Direct nephrotoxic effects produced by venoms of Sri Lankan cobra, Russell's viper and hump nosed viper

Nephrotoxicity is the principal cause of death following Russell 's viper envenomation. Envenomation following the bite of several other snakes is also known to cause nephrotoxicity. The nephrotoxicity can be due to direct effects of venom or secondary to circulatory disturbances (eg. ischaemia), which these patients often manifest. As separating out the contributions of direct toxic effects and ischaemic effects are difficult in the in vivo situation, experiments were carried out using the kidney slice model to study and compare the direct toxic effects of venom of cobra, Russell's viper and hump nosed viper. The effect of cobra venom (CV) on rat kidney slices and the effects of Russell's viper venom (RV V) and hump nosed viper venom (HNVV) on rabbit kidney slices were examined. Healthy male animals were anaesthetized and kidneys were harvested. Kidneys were decapsulated, bisected and sliced. Rat kidney slices were incubated with CV and rabbit kidney slices were incubated with RVV and HNVV for different time periods. Rat and rabbit kidney slices were incubated with 0.9% sodium chloride as the control. At the end of each observation period kidney slices were preserved for light and electron microscopy (LM and EM). When CV was used, complete necrosis was seen in proximal and distal convoluted tubular cells (PCT and DCT). When rabbit kidney slices were incubated with RVV for 4 hours there was com­ plete necrosis of glomeruli and PCT with the pres­ ervation of the basement membrane. LM and EM changes were mostly confined to PCT when HNVV was used. The results of this experiment provide evidence that the venoms studied produce direct damage on renal tissue. Different areas of the nephron are differentially susceptible to the effects of the three venoms.


Introduction
The death rate due to snake bite envenomation in Sri Lanka is one of the highest in the world (I). These fatalities are often caused by cobra, Russell's viper and the common krait. Though cobra and krait venom are more potent than Russell's viper venom, Russell's viper is the most dreaded snake in the island (2) and it is responsible for 40% of deaths due to envenomation (1).
Although death following Russell's viper envenomation could be due to different reasons (shock, haemorrhage, disseminated intravascu lar coagulation, neurotoxicity, respiratory failure or acute renal failure), nephrotoxicity leading to acute renal failure has been identified as the principal cause (1). In one study it was found that envenomation by Russell's viper contributes for 49% of cases of acute renal failure reported at the National Hospital of Sri Lanka (3). Envenomation following the bite of several other snakes (e.g. cobra, pit vipers and sea snake) is also known to cause nephrotoxicity (4,5).
The development of nephrotoxicity in envenomation can be due to direct effects of venom or secondary to circulatory disturbances (ischaemia), which these patients often show. Separating out the contribution of direct

Animals
The protocol reported here is part of a detailed investigation on effects of venom on renal structure and function (7,8,9). In this protocol studying the direct toxic effects of venom, CV was tested in rats and RVV and HNVV were tested in rabbits. The kidneys were decapsulated and bisected using sterile instruments while keeping in 0.9% sodium chloride. Uniform slices of 0.3 mm thickness were obtained from the bisected kidneys using a tissue sheer. Rat kidney slices were incubated with 10 mg/ml of CV and rabbit kidney slices were incubated with 10 mg/ml of RVV and HNVV separately for 1,2,3 or 4 hours in a water bath at 37 °C (n=5 for each stage). This dose of venom (10 mg/ml) was decided based on the experiments by Soe-Soe et al., (6). In their experiments varying doses of RVV (Vipera russelli russelli) were used (300ng/ml, 2.5 mg/ml, 5 mg/ml and 10 mg/ml). They found effects only at doses of 5 mg/ml and 10 mg/ml. Hence, for our experiments only the 10 mg/ml dose was used. Kidney slices incubated with 0.9% sodium chlo ride were used as controls. After incubating for different time periods (1 hour, 2 hours, 3 hours or 4 hours), kidney slices were fixed in modified Boins solution or with 2% gluteraldehyde for light microscopy (LM) or electron microscopy (EM) respectively. Pathological changes in kidney slices were expressed as percentages. The percentage of change (degeneration or necrosis) were categorized as described below after examining ten fields under low and high dry power (10 X 20, 10X40) in two slides.

Effects of RVV
In the rabbit kidney slices incubated with RVV, glomerular tuft of the surface glomeruli showed necrosis and rest of the glomeruli had pyknotic nuclei at 3 and 4 hours after incubation. 2 and 3). However, the basement membrane of the cells was preserved. (Figure 4).

Appearance of control specimens
Glomeruli in the control specimens were normal in appearance. Necrotic changes were not observed in the PCT or DCT cells. Electron microscopically swollen mitochondria and disappearance of cristae were noticed in a few PCT cells. The variation in the direct nephrotoxic effects of venoms may be due to the different amounts of phospholipase A 2 present in them or due to dif ferences in their iso-enzymes (12).

Figure I. Electron micrograph appearance of rat kidney slices when incubated with cobra venom for 4 hours. Complete destruction of the ultrastructure of the PCT cells with the preservation of the basement membrane is seen (arrowhead).
Magnification X 10000, bar = I mm.

Figure 2. Electron micrograph appearance of the completely destroyed ultrastructure of a glomeru lus of a rabbit kidney slice incubated with Russell s viper venom for 4 hours. Basement membrane is preserved (arrowhead). Clumping of chromatin in the nucleus of a white blood cell is seen (arrow).
Magnification X 10000, bar = 1 mm.

Figure 4. Electron micrograph appearance of a PCT cell of a rabbit kidney slice when incubated with hump nosed viper venom for 4 hours. Appearance of myelin bodies; concentrically laminated elec tron-dense bodies which represents altered lysosomes (arrow heads), dense bodies (small arrows)
and vacuoles (large arrows) are seen. Magnification X 10000, bar = I mm.