FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / As serpentes do gÃnero Bothrops sp. sÃo responsÃveis pelo maior nÃmero de acidentes ofÃdicos no Brasil, de acordo com o MinistÃrio da SaÃde. A insuficiÃncia renal aguda (IRA) à a complicaÃÃo grave mais associada nestes tipos de acidentes. A serpente Bothrops alternatus (Rhinocerophis alternatus) à uma espÃcie de importÃncia mÃdica responsÃvel por acidentes ofÃdicos, com predominÃncia no Sul do Brasil. Desta forma, investigou-se alteraÃÃes renais induzidas pelo veneno total de Bothrops alternatus (vBalt) em sistema de rim isolado de rato e em cultura de cÃlulas de tÃbulo proximal renal das linhagens LLC-MK2 (Macaco) e HK-2 (Humanas). No sistema de perfusÃo renal, utilizou-se ratos Wistar (250 a 300g), com dois grupos experimentais (1Âg/mL e 3 Âg/mL de vBalt; n=6). Foi verificado diminuiÃÃo na pressÃo de perfusÃo (PP) na concentraÃÃo de 1 μg/mL aos 90 e 120 min e na de 3 μg/mL aos 60, 90 e 120 min. Para ambas as concentraÃÃes, a resistÃncia vascular renal (RVR) e o fluxo urinÃrio (FU) diminuÃram aos 90 e 120 minutos e o ritmo de filtraÃÃo glomerular (RFG) mostrou-se reduzido a partir dos 60 min. Quando se analisou o percentual de transporte de eletrÃlitos, observou-se que o transporte tubular de sÃdio (%TNa+) foi reduzido aos 120 min para concentraÃÃo de 1 Âg/mL, e aos 90 e 120 min para a de 3 Âg/mL; o transporte tubular de cloreto (%TCl-) tambÃm foi reduzido aos 120 min para 1 Âg/mL e aos 60, 90 e 120 min para a concentraÃÃo de 3 Âg/mL; quando analisou-se os transportes tubulares proximais foi visto que o transporte tubular proximal de sÃdio (%TpNa+) e o transporte tubular proximal de cloreto (%TpCl-) diminuÃram a partir dos 60 min atà o fim o experimento, para concentraÃÃo de 1 Âg/mL, e aos 60, 90 e 120 para a de 3 Âg/mL. A anÃlise histopatolÃgica demonstrou presenÃa de alteraÃÃes morfolÃgicas microscÃpicas significativas, tais como acÃmulo de proteÃnas a nÃvel tubular e glomerular, dano glomerular e degeneraÃÃo hidrÃpica em ambas as concentraÃÃes estudadas. Na avaliaÃÃo do perfil do estresse oxidativo, observou-se que os nÃveis de glutationa reduzida (GSH) encontravam-se diminuÃdas em ambas as concentraÃÃes: 1Âg/mL (643,8  36,31 Âg/g de tecido) e 3Âg/mL (757,4 Â126,7 Âg/g de tecido), jà os nÃveis de malondiÃldeido (MDA) se encontravam aumentados em ambas as concentraÃÃes: 1Âg/mL (16,42  3,561 Âg/g de tecido) e 3Âg/mL (15,37Â2,237 Âg/g de tecido). Os nÃveis de nitrito tambÃm estavam aumentados em relaÃÃo ao controle: 1Âg/mL (309,4Â25,29 nM/g de tecido) e 3Âg/mL (330,7  29,19 nM/g de tecido). No cultivo de cÃlulas LLC-MK2 e HK-2, o vBalt diminuiu a viabilidade celular em altas concentraÃÃes (50, 100 e 200Âg/mL) apenas para a LLC-MK2, por outro lado, nas concentraÃÃes reduzidas (0,19, 0,39, 0,78, 1,56, 3,12, 6,25, 12,5, 25Âg/mL), observou-se um aumento na viabilidade celular, apresentando uma IC50 de 221,3μg/mL, para linhagem LLC-MK2. Na anÃlise do tipo de morte celular induzida, utilizou-se a citometria de fluxo com Anexina V-PE e 7-AAD, foi observado que a morte celular ocorreu de forma predominante por necrose. Esses resultados demonstraram que o veneno de Bothrops alternatus alterou todos os parÃmetros e funÃÃes renais avaliados no modelo de perfusÃo renal, demonstrando sua aÃÃo nefrotÃxica direta e seus efeitos citotÃxicos nos cultivos celulares estudados. / The snakes of the genus Bothrops sp. are responsible for the highest number of ophidian accidents in Brazil, according to the Ministry of Health. Acute kidney injury (AKI) is the severe