Action of cobra venom on the renal cortical tissues: electron microscopic studies

Abstract

The effect of intramuscular (IM) injection of a sub-lethal dose of Naja haje cobra venom (0.015 mug/gm body weight) on the ultrastructure of renal cortical tissues of rabbits was studied at different time intervals after envenomation. Succinic dehydrogenase (SDH) enzyme activity was also detected in the renal cortical tubules. Three hours after venom injection, slight changes were seen in visceral cells and glomerular endothelia. The cortical tubular epithelia revealed an increase of lysosomal structures, cytoplasmic vacuolization, and nuclear irregularity. Severe ultrastructural changes were recorded 6 hours after envenomation, including hypertrophied parietal cells, blebbed visceral epithelial cells, and foot process disorganization. Dilated glomerular capillaries lined by hypertrophied endothelial cells and signs of mesangiolysis were also observed. The proximal tubular epithelia showed vesiculated endoplasmic reticulum and numerous lysosomal vacuoles, while the distal tubular epithelia were mostly necrotic. These alterations were more intense 12 hours after venom injection. Parietal and visceral cells showed similar changes to those of the six-hour group. Most of the glomerular endothelial cells lost their integrity and diffused into the capillary lumen. Mesangiolysis was also observed. The proximal tubular epithelial cells revealed severely affected cellular organelles, dilated brush borders, disruption of the basal membrane infoldings, and cellular necrosis. The distal tubular epithelia lost most of their cytoplasmic electron density and cellular organelles. Necrotic cells were also seen. Generalized mitochondrial alterations were observed in all renal cortical cell types accompanied by a marked depletion of SDH enzymatic activity in those cells. It is believed that these venom-induced non-specific lesions could be due to secondary synergistic action of more than one venom toxin, which mainly target the mitochondria in all components of renal cortical tissues. This could lead to an ischemic condition that could be responsible for the appearance of these ultrastrucural alterations.

snake venom; nephrotoxicity; kidney; ultrastructure

ACTION OF COBRA VENOM ON THE RENAL CORTICAL TISSUES: ELECTRON MICROSCOPIC STUDIES

T. R. RAHMY¹

1 Department of Biology, Faculty of Science, United Arab Emirates University

ABSTRACT:The effect of intramuscular (IM) injection of a sub-lethal dose of Naja haje cobra venom (0.015 mg/gm body weight) on the ultrastructure of renal cortical tissues of rabbits was studied at different time intervals after envenomation. Succinic dehydrogenase (SDH) enzyme activity was also detected in the renal cortical tubules. Three hours after venom injection, slight changes were seen in visceral cells and glomerular endothelia. The cortical tubular epithelia revealed an increase of lysosomal structures, cytoplasmic vacuolization, and nuclear irregularity. Severe ultrastructural changes were recorded 6 hours after envenomation, including hypertrophied parietal cells, blebbed visceral epithelial cells, and foot process disorganization. Dilated glomerular capillaries lined by hypertrophied endothelial cells and signs of mesangiolysis were also observed. The proximal tubular epithelia showed vesiculated endoplasmic reticulum and numerous lysosomal vacuoles, while the distal tubular epithelia were mostly necrotic. These alterations were more intense 12 hours after venom injection. Parietal and visceral cells showed similar changes to those of the six-hour group. Most of the glomerular endothelial cells lost their integrity and diffused into the capillary lumen. Mesangiolysis was also observed. The proximal tubular epithelial cells revealed severely affected cellular organelles, dilated brush borders, disruption of the basal membrane infoldings, and cellular necrosis. The distal tubular epithelia lost most of their cytoplasmic electron density and cellular organelles. Necrotic cells were also seen. Generalized mitochondrial alterations were observed in all renal cortical cell types accompanied by a marked depletion of SDH enzymatic activity in those cells. It is believed that these venom-induced non-specific lesions could be due to secondary synergistic action of more than one venom toxin, which mainly target the mitochondria in all components of renal cortical tissues. This could lead to an ischemic condition that could be responsible for the appearance of these ultrastrucural alterations.

KEY WORDS: snake venom, nephrotoxicity, kidney, ultrastructure.

INTRODUCTION

Nephropathy induced by cobra factor was mentioned by Nangaku et al. (18). In a previous study, a sub-lethal dose of the Egyptian cobra venom was found to induce a deleterious action on the histological and histochemical patterns of animal renal tissues (24). This action was attributed to the effect of different venom toxins, such as myotoxins (26), cytotoxins (8), phospholipases (28), and cardiotoxins (11).

