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Isabel Cristina Oliveira de Morais

Isabel Cristina Oliveira de Morais

Federal University of Ceara, Brazil

Title: L-Aminoacid Oxidase from Bothrops leucurus Venom Induces Nephrotoxicity via Apoptosis and Necrosis

Biography

Master in Pharmacology from the Federal University of Ceará (2011). PhD in Pharmacology from the Federal University of Ceará (2015), researching the renal effects and cellular fraction L amino acid oxidase (LAAO) isolated from the venom of Bothrops leucurus. Trained on his doctorate sandwich methodologies for signaling of cell death such as flow cytometry, western blott, PCR and immunofluorescence in the center of Investigacion Principe Felipe- CIPF- Valencia-Spain under the supervision of researcher Dr. Mar Orzaez Calatayud. It has experience in Pharmacy, with emphasis in Pharmacology, Physiology, Toxicology, studying mainly toxins in the search for new therapeutic targets.

Abstract

The pit viper Bothrops leucurus (White-tailed-jararaca) is a poisonous snake habituating area in the northeast of Brazil. The biological effects due envenomation have similar profile than those observed with other Bothrops, such as coagulant activity, hemorrhagic, fibrinolytic, and acute renal failure (ARF). ARF is a common complication caused by Bothrops snakebite with relevant morbidity and mortality. Pathogenesis of ARF in snakebite envenomation may be related to hypovolemia and hypoperfusion secondary to cardiovascular disturbances, deposit of fibrin in the glomerular capillaries leading thrombotic microangiopathy and high venom concentration at the renal tissue, direct venom action on the tubular cells and oxidative stress. Recently, we observed that Bothrops leucurus venom induces nephrotoxicity in the isolated perfused kidney of rats associated with cytotoxicity against renal tubular epithelia cells. In this study, it was evaluated the direct nefrotoxicity of a main component of B. leucurus venom called L-aminoacid oxidase (LAAO-Bl) by using tubular epithelial cell lines MDCK and HK-2. In these cells treated with LAAO-Bl, 1.56 – 100 µg/mL for 12 h, there was a decrease in their viability in a concentration-dependent manner. We next evaluated if necrosis was implicated in the cellular viability decrease observed by analyzing lactate dehydrogenase (LDH) release. In MDCK cells LDH release was not observed after 12 h of LAAO-Bl exposure while LAAO-Bl induced an apparent membrane rupture in HK-2 cells at the highest concentrations studied when compared with untreated cells. Annexin V/PI staining was applied to detect apoptotic/necrotic cells after LAAO-Bl treatment. In MDCK cells, LAAO-Bl significantly increased the percentage of early apoptotic (Annexin-V+, PI-), necrotic (Annexin-V-, PI+) and secondary necrotic cells (Annexin-V+, PI+) when compared with control untreated cells. In HK-2 cells, in accordance with data obtained in the LDH-release assay, the Annexin-V-PI loading cell analysis demonstrated an increase in necrotic (PI+ cells) and secondary necrotic cells (Annexin-V+, PI+) in a concentration-dependent manner. MDCK and HK-2 apoptosis induction was accompanied with Ca2+ release from the endoplasmic reticulum, reactive oxygen species (ROS) generation, mitochondria dysfunction with enhanced expression of Bax protein levels, caspase-3 and caspase-7 activation, suggesting that LAAO-Bl causes nephrotoxicity by acting in multiple cell death pathways. LAAO-Bl (10 µg/mL) exerts significant effects on the isolated kidney perfusion increasing perfusion pressure and urinary flow and decreasing the glomerular filtration rate and sodium, potassium and chloride tubular transport. Taken together our results suggest that LAAO-Bl is responsible for the nephrotoxicity observed in the envenomation by snakebites.