Study of effects renal and mechanisms of cell death induced by snake venom of Bothrops leucurus

ICO MORAIS1, AFC TORRES2, GJS PEREIRA3, TP PEREIRA2, ARC JORGE1, AH BINDÁ1, SS SMAILI3, HSA MONTEIRO1, MH TOYAMA4, AMC MARTINS2. 1Federal University of Ceará, Department of Physiology and Pharmacology, 60430-970, Brazil, 2Federal University of Ceará, Department of Clinical and Toxicological Analysis, 60430-370, Brazil, 3Federal University of São Paulo, Department of Pharmacology, 04044-020, Brazil, 4São Paulo State University, Department of Biochemistry, 11330-900, Brazil

Bothrops venoms consist of complex mixture of active substances, mainly peptides and proteins, which interfere with many physiologic processes. Acute renal failure (ARF) is one of the most serious complications of Bothrops snakebites. Recent studies suggest that of ARF pathogenesis is multifactorial and involves hemodynamic disturbances, immunologic reactions and direct nephrotoxicity. The aim of the work was to investigate the effects of the Bothrops leucurus venom (VBl) in the renal perfusion system and on renal epithelial cells. The Bothrops leucurus venom (VBl) (10μg/mL) was added to the perfusion system 30 min after the beginning of each perfusion and effects were compared to a control group perfused with modified Krebs–Henseleit solution alone at 60, 90 and 120 minutes of experiment. VBl (10μg/mL) decreased the perfusion pressure at 90 and 120 min. The glomerular filtration rate and urinary flow decreased at 60 and 90 min. The renal vascular resistance decreased at 120 min. It was also observed a decrease on percentual tubular transport of sodium (%TNa+) at 120 min and of chloride (%TCl- ) at 60 and 90 min. We evaluated the cytotoxic potential of venom using culture of renal tubular cells (MDCK). The treatment for 24 h with VBl (1.25 μg/mL) decreased the cell viability in 50% in relation to untreated cells. Flow cytometric analysis showed that the necrotic cell population increased significantly in a concentration-dependent fashion after 24h exposure, with low percentage of apoptotic cells only in lower concentrations. We assessed whether there was increased expression of genes considered markers of apoptosis and founded a increase in caspase 3 and caspase 8 expression observed with the lowest concentration tested (1μg/mL, 24h). The treatment of MDCK cells with 1.25μg/mL (CI50) of VBl promoted the mitochondrial depolarization in majority of the mitochondria investigated (57.6%). Since the change in ∆Ψm and apoptosis can be associated with increases in cytosolic Ca2+ we have also observed to the Ca2+ changes induced by the venon, our results showed that there is an increase in cytosolic Ca2+ after addition of VBl indicating that Ca2+ may participate in the venom of B. leucurus effect. These findings may be important aspects of the process of nephrotoxicity mediated by B. leucurus venom. The characterization of the effects in the isolated kidney and renal tubular cells gives strong evidences that the acute renal failure induced by this venom is result of the direct nephrotoxicity.