It was found that myotoxin probably causes renal damage due to myoglobin cast nephropathy (22). Venom phospholipase is known to be toxic to cells and believed to be responsible for disturbing the cell membrane permeability (16). Phospholipase may also alter the mitochondrial respiratory functions (10) and induce a hemolytic activity (17). Cobra cytotoxins (31) and cardiotoxins (21) may also disturb different cell types. Cytotoxin lytic activity in synergy with various phospholipases of cobra venom was also reported (5).

This study aimed to demonstrate the nephrotoxic action of sub-lethal dose cobra venom at the ultrastructural level and to detect the activity of a mitochondrial-related enzyme during envenomation. This introduces a new approach about venom action on animal kidney.

MATERIALS AND METHODS

SNAKE VENOM. Lyophilized venom from the Egyptian cobra Naja haje was reconstituted in saline solution (0.85% NaCl) to prepare weight-matched concentrations for use on experimental animals.

EXPERIMENTAL ANIMALS. Healthy male rabbits (Oryctolagus cuniculus) aged between 26 and 28 weeks and weighing 1kg + 100 gm were kept under ideal lab conditions, with daily observation before and throughout the experiment. The rabbits were divided into four groups of 5 animals each. The first group, control, animals were intramuscularly (IM) injected with 0.1ml saline solution and sacrificed 12h after injection. Groups two, three, and four were IM injected with sub-lethal doses (0.015 mg/gm b.wt.) of cobra venom (34) and sacrificed under ether anesthesia 3, 6, and 12h after envenomation respectively.

ELECTRON MICROSCOPIC PREPARATIONS. After abdominal dissection, a sample from the right kidney of each rabbit was removed and prepared to small pieces (1x1x3 mm). They were rapidly fixed with 0.1M phosphate buffered fixative solution (4% formaldehyde + 1% glutaraldehyde, pH 7.4) overnight at 4°C (12). The tissues were then washed in 0.1M phosphate buffer, fixed for 1h in 0.1M phosphate buffered 1% osmium tetroxide (at room temperature), and washed several times in the buffer. Dehydration in an ascending series of ethyl alcohols was followed by two changes of propylene oxide. The tissues were transferred to a mixture of propylene oxide and resin and embedded in a mixture of resin (Agar 100 Epoxy resin + DDSA + MNA + DMP-30) overnight. Two changes of resin were then applied, and the samples were left in the oven at 60°C for 24 h for polymerization into plastic blocks. The blocks were then sectioned at a thickness of 60-70 nm and mounted on copper grids. The grids were stained with uranyl acetate and lead citrate and left to dry. Selected areas were photographed using a CM10 Philips Electron Microscope at the School of Medicine, United Arab Emirates University.

DETERMINATION OF SUCCINIC DEHYDROGENASE (SDH) ENZYMATIC ACTIVITY. Small kidney pieces (1x1x3 mm) from each rabbit were rapidly fixed with a phosphate buffered mixture of 4% formaldehyde + 1% glutaraldehyde, pH 7.2, at 4°C for 30 min. Specimens were plunged immediately into a ferricyanide reaction mixture for incubation (4). The samples were incubated at 37°C for 1h in the dark and then washed in 0.05M phosphate buffer, pH 7.6. Samples were fixed for 1 h and 30 min. in buffered 1% osmium tetroxide and processed for EM examination.

RESULTS

ULTRASTRUCTURAL OBSERVATIONS

Control group. Renal cortical tissues of control animals revealed normal ultrastructural patterns of renal corpuscles and tubules (Figure 1).

Figure 1
. Epithelial cell of a proximal convoluted tubule from a control animal. BB: Brush borders.

The three-hour group. Three hours after envenomation, the renal corpuscles (Figure 2) showed slightly swollen parietal epithelia resting on wrinkled but intact tubular capsules. Hypertrophied visceral cells were observed containing irregular nuclei and mitochondria with broken cristae. Loss of the foot-like aspect of the foot processes (pedicles) was also seen. The glomerular capillaries were occluded with electron dense substances and remnants of ruptured cells. The lining endothelial cells showed cytoplasmic blebbing and nuclear irregularity. The mesangial cells also showed irregular nuclei.

Figure 2.
A renal corpuscle of a 3 hour-animal showing parietal epithelia (P), hypertrophied visceral cells (V), disorganized pedicles (F), blebbing (B), and irregular nuclei (N) of glomerular endothelia. Note electron-dense substances (E) in the glomerular capillaries.

