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Oxidative Stress and Apoptosis are Markers in Renal Toxicity Following Egyptian Cobra (Naja haje) Envenomation.

Byline: Mohamed A. Dkhil Saleh Al-Quraishy Abdel Razik H. Farrag Ahmed M. Aref Mohamed S. Othman and Ahmed E. Abdel Moneim

Abstract Snakebite is a serious and important problem in tropical and subtropical countries including Egypt. The venom of Egyptian cobra (Naja haje; L.) is complex and it has been considered as a good source of short neurotoxins and several cytotoxins. In this study oxidative stress inductions as well as apoptotic effects of the Egyptian cobra crude venom at a dose of 0.025mg/kg (intraperitoneal injection; i.p.) has been investigated in kidney of rats after 4 h. Twelve rats divided into 2 groups Group I served as control group Group II received i.p. injection of 0.025mg/kg of crude venom. The venom enhanced lipid peroxidation and nitric oxide productions in the kidney with concomitant reduction in glutathione content and superoxide dismutase catalase glutathione peroxidase glutathione reductase and glutathione-S-transferase activities were inhibited.

Moreover the venom induced a renal injury as indicated by histopathological changes in the kidney tissue with an elevation in serum creatinine and urea. In addition the renal ultrastructural changes were in the form of blebbing of visceral epithelial cells and foot process disorganization. Also the glomerular capillaries lined by hypertrophied endothelial cells. These findings were associated with the pro-apoptotic action in the kidney. The results suggest that Egyptian cobra venom stimulates oxidative stress to induce apoptosis in renal tissue through inhibition of mitochondrial respiration in male rats.

Keywords: Egyptian cobra venom renal toxicity oxidative stress apoptosis.

INTRODUCTION

Snakebite envenomation is known to man since antiquity and many references to snakebite are found in the oldest medical writings. There are more than 2.5 million venomous snake bites annually with greater than 125000 deaths. The risk is highest in the tropics and West Africa predominantly among rural population (Gutierrez et al. 2006). Snake venoms are a mixture of complex toxins that may be independent synergistic or antagonistic (Al- Quraishy et al. 2014). Broadly there are two types of toxins namely neurotoxins which attack the central nervous system and hemotoxins which target the circulatory system (Markland 1998). It is important to understand that the actual mixture of toxins in the venom will vary by individual species and also by age and season (Al-Sadoon et al. 2013).

The venom of Egyptian cobra (Naja haje) is complex and it has been a good source of short neurotoxins and several cytotoxins. From this venom the purification and the primary structure of two short neurotoxins and of 14 cytotoxins have been reported. Besides the toxins N. haje venom contains also low-molecular-weight polypeptides usually of relative low toxicity and of completely distinct immunochemical properties (Joubert and Taljaard 1978). Although nearly all snakes with medical relevance can induce nephropathy leading to acute renal failure (ARF) it is unusual except with bites by Egyptian cobra. This action was attributed to the effect of different venom toxins such as myotoxins cytotoxins phospholipases and cardiotoxins (Rahmy 2001). It was found that myotoxin probably causes renal damage due to myoglobin cast nephropathy.

Venom phospholipase is known to be toxic to cells and believed to be responsible for disturbing the cell membrane permeability

(Mukherjee and Maity 1998). Phospholipase may also alter the mitochondrial respiratory functions and induce a hemolytic activity (Mukherjee et al. 1998). Cobra cytotoxins and cardiotoxins may also disturb different cell types (Rahmy 2001). Cytotoxin lytic activity in synergy with various phospholipases of cobra venom was also reported (Chaim-Matyas et al. 1995).

Nevertheless the oxidative stress induced by the venom of N. haje was not sufficiently covered in the available literature (El Hakim et al. 2011). Thus it is so special interest to examine the possible effects of LD50 of the crude venom in kidney of rats after 4 h of envenomation.

MATERIALS AND METHODS

Experimental animals

Adult male Wistar albino rats weighing 120 150 g were obtained from the Holding Company for Biological Products and Vaccines (Vacsera Cairo Egypt). After an acclimatization period of one week the animals were divided into two groups (6 rats per group) and housed in wire bottomed cages under standard conditions of illumination with a 12 h light-dark cycle at 251C. They were provided with water and a balanced diet ad libitum. We have followed the European Community Directive (86/609/EEC) and national rules on animal care that was carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals 8th edition.

