Porthidium Lansbergii Hutmanni) Venom on Renal Ultrastructure in Experimental Mice Acta Scientiae Veterinariae, Vol

Acta Scientiae Veterinariae ISSN: 1678-0345 [email protected] 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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ORIGINAL ARTICLE ISSN 1679-9216 (Online) Pub. 941

Effects of Lansberg’’’ s Hognose Pit Vipers (Porthidium lansbergii hutmanni) Venom on Renal Ultrastructure in Experimental Mice*

Alba Vargas1, Héctor Finol2, María Girón3, Héctor Scannone1,,, Irma Fernández1 & Alexis Rodriguez-Acosta3

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 [[email protected]]. 1 A.Vargas,,, H.Finol, M.Girón, et al... 2011. Effects of Lansberg’s Hognose Pit Vipers (Porthidium lansbergii hutmannii) Venom on Renal Ultrastructure in Experimental Mice. Acta Scientiae Veterinariae 39(1): 941. weight) of CVPlh and sacrificed at 3, 6, and 24 h later. INTRODUCTION Two mice injected with saline were use as normal controls. Porthidium lansbergii hutmanni species occurs Ethical statement at low and moderate elevations down 800 meters altitude Expert personnel prepared all the experimental in Margarita Island, Venezuela. It appears to be restricted procedures relating to the use of live animals. The to this island. Two different species; Porthidium Venezuelan pertinent regulations as well as lansbergii rozei and P. lansbergii lansbergii live in the institutional guidelines, according to protocols mountains surrounding the Cordillera de La Costa in approved by the Institute of Anatomy of the mainland Venezuela [4], located in the extreme Universidad Central de Venezuela and the norms northwestern and northeastern portion of the country. The obtained from the guidelines for the care and use of epidemiological importance at Margarita Island is laboratory animals, published by the US National unknown because the accidents are reported in Institute of Health [1] were followed. conjunction with other viper snakes envenomations. The mainly systemic consequence and the most frequent Determination of protein concentration complication in fatal cases of snakebites in Venezuela is Protein concentrations of all tested venoms were acute renal failure (ARF) secondary to acute tubular measured by the Lowry method [9], using bovine necrosis [5, 14]. In general, glomerular changes in human serum albumin1 as standard protein. victims of snakebite envenoming have concerned modest consideration, and it has not had methodical study of this Lethality assay problem. There is not literature description of Porthidium The Spearman-Karber [20] method was used venom activity on the renal structure. Otero et al. (2002) to calculate the P.l. hutmanni crude venom lethal dose fifty (LD ). Six groups of five mice (18-22 g) were in a report of Viperidae envenomed patients described 2 50 cases of envenoming by Porthidium nasutum that did intraperitoneally injected with 0.1 mL of several not have clinically renal damage. The purpose of this study concentrations of venom and the number of mice that was to determine how Porthidium lansbergii hutmanni died was counted after 48 h. venom injection into mice could lead to severe renal injury. Transmission electron microscopy Kidney biopsies were obtained at 3, 6, and 24 MATERIALS AND METHODS h after venom injection from envenomed mice under Porthidium lansbergii hutmanni venom anesthesia2. Samples were immediately fixed in situ Crude venom from Porthidium lansbergii 3 with 3% glutaraldehyde and 1% OsO4 (both fixatives hutmanni (CVPlh) was obtained milking every 30 days diluted in pH 7.4, 320 mM phosphate buffer saline), twelve mature snakes, captured in Juan Griego and dehydrated in ethanol and embedded in LX-112 Porlamar, Margarita Island (Venezuela) and resin4. Ultrathin sections were stained with uranyl maintained in captivity in the Serpentarium of the acetate3 and lead citrate3 and observed in a Hitachi Laboratory of Investigations, Pharmacy Faculty of the H-500 transmission electron microscope with an Universidad Central de Venezuela. The extracted accelerating voltage of 100 KV. venom was dehydrated in vacuum desiccators, maintained with calcium chloride1 at 4ºC until its total SDS-PAGE crystallization. Electrophoresis using a MINI-PROTEAN II (BioRad, USA) chamber was performed. Sodium Mice dodecyl sulphate-polyacrylamide gel electrophoresis Fifteen male mice INH strain weighing 18-22 was carried out according to the Laemmli method [8] g from the Instituto Nacional de Higiene “Rafael using 12.5% gels under reducing conditions2. Rangel”, Caracas, Venezuela were used. The mice Molecular weight markers5 were run in parallel and boxes were kept in a colony room maintained at 23 gels were stained with Comassie Blue R-2501. ºC on a 12/12-h lumen/dark cycle. CVPlh samples were dissolved in 1:1 proportion Envenomed mice in the solubilizer solution 0.5 M tris-HCl buffer (pH 6.8) with 10% (v/v) SDS1, 10% (v/v) b-mercaptoethanol1, Fifteen mice (18-22 g) were intramuscularly injected with 0.1 mL of a sub-lethal dose (2.0 mg/kg body 2 A.Vargas,,, H.Finol, M.Girón, et al... 2011. Effects of Lansberg’s Hognose Pit Vipers (Porthidium lansbergii hutmannii) 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 injection, in this section a region formerly occupied by the interdigitations was observed; vacuoles (V) and vesicles (v), in Figure 3. Electron-micrographs of mice kidney after 6 h CvPlh addition to swollen mitochondria (stars) with variable number of injection, panoramic view of proximal convoluted tubule section cristae in different orientations were noticed. The capillary showed showing pleomorphic mitochondria with calcic deposits (arrows) widened fenestrae (arrows). In the tubule at the right position, an and an autophagolysosome (rhombus) with mylenic figures inside area of interdigitations with membranous debris (rhombus) was (X 20.000). observed (X 16.000). 3 A.Vargas,,, H.Finol, M.Girón, et al... 2011. Effects of Lansberg’s Hognose Pit Vipers (Porthidium lansbergii hutmannii) Venom on Renal Ultrastructure in Experimental Mice. Acta Scientiae Veterinariae 39(1): 941. After 24 h CvPlh injection, in the glomerule, glomerular structures and capillaries cluster compatible fenestrated areas of the capillary endothelia lost and with the renal dysfunction caused by snakebite described widened basal membrane in some regions (parallelogram) elsewhere [3]. The predominant renal lesions observed were seen. Podocytes irregular form and dimensions was in the experimental animals were tubular degeneration observed (Figure 5). 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