The tubular epithelial cells of the proximal (Figure 3) and distal convoluted tubules revealed focal decrease in the electron density of the cytosol and mitochondria with dense materials. High magnification (Figure 4) showed that the mitochondrial cristae were mostly intact, a few, being partially broken. Increased numbers of lysosomal-related structures and cytoplasmic vesiculation and vacuolization were observed in the proximal tubular epithelia (Figure 3). Some nuclei showed irregular outer lining, but the nuclear materials were generally of normal appearance. The brush borders and basal membrane infoldings of the renal tubules were normal (Figure 3).

Figure 3.
Epithelial cell of a proximal convoluted tubule 3 h after venom injection. Note lower electron-dense cytosol (C), lysosomal-related structures (L), cytoplasmic vesiculation (V), intact brush border (B), and basal membrane infoldings (I).

Figure 4.
Highly magnified mitochondria with mostly intact mitochondrial cristae, with some partially broken cristae (arrows) 3 h after venom injection.

The six-hour group. The renal tissues showed severe ultrastructural changes 6 h after venom injection. There were noticeably hypertrophied parietal cells, containing swollen mitochondria with broken cristae and vesiculated cytoplasm (Figure 5). The visceral epithelial cells showed cytoplasmic blebbing, dilated endoplasmic reticulum, and foot process disorganization. Dilated glomerular capillaries were seen containing scattered electron dense granules (Figure 5) and lined by hypertrophied glomerular endothelial cells, with poorly recognized cell membranes. Signs of mesangiolysis were also visible.

Figure 5.
Hypertrophied parietal cell (PR) containing swollen mitochondria with damaged cristae (M) 6 h after venom injection. Note cytoplasmic blebbing (B), fragmentation (F) of visceral cells, and loss of the pedicle foot-like aspect (P).

The epithelial lining cells of the proximal convoluted tubules showed swollen mitochondria with broken cristae, vesiculated endoplasmic reticulum, depleted cytosol (Figure 6), and numerous lysosomes. At high magnification, most of the mitochondrial cristae were broken, especially at their luminal ends, or completely lost at certain points. However, intact cristae were represented in some mitochondria (Figure 7). The basal membrane infoldings and the brush borders were slightly swollen (Figure 6). The epithelial lining cells of the distal convoluted tubules were mostly necrotic, with pyknotic nuclei and heavily condensed mitochondria.

Figure 6.
Epithelial lining cells of a proximal convoluted tubule 6 h after envenomation, showing depleted cytosol (C), mitochondria with broken cristae (M), fragmented rough endoplasmic reticulum (R), and slightly swollen brush borders (B).

Figure 7.
Injured mitochondria showing damage (arrows) and loss of mitochondrial cristae (double arrows) 6 h after envenomation.

Twelve-hour group. Twelve hours after venom injection, the renal corpuscles showed extremely enlarged parietal epithelia containing pyknotic nuclei, swollen mitochondria with broken cristae, vesiculated endoplasmic reticulum, and vacuolated cytoplasm (Figure 8). The visceral epithelial cells showed large empty vacuoles, mitochondria with broken cristae (Figure 8 and Figure 9), and dilated fragments of endoplasmic reticulum (Figure 9). Noticeable loss of the foot-like aspect of the pedicles was seen at certain points (Figure 8 and Figure 9). The glomerular capillaries enclosed remnants of dead cells and most of their lining endothelial cells lost their integrity and diffused with the surrounding matrix of the capillary lumen (Figure 9). Other endothelial cells suffered from marked blebbing and cellular swelling, which constricted the glomerular capillary lumen (Figure 9). They contained large irregular vacuoles (Figure 8), swollen mitochondria with completely broken cristae, dilated endoplasmic reticulum, and irregular nuclei (Figure 9). The mesangial matrix showed loss of electron density and focal areas of dissolution. The mesangial cell underlying basement membranes were disintegrated at certain points, and the mesangial cells showed irregular nuclei and a loss of mitochondrial cristae (Figure 9).

Figure 8.
Twelve hours after envenomation, a parietal cell with pyknotic nucleus (N), distorted mitochondrial cristae (M), and vacuolated cytoplasm (V). Note blebbing (B), vacuolization (VC), and mitochondrial deterioration (D) of visceral cells and cellular debris (G) in the glomerular capillaries.

Figure 9.
Broken mitochondrial cristae (M) and dilated fragments of endoplasmic reticulum (R) in different cells of a renal corpuscle 12 h after envenomation. Note blebbing (B) of visceral cells, abnormal foot process (F), swollen endothelia (G), mesangiolysis (arrows), nuclear irregularity (N), and disintegrated basement membranes (double arrows).