Experimental protocol

Venom was milked from adult snakes collected from the western Nile delta in Egypt in September dried and reconstituted in saline solution prior to use. LD50 of venom was determined as described by Meier and Theakston (1986). To study the effect LD50 of the venom in kidney of rats after 4 h rats were divided into two groups six rats of each. Group I served as a control and received saline (0.2 ml saline/ rat) by intraperitoneal (i.p.) injection. Group II was injected i.p. with LD50 dose of N. haje venom in saline (0.025mg/kg). The animals of the two groups were sacrificed and blood samples were collected by cardiac puncture. The blood stranded for half an hour and then centrifuged at 3000 g for 15 min at 4C to separate serum and stored at -70C until analysis. The left kidney was weighed and homogenized immediately to give a 50% (w/v) homogenate in ice-cold medium containing 50 mM Tris-HCl pH 7.4. The homogenate was centrifuged at 3000 g for 10 min at 4C.

The supernatant (10%) was used for the various biochemical determinations. Right kidney was cut into small pieces and kept for the histological and molecular studies.

Biochemical estimations

Kidney function test Serum uric acid urea and creatinine contents were determined colorimetrically by commercially available diagnostic kits (Biodiagonstic-Egypt) as per manufacturer's instructions.

Oxidative stress markers

Lipid peroxidation (LPO) in kidney was determined by using 1 ml of trichloroacetic acid 10% and 1 ml of thiobarbituric acid 0.67% and were then heated in a boiling water bath for 30 min. TBARS were determined by the absorbance at 535 nm and expressed as malondialdehyde (MDA) formed (Ohkawa et al. 1979). Also nitric oxide was determined in acid medium and in the presence of nitrite the formed nitrous acid diazotized sulfanilamide is coupled with N-(1naphthyl) ethylenediamine. The resulting azo dye has a bright reddish-purple color that can be measured at 540 nm (Green et al. 1982). In addition the renal glutathione (GSH) was determined by the reduction of Elman's reagent (55` dithiobis (2-nitrobenzoic acid) DTNB) with GSH to produce a yellow compound . The reduced chromogen directly proportional to GSH concentration and its absorbance can be measured at 405 nm (Ellman 1959).

Enzymatic antioxidant status

The homogenates of kidney were used to determine the activities of superoxide dismutase (SOD) (Nishikimi et al. 1972) catalase (CAT) (Aebi 1984) glutathione peroxidase (GPx) (Paglia and Valentine 1967) glutathione-S-transferease (Habig et al. 1974) and glutathione reductase (GR) (Factor et al. 1998). Flow cytometry

Pieces of kidney were prepared by manual disaggregation procedure. Briefly a few drops of RPMI were added to tissue and then minced until complete tissue disaggregation was achieved. Suspended cells were filtered using a 50 m pore size mesh and then centrifuged at 1000 g for 10 min. Cells were resuspended in phosphate buffer counted and washed by calcium buffer then centrifuged at 1500 g for 5 min. The pellet was resuspended and then cells were counted. Annexin- PI apoptotic assay was carried out using IQP-120F Kit (IQ Products Groningen Netherlands). FAC scan Becton-Dickinson (BD) flow-cytometer was used and data were analyzed using cell Quest software.

Western blot analysis

Total proteins were extracted using RIPA buffer. Protein determination was applied by a Lowry method (Lowry et al. 1951). Denatured proteins (20 g) were size fractionated by 12.5% SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose membrane at 30 V for 1 h. The blots were blocked for 1.5 h at room temperature in fresh blocking buffer (0.1% Tween-20 in Tris- buffered saline pH7.4 containing 5% BSA). The membrane was incubated overnight at 4C with primary antibodies against Bax GPx GR SOD and mitochondrial respiratory complexes at dilution of 1:200. AY-actin was used as a loading control. The membrane was incubated for 2 h with HRP- conjugated secondary antibodies at a dilution of 1:1000. Chemiluminescent signals were captured using an ECL-plus chemiluminescent kit (GE Healthcare UK) and Kodak Luminescent Image Analyzer (Kodak In-Vivo Imaging Systems F).