Figure 5. Electron-micrographs of mice kidney after 24 h CvPlh complexity of the usual interdigitating pattern of cell–cell injection, in the glomerule, fenestrated areas of the capillary endothelia connections and an evident diminution of filtration capacity. lost (circle) and widened basal membrane in some regions Comparable modifications have been found in human (parallelogram) were observed. Podocytes irregular form and glomerulonephritis linked with nephrotic syndrome[6] and dimensions was noticed (X 20.000). in nephrosis induced by antibiotics, which are toxic to Sodium dodecyl sulphate-polyacrylamide gel visceral epithelial cells [15, 19] as well as in renal lesions electrophoresis (SDS-PAGE) caused by Uracoan rattlesnake venom [5], where CvPlh SDS-PAGE profile is shown in Figure 6. important podocytes alterations were described. Our Under native conditions approximately 14 bands results reproduced these anomalous features and offer distributed from 7.6 to 215.0 kDa were observed. 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 Figure 6. Sodium dodecyl sulphate-polyacrylamide gel Trimeresurus flavoviridis [10]. The high proteolytic electrophoresis of Porthidium lansbergii hutmanni crude activity of the last venom was thought to produce most of venom. (CvPlh), MWM: molecular weight markers. the harm to the glomerular mesangium [16] and could DISCUSSION also be responsible for CvPlh-induced mesangiolysis. The disorder usually causes edema, and nephrotic Lansberg’s hog-nosed pit viper is a nocturnal syndrome and it may progress to renal failure. It is seen animal. They are not violent snakes (excluding their prey) more commonly in Crotalus envenomed patients who but will respond energetically if threatened. develop acute renal failure. All renal structures can be The results of this study show that in mice CvPlh involved. Tubular necrosis is the important pathological causes ultrastructural changes in the tubular and 4 A.Vargas,,, H.Finol, M.Girón, et al... 2011. Effects of Lansberg’s Hognose Pit Vipers (Porthidium lansbergii hutmannii) Venom on Renal Ultrastructure in Experimental Mice. Acta Scientiae Veterinariae 39(1): 941 counterpart of acute renal failure. This is the first time main components of Viperidae venoms are found and that these kinds of lesions have been observed in most likely equivalent to metalloproteinases (40-64 kDa),

Porthidium genus. serine proteinases (20-40 kDa) and phospholipases A2

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

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