Tubular injury was very severe in cortical tissues. The lining epithelia of the proximal convoluted tubules displayed increased lysosomal-related structures and vesiculated endoplasmic reticulum (Figure 10). Highly swollen mitochondria with increased crista fragmentation were observed. The cristae were partially and/or completely broken, with these remnants intermixed with fine electron-dense dusty granules (Figure 11). The basal membrane infoldings were either highly dilated or ruptured at some points (Figure 10), while the brush borders were mostly dilated. Some tubular epithelial cells represented cellular necrosis (Figure 10).

Figure 10.
Necrotic proximal tubular epithelial cell 12 h after envenomation. Note pyknotic nucleus (N), lysosomal vacuoles (L), dilated basal membrane infoldings (I), and swollen mitochondria (M).

Figure 11.
Injured mitochondria showing an increased degree of damaged cristae (arrows) 12 h after venom injection.

The lining epithelial cells of the distal convoluted tubules were swollen and lost most of their cytoplasmic electron-dense appearance (Figure 12). Blebbing of the luminal parts of some epithelial cells was seen (Figure 12). The epithelial cells showed various forms of mitochondrial injury, including extensive mitochondrial crista breakdown, mitochondrial condensation with heavy electron-dense matrix, highly swollen mitochondria that had lost all their cristae and appeared as large bags containing fine electron-dense granules and rupture of the mitochondrial membrane (Figure 12). The endoplasmic reticulum was either broken or fragmented into small vesicles (Figure 12). Some tubular cells lost most of their organelles and appeared to contain variable-sized irregular vacuoles. Nuclear pyknosis, karyolysis, and envelope dilatation were also seen along with necrotic cells.

Figure 12.
Epithelial lining cell of a distal tubule 12 h after envenomation, showing loss of the cytoplasmic electron density, protrusion of the luminal portion (P), mitochondrial injury (M), broken endoplasmic reticulum (R), and broken basal membrane infoldings (I).

Succinic dehydrogenase (SDH) enzyme activity. SDH activity in control tubular epithelial cells was indicated by dark electron-dense spots homogeneously distributed on the mitochondrial membranes as well as within the mitochondria (Figure 13). Three hours after envenomation, SDH activity was represented by clumps of electron-dense spots at certain locations of the mitochondria (Figure 14). Six hours after venom injection, a marked depletion of SDH activity was indicated by the disappearance of enzymatic activity in many mitochondria. The enzyme activity was represented by a few irregularly distributed low electron-dense spots within the mitochondria. A thin layer of electron-dense SDH activity was observed at the boundaries of some mitochondria (Figure 15).

Figure 13.
SDH activity in control tubular epithelial cells in the form of dark electron-dense spots (arrows).

Figure 14.
Clumps of electron dense spots (arrows) representing SDH activity 3 h after envenomation.

Figure 15.
Marked depletion of SDH activity 12 h after of envenomation. Note low electron-dense spots (arrows) and thin layers of electron-dense materials at the mitochondrial boundaries (double arrows).

DISCUSSION

The results of this study showed a broad spectrum of ultrastructural changes in all envenomed animal renal cortical components. These changes were severely increased the longer the time interval after envenomation. Similar changes were recorded in envenomings with other snake venoms (1,23).

The swelling of the parietal and visceral epithelia was a characteristic sign of glomerulopathy (30). It is believed that the progression of cellular swelling during envenomation is responsible for the induction of the observed fragmentation and blebbing of the visceral epithelia. Visceral epithelial edema, blebbing, vesiculation, dilatation of the endoplasmic reticulum and mitochondria, as well as occasional loss of the pedicle foot-like aspect were consistent findings of Ponraj and Gopalakrishnakone (23) in envenomings caused by Pseudoechis australis.

Loss of the foot-like aspect of the foot process and endothelial cell blebbing due to cobra venom factor injection was reported by Rehan et al. (27). They attributed these changes to intravascular activation of the complement, which causes glomerular injury by producing proteinuria. Nangaku et al. (18) added that in cobra factor envenomation, a terminal product of the complement cascade activation plays an important role in the pathogenesis of the immune renal microvascular endothelial injury. Martins et al. (13) added that Crotalus durissus cascavella venom induced a direct action on the kidneys, but also an indirect action by the release of mediators from endothelial cells.