Histopathology and p53 and caspase-3 immunohistochemistry

Small pieces of the kidney were quickly removed then fixed in 10 % neutral buffered formalin. Following fixation specimens were dehydrated embedded in wax and then sectioned to 5 microns thickness. For histological examinations sections were stained with haematoxylin and eosin. Immunolocalization techniques for p53 and caspase- 3 were performed on 3-4 m thickness sections according to Pedrycz and Czerny (2008). For negative controls the primary antibody was omitted. In brief mouse anti-p53 or mouse anti-caspase-3 (diluted1:200 Santa Cruz Biotechnology Santa Cruz CA USA) were incubated with sections for 60 min. Primary antibodies were diluted in Tris buffered saline (TBS)/1% BSA. Then a biotinylated secondary antibody directed against mouse immunoglobulin (Biotinylated Link Universal DakoCytomation kit supplied ready to use) was added and incubated for 15 min followed by horse radish peroxidase conjugated with streptavidin

(DakoCytomation kit supplied ready to use) for a further 15 min incubation. At the sites of immunolocalization of the primary antibodies a reddish to brown colour appeared after adding 3- amino-9-ethylcarbasole (AEC) (DakoCytomationkit supplied ready to use) for 15 min. The specimens were counterstained with hematoxylin for 1min and mounted using the Aquatex fluid (Merck KGaA Germany).

Scanning electron microscopic (SEM) study

Kidneys were removed and cortical slices were cut into big pieces which were immersed in Karnovsky's solution (2% glutaraldehyde 2% paraformaldehyde in 0.1 M phosphate buffer pH 7.4) at 4C overnight. After rinsing in phosphate buffer for 1 h the specimens were post-fixed in buffered 1% OsO4 at 4C in the dark for 2 h and then immersed in a 2.3 M sucrose solution at 4C 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. After identifying the fractured surface specimens were mounted on stubs sputtered with gold for 120 s and examined and photographed with a Zeiss DSM 940A SEM operated at 10 kV.

Transmission electron microscopic (TEM) study

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 h at room temperature. After rinsing in a sugar-saline solution (0.15 M NaCl 0.2 M sucrose) the specimens were post-fixed with 1% OsO4 at 4C in the glucose-saline solution in the dark for 2 h 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 60C for 48 h. Thin sections (6070 nm) were double-stained with uranyl acetate and lead citrate and were observed and photographed with a LEO 906 TEM operated at 60 kV.

Statistical analysis

The obtained data were presented as means standard deviation of the mean. Statistical analysis was performed using an unpaired Student's t-test using a statistical package program (SPSS version 17.0). Differences between groups were considered significant at Pless than 0.05.

RESULTS

Kidney function parameters were affected by a single i.p. injection of Egyptian cobra (0.025 mg/kg). The levels of serum urea and creatinine were increased significantly (27.7 and 44.0% respectively) in envenometed rats (Table I). However the level of uric acid was significantly reduced (22.2%; Pless than 0.05) after 4 h of venom injection compared to the control rats.

Table I.- Changes in kidney function of rats induced by Egyptian cobra venom after 4 h.

Parameters###Control###Intoxicated

###rats###rats###

Serum uric acid (mg/dL)###69.639.81###54.146.61

Serum urea (mg/dL)###3.210.40###4.10.46

Serum creatinine (mg/dL)###0.500.03###0.720.03

Table II shows the potential oxidative stress effects of Egyptian cobra venom in the kidney homogenate. This was evidenced by the significant increase in the level of both of the lipid peroxidation and nitric oxide by 78.5 and 82.2% respectively. The elevation in nitric oxide content of the kidney could be explained by up-regulation in iNOS gene that measured with RT-PCR (data not shown). Also the glutathione level was significantly reduced by about 55% in the kidneys (Table II) of rats receiving the venom. Moreover there was a reduction in GR GST and GPx by approximately 28.6 60.9 and 45.7% respectively. Interestingly the reduction in GR and GPx activities were accompanied by concomitant significant effect on its expression (Pless than 0.05) (Fig. 1). The reduction in GR and GPx proteins expression were 24.1 and 15.1% respectively. The reduction in GPx enzyme activity was also tested with RT-PCR method where GPx gene was down-regulated as compared with that of the control (data not shown).

Table II.- Changes in kidney oxidant/antioxidant state of rats induced by Egyptian cobra venom after 4 h.

Parameters###Control###Intoxicated

###rats###rats

Tissue MDA (nmol/g tissue)###729.7050.21###1302.8281.66

Tissue NO (mol/g tissue)###100.947.73###183.8913.10

Tissue GSH (mmol/g tissue)###67.332.89###39.711.33

Tissue GPx (U/g tissue)###2075.03317.67###1125.77203.39

Tissue GR (mol/h/g tissue)###140.6722.01###100.4823.62

Tissue GST (mol/h/g tissue)###0.230.06###0.090.01

Tissue SOD (U/g tissue)###2.520.04###1.210.16

Tissue CAT (U/g tissue)###1.510.16###0.960.12

Severe inhibition of SOD and CAT activities (108.3 and 36.4% respectively) were observed in the kidneys (Table II) of Egyptian cobra venom- injected rats compared to these of the controls. The inhibition in SOD was accompanied by a significant reduction in the expression of SOD after 4 h Egyptian cobra envenomation by approximately 49% (Pless than 0.05).