complication most associated in these types of accidentes. The Bothrops alternatus snake is a species of medical importance responsible for ophidian accidents, predominantly in Southern Brazil. Thus, it was investigated renal changes induced by the total venom of Bothrops alternatus (Baltv) in isolated rat kidney system and kidney proximal tubule cells culture from LLC-MK2 (Monkey) and HK-2 (Human) lines. In the kidney perfusion system, Wistar rats (250 to 300 g) were used, with two experimental groups (1 μg/mL and 3 μg/mL vBalt; n = 6). There was a decrease in the perfusion pressure (PP) at the concentration of 1 μg/mL at 90 and 120 min and of 3 μg/mL at 60, 90 and 120 min. For both concentrations, renal vascular resistance (RVR) and urinary flow (UF) decreased at 90 and 120 minutes and the glomerular filtration rate (GFR) was reduced from 60 min. When the percentage of electrolyte transport was analyzed, it was observed that the tubular sodium transport (% TNa+) was reduced at 120 min in the concentration of 1 μg/mL, and at 90 and 120 min to 3 μg/ml; The tubular chloride transport (%TCl-) was also reduced at 120 min to 1μg/ml and at 60, 90 and 120 min for the concentration of 3 μg/mL; when the proximal tubular transport was analyzed, it was seen that the proximal tubular sodium transport (%TpNa+) and proximal tubular chloride transport (%TpCl-) decreased from 60 min until the end of the experiment to the concentration of 1 μg/mL, and at 60, 90 and 120 to 3 μg/mL. The histopathological analysis demonstrated the presence of significant microscopic morphological alterations, such as accumulation of proteins at the tubular and glomerular level, glomerular damage and hydropic degeneration at both concentrations studied. In the evaluation of the oxidative stress profile, it was observed that the levels of reduced glutathione (GSH) were decreased in concentrations: 1μg/mL (643.8  36.31 μg/g tissue) and 3μg/mL (757.4  126.7 μg/g tissue), with malondialdehyde levels (MDA) increased at concentrations of 1 μg/mL (16.42  3.561 μg/g tissue) and 3 μg/mL (15.37  2.237 mg /g tissue). Nitrite levels also increased in relation to the control: 1μg/mL (309.4  25.29 nM /g tissue) and 3μg/mL (330.7  29.19 nM/g tissue). In the culture of LLC-MK2 and HK-2 cells, the Baltv decreased cell viability at high concentrations (50, 100 and 200 μg / mL) only for LLC-MK2, on the other hand, in reduced concentrations (0,19, 0,39, 0,78, 1,56, 3,12, 6,25, 12,5, 25Âg/mL), an increase in cell viability was observed, presenting an IC 50 of 221.3 μg/mL to the LLC-MK2 line. In the analysis of type of induced cell death, flow cytometry was used with Annexin V-PE and 7-AAD, and it was observed that cell death occurred predominantly due to necrosis. These results demonstrated that the venom of Bothrops alternatus altered all renal parameters and functions evaluated in the kidney perfusion model, demonstrating its direct nephrotoxic action and its cytotoxic effects in the studied cell cultures.
| Identifer | oai:union.ndltd.org:IBICT/oai:www.teses.ufc.br:11943 |
| Date | 18 January 2017 |
| Creators | Francisco Assis Nogueira JÃnior |
| Contributors | Helena Serra Azul Monteiro, Krishnamurti de Morais Carvalho, Roberta Jeane Bezerra Jorge |
| Publisher | Universidade Federal do CearÃ, Programa de PÃs-GraduaÃÃo em Farmacologia, UFC, BR |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFC, instname:Universidade Federal do Ceará, instacron:UFC |
| Rights | info:eu-repo/semantics/openAccess |
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