In addition, hypertrophied glomerular endothelia that lost most of their integrity and diffused into the lumen of the glomerular capillaries were also reported due to injection of Cerastes cerastes venom (1) and a myotoxin from Pseudoechis australis venom (23). Sugimoto et al. (32) related changes in the glomerular tuft to the action of venom proteinase. The toxic effect of Russell's viper venom was also mentioned as directed primarily against glomerular and vascular structures, inducing lysis of vascular walls and mesangial cells and disintegration of confluent mesangial cell layers (36). Mortia et al. (15) believed that mesangiolysis results from endothelial injury with lysis of mesangial anchor points. This could link the mesangial changes observed in this paper to glomerular injury. In addition, the observed nuclear changes in both glomerular and mesangial cells may indicate that these cells were tending toward cellular necrosis.

On the other hand, glomerular changes and reduction of the glomerular filtrate rate induced by snake venoms were believed to cause of tubular lesions (13,36). In this study, various forms of tubular lesions were recorded. Oron et al. (19) reported that cytotoxin from cobra venom caused mitochondrial deformation followed by cytoplasmic vacuolization and increase in lysosomal vacuoles. Therefore, the endoplasmic reticulum assumed a microsomal-like appearance.

Mitochondrial alterations were detected in all cortical cells of the envenomed animals. Ownby et al. (20) reported that cobra venom cardiotoxin could induce swollen and damaged mitochondria. Barraviera et al. (3) showed that the effect of venom on the tissues could be by an effect on mitochondria or a cytokine effect on the cells. Oron et al. (19) added that cobra cytotoxin may affect mitochondria either directly by interaction with the mitochondria after penetrating the cell, or indirectly by stimulating intracellular processes after binding to the cell membrane. Hung and Gopalakrishnakone (9) reported that phospholipase A2 from cobra venom is involved in causing degeneration, vesiculation, and distortion of the mitochondria. Valente et al. (35) considered mitochondrial swelling as an indication of phospholipase A2 attack. They added that phospholipase A2 led to mitochondrial swelling, permeabilization of the mitochondrial membrane, and disruption of oxygen consumption. They suggested that free fatty acids are directly responsible for the observed effects induced by phospholipase A2.

Structural alterations in the mitochondria were accompanied by marked inhibition of SDH enzymatic activities. Shah et al. (29) mentioned that Crotoxin (neurotoxin phospholipase) produces structural alterations and marked enzymatic reduction of mitochondria. The effect of phospholipase A2 on the mitochondria oxidative functions was reported by Khole and Khole (10). Venom is believed to be an inhibitor of mitochondrial oxygen uptake, inhibiting ATP synthesis and ATP hydrolysis. It may also induce formation of reactive oxygen species by enhancing auto-oxidation of mitochondrial components and inhibiting the electron transport chain (7).

Such structural and enzymatic alterations of the mitochondria would probably lead renal cells being deprived of their required energy and induce a possible secondary ischemic tubular epithelial necrosis (6). This could explain the occurrence of tubular cellular necrosis and degeneration of most cellular organelles 6 to 12 hours after venom injection. Similar observations were described by Abd El-Aal and Fares (1), whotested the effect of Cerastes cerastes venom on the kidney ultrastructure. Also, a study on the effect of Crotalus venom on isolated kidney revealed that the proximal convoluted tubule was the major site for venom toxic effect, and venom phospholipase A2 was the main toxic component responsible for inducing this effect (13). Degeneration and necrosis of the proximal tubules in Russell's viper envenomation have been reported as being due to a common nephrotoxic protein component present in the venom (33).

The increased number of lysosomal-related structures in the proximal tubular epithelia is in agreement with Aung et al. (2) in Russell's viper envenomation. They found that with an increased time interval after envenomation, the activities of all lysosomal enzymes generally increased, and the lysosomal membrane integrity was apparently reduced. This could indicate a defensive action of renal cells during the progression of venom toxicity (25).

Brush border swelling and basal membrane infolding swelling and distortion could indicate a tubular function disturbance during envenomation. Morduchowicz et al. (14) revealed a direct stimulatory effect of angiotensin II, which is known to be released during snake envenomation on the brush border membranous system. In addition, nephrotoxicity due to Russell's viper venom included absolute disturbance in the tubular reabsorption of sodium and water (36).

It believed that this wide range of non-specific renal cortical lesions induced by cobra venom could be due to secondary synergistic actions of more than one venom toxin. An overlapping of toxic and ischemic lesions may have occurred and could deprive the renal cells from their energy requirement, as indicated by the mitochondrial alterations in all components of the cortical renal tissues.

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CORRESPONDENCE TO:

T. R. RAHMY - Department of Biology, Faculty of Science, United Arab Emirates University, Al Ain 17551, United Emirates University.

Publication Dates

• Publication in this collection
  02 Apr 2001
• Date of issue
  2001