Figure 1 shows alterations in the activities of mitochondrial respiratory complexes. Complex II III and IV activities in the kidney tissue from envenomated rats were highly increased (40100% from control; Pless than 0.05). However complex V showed a significant inhibition (32%; Pless than 0.05) in its activity. In the present investigation the percentage of both of the apoptotic and necrotic cells in the kidney of Egyptian cobra venom-injected rats were 28.3 and 32.06% respectively up to the control (Fig. 2) while the viable cells were decreased down to - 21.4% compared to the control samples.

Microscopic examination of the renal tissue shows that the venom induced a severe glomerular degeneration and coagulative necrosis. Also the urinary spaces appeared wider as compared with the control one. Moreover most of the renal tubules were degenerated and filed with cellular debris (Fig. 3B). Immunohistochemical findings in control kidneys showed a week immunoreactivity of both of caspase-3 and p53 (Fig. 3CE respectively) while a strong positive reaction was detected in the sections of the venom injected group (Fig. 3D and F respectively).

The ultrastructure of the glomeruli of the control animals showed capillary loops which embodied blood cells and precipitated plasma proteins. The capillaries are lined with a thin layer of flattened and fenestrated endothelial cells. The nuclei of such cells can be seen bulging into the capillary lumina. Mesangial cells and mesangial substance provides support for the capillary loops. The podocytes form the outer layer of the capillary wall each has a cell body from which arise several primary processes. Each primary process gives rise to numerous secondary processes called pedicels that embrace the glomerulus capillaries. The fenestrated capillary endothelium is closely applied to the glomerular basement membrane and on the opposite side of the basement membrane are the podocytes secondary processes (pedicels) separated from each other by slight pores of approximately uniform width (Fig. 4A).

The electron micrographs of the convoluted tubules of control animals reveal profuse tall microvilli which represent the brush border seen with light microscopy. The plasma membrane at the bases of the cells of the tubules exhibits deep basal infoldings into the cells. These infoldings set up basal compartments that embody elongated mitochondria (Fig. 4B).

In the venom injected group the glomeruli appeared with proliferated mesangial cells and most of the foot processes of the epithelial cells were discrete but few of these processes were fused together (Fig. 4C). On the other hand the convoluted tubule showed deformity of the nuclei mitochondria and the enfolding of the basement membrane. Extensive degenerative changes represented by lysis of many cell contents were notice (Fig. 4D).

SEM examination of the glomeruli tufts of normal rats showed many erythrocytes with various shapes in the capillary networks and interdigitating foot processes (Fig. 5A). The convoluted tubules of normal rats contained basement membranes mitochondria nuclei infoldings and lamina (Fig. 5B). On the other hand glomeruli of the venom injected rats were enlarged due to loss of mesangial cells and matrix. Also the glomeruli appeared with a segmental ballooning lesion with honeycomb-like appearance on the surface (Fig. 5C). In addition the convoluted tubules were damaged (Fig. 5D).

DISCUSSION

Several works dealing with the effects of snake venoms in blood cells marrow cells and in cells from other organs of animals like muscle liver kidney and skin showed varying results depending on the experimental concentrations exposure time site of injection and type of toxin (Maria et al. 2003; Fox and Serrano 2008; Tohamy et al. 2014). In the present investigation we explored the systemic physio-pathological changes induced by the Egyptian cobra venom in kidney as a vital organ. The high levels of creatinine and urea indicate an impairment of renal function. Similar observations were reported in rats following administration of various snake venoms (Omran and Abdel-Nabi 1997; Schneemann et al. 2004). Such increased vascular permeability together with renal damage would further aggravate the accompanying hypoproteinemia. Furthermore the rise in serum urea and creatinine associated with the reduction of serum uric acid level observed in the present study

Supports the proposed impairment of renal function. Similar observations were reported following various viper envenomation of rats (Sant and Purandare 1972; Omran and Abdel-Nabi 1997). The proteolytic and phospholipase A2 activities of Egyptian cobra venoms have important cytotoxic effects (Kerns et al. 1999; Rowan et al. 1991; Barrington et al. 1986; Stefansson et al. 1990) and could contribute to the nephrotoxicity seen in the current study.

