] have significantcytotoxic consequences in many cell types [13] andcould contribute to the nephrotoxicity seen in thecurrent study. A theoretical cytotoxin-like activity hasbeen estimated for members of the same family(Viperidae) myotoxins acting on skeletal muscle cells invivo [7]. Following fixing to an unknown place on thecell plasma membrane, the myotoxins would go in thebilayer through hydrophobic interactions, producingenhanced permeability to ions and macromolecules. Animportant calcium influx would almost certainly be themost significant consequence of membrane disorder andwould be dependable for the beginning of an array ofcritical 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 showedprotein bands with molecular masses between 7.0 and206 kDa (Fig. 6), corresponding to the range in which the

main components of Viperidae venoms are found andmost 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 timedemonstrates that intramuscular administration of P.l.hutmanni venom causes glomerular and tubular kidneychanges and that this contribute to the nephrotoxicity inARF. It seems likely that mesangiolysis, mitochondria andmicrovascular damages are a consequence of the highproteolytic and PLA2 activities of this venom. Studiesare being designed to identify the fraction(s)venomresponsible(s) for such ultrastructural changes.

SOURCES AND MANUFACTURERS1Sigma Aldrich. St. Louis, MO, USA.2Merck. Darmstadt , Germany).3Electron Microscopy Sciences. Hatfield, PA, USA.4Ladd Research Inc. Williston, VT , USA.5Bio-Rad. USA.

Conflict of interest. The authors declare that there are noconflicts of interest concerned with this work.

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A.VA.VA.VA.VA.Vararararargasgasgasgasgas,,,,, H.F H.F H.F H.F H.Finol,inol,inol,inol,inol, M.G M.G M.G M.G M.Girón,irón,irón,irón,irón, et alet alet alet alet al..... 2011. 2011. 2011. 2011. 2011. Effects of Lansberg’s Hognose Pit Vipers (Porthidium lansbergii hutmannii)Venom on Renal Ultrastructure in Experimental Mice. Acta Scientiae Veterinariae 39(1): 941.

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