The ultrastructural alterations induced by the snake venom in the trilaminar structure of the glomerular basement membrane (GBM) suggest the involvement of physicoelectrostatic barrier sites. Podocytes and endothelial cells retain heparin sulfate (Stow et al. 1985) in the lamina rara external and internal of the GBM and the feet processes are coated with sialoproteins that contribute to the anionic charges of the filtration barrier (Kerjaschki et al. 1984). This ionic barrier of glomerular filtration was shown to be destroyed by Vipera russelli venom in isolated rat kidneys (Willinger et al. 1995). Furthermore since the glomerular and renal tubule epithelial cells are strategically interposed between the extra and 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.

Snakebites are most often accompanied by signs of inflammation and local tissue damage (Nelson 1989). Neutrophils and macrophages are induced to produce superoxide radical anion which belongs to a group of reactive oxygen species (ROS) and this reacts with cellular lipids leading to the formation of lipid peroxides and the observed necrosis (Valko et al. 2007). As the origin of oxidative stress is the mitochondrial respiratory electron transport chains (Fletcher et al. 1991) it is possible that mitochondrial death mediates venom- induced cellular damage (Haffor and Al-Sadoon 2008; Abdel Moneim et al. 2014a).

It is generally accepted that a marked increase in Ca2+ could lead to ROS production. In many models of cell death it has also been proposed that ROS production is an early event in the process of apoptotic cell death. Furthermore it had been proposed that ROS can also modulate gene expression by acting on transcription factors in a variety of families like NF-B activator protein 1 (AP-1) and AP-2 (Ray et al. 2012). Also N. haje venom contains cardiotoxins (CTX) a group of highly basic polypeptides of approximately 60 amino acids present in the many snakes have ability to increase H2O2 production. All of these findings suggested that the renal cytotoxic effects of venom were apparently triggered by oxidative stress mediated by H2O2 and superoxide anion production. Generation of H2O2 in the mitochondria may result in mitochondrial peroxidation (Al-Quraishy et al.2014).

Superoxide dismutase and catalase are considered as the primary antioxidant enzymes since they are involved in the direct elimination of active oxygen species (Abdel Moneim et al. 2014b). Dai et al. (2012) have reported that Najasp. venoms have the SOD activity. Apart from this in the past few years several peptides have been reported to exert deferent mechanisms of action in free radical mediated oxidative sequences by radical scavenging and metal ion chelation (Sri Balasubashini et al. 2006; Tohamy et al. 2014).

Many L-amino acid oxidase demonstrate apoptosis-inducing activity (Zhang and Wu 2008) and it is partially due to the generation of hydrogen peroxide. Hydrogen peroxide belongs to ROS and it is widely accepted that mitochondrial perturbation has been associated with the increased production of ROS (Othman et al. 2014).

Expression of p53 as proapoptotic protein was significantly activated by Egyptian cobra venom. Moreover our results showed that caspase-3 was most potently activated by the venom. Moreover the present data suggest that the activation of caspase-3 is critical in snake venom- induced kidney damage. Also p53 plays multiple roles in cell cycle control differentiation genomic stability angiogenesis and apoptosis (Maxwell and Davis 2000). Miao et al. (1999) showed that the mRNA of p53 increased within 3 and 6 h after vascular endothelial cells were treated with rattle snake venom. Our results indicated that p53 plays an important role in apoptosis induced by Egyptian cobra venom in the kidney and they are in the same pathway in apoptotic signal transduction in the kidney.

CONCLUSIONS

The data presented herein suggested several aspects of the mechanisms of Egyptian cobra venom-induced renal toxicity. We proposed that: (i) Egyptian cobra venom induced apoptosis in kidney via activation of caspase-3 and modulation of the protein levels of Bax and Bcl-2; (ii) Egyptian cobra venom-activated p53 induced apoptosis; (iii) oxidative stress involved in transmitting apoptotic signals in Egyptian cobra venom-treated rats. These results provide valuable insight into the toxicity of Egyptian cobra venom and deepen our previous understanding of its molecular mechanisms of action.

Conflict of interests statement

The authors declare that there is no conflict of interests regarding the publication of this article.

ACKNOWLEDGEMENTS

The authors would like to extend their sincere appreciations to the Deanship of Scientific Research at King Saud University for its funding this Research group NO. (RG -1435-198).

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Publication:Pakistan Journal of Zoology
Article Type:Report
Date:Dec 31, 2014
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