RESUMO
ANG II has many biological effects in renal physiology, particularly in Ca2+ handling in the regulation of fluid and solute reabsorption. It involves the systemic endocrine renin-angiotensin system (RAS), but tissue and intracrine ANG II are also known. We have shown that ANG II induces heterodimerization of its AT1 and AT2 receptors (AT1R and AT2R) to stimulate sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity. Thus, we investigated whether ANG II-AT1R/AT2R complex is formed and internalized, and also examined the intracellular localization of this complex to determine how its effect might be exerted on renal intracrine RAS. Living cell imaging of LLC-PK1 cells, quantification of extracellular ANG II, and use of the receptor antagonists, losartan and PD123319, showed that ANG II is internalized with AT1R/AT2R heterodimers as a complex in a microtubule-dependent and clathrin-independent manner, since colchicine-but not Pitstop2-blocked this process. This result was confirmed by an increase of ß-arrestin phosphorylation after ANG II treatment, clathrin-mediated endocytosis being dependent on dephosphorylation of ß-arrestin. Internalized ANG II colocalized with an endoplasmic reticulum (ER) marker and increased levels of AT1R, AT2R, and PKCα in ER-enriched membrane fractions. This novel evidence suggests the internalization of an ANG II-AT1/AT2 complex to target ER, where it might trigger intracellular Ca2+ responses.
Assuntos
Angiotensina II/metabolismo , Membrana Celular/metabolismo , Endocitose , Retículo Endoplasmático/metabolismo , Rim/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 2 de Angiotensina II/farmacologia , Animais , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Rim/efeitos dos fármacos , Células LLC-PK1 , Microtúbulos/metabolismo , Complexos Multiproteicos , Fosforilação , Proteína Quinase C-alfa/metabolismo , Transporte Proteico , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 2 de Angiotensina/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Suínos , beta-Arrestinas/metabolismoRESUMO
We have previously reported decreased expression and activities of lysosomal cathepsins B and L in diabetic kidney. Relevant morphological changes were observed in proximal tubules, suggesting that these cells are implicated in the early stages of the disease. The aim of the present study was to investigate the mechanisms that lead to these changes. The effects of high glucose (HG) and advanced glycation end products (AGEs) on cell viability, lysosomal enzymes and other effectors of cell signaling of cultured kidney cells were studied. HG increased viable mesangial cells (ihMC) in 48 h, while epithelial tubular cells were not affected (LLC-PK1 and MDCK). In contrast, the number of viable cells was markedly decreased, for all cell lines, by AGE-BSA. Concerning lysosomal enzymes, the main cysteine-protease expressed by these cells was cathepsin B, and its concentration was much higher in epithelial than in mesangial cells. Exposure to HG had no effect on the cathepsin B activity, but AGE-BSA caused a marked decrease in LLC-PK1, and increased the enzyme activities in the other cell lines. The levels of nitric oxide (NO) was increased by AGE-BSA in all cell lines, suggesting oxidative stress, and Western blotting has shown that, among the investigated proteins, cathepsin B, mTOR and transcription factor EB (TFEB) were the most significantly affected by exposure to AGE-BSA. As mTOR induces anabolism and inhibits autophagy, and TFEB is a master transcription factor for lysosomal enzymes, it is possible that this pathway plays a role in the inhibition of lysosomal enzymes in proximal tubule cells.
Assuntos
Glucose/farmacologia , Produtos Finais de Glicação Avançada/farmacologia , Rim/citologia , Animais , Bovinos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cães , Humanos , Lisossomos/enzimologia , Soroalbumina Bovina/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Somatic ACE (sACE) is found in glomerulus, proximal tubule and excreted in urine. We hypothesized that N-domain ACE can also be found at these sites. ACE profile was analyzed in mesangial (IMC), proximal (LLC-PK1), distal tubule (MDCK) and collecting duct (IMCD) cells. Cell lysate and culture medium were submitted to gel filtration chromatography, which separated two peaks with ACE activity from cells and medium, except from distal tubule. The first had a high molecular weight and the second, a lower one (65 kDa; N-domain ACE). We focused on N-domain ACE purification and characterization from LLC-PK1. Total LLC-PK1 N-domain ACE purification was achieved by ion-exchange chromatography, which presented only one peak with ACE activity, denominated ACE(int2A). ACE(int2A) activity was influenced by pH, NaCl and temperature. The purified enzyme was inhibited by Captopril and hydrolyzed AngI, Ang1-7 and AcSDKP. Its ability to hydrolyze AcSDKP characterized it as an N-domain ACE. ACE(int2A) also presented high amino acid sequence homology with the N-terminal part of sACE from mouse, rat, human and rabbit. The presence of secreted and intracellular N-domain ACE and sACE in IMC, LLC-PK1 and IMCD cells confirmed our studies along the nephron. We identified, purified and characterized N-domain ACE from LLC-PK1.
Assuntos
Regulação Enzimológica da Expressão Gênica , Peptidil Dipeptidase A/biossíntese , Peptidil Dipeptidase A/isolamento & purificação , Animais , Cromatografia por Troca Iônica , Cães , Humanos , Células Madin Darby de Rim Canino , Células Mesangiais/enzimologia , Camundongos , Oligopeptídeos/química , Peptidil Dipeptidase A/química , Estrutura Terciária de Proteína , Coelhos , Ratos , Especificidade por SubstratoRESUMO
The aim of the study was to characterize the cell damage mechanisms involved in the pathophysiology of cytotoxicity of polymyxin B in proximal tubular cells (LLC - PK1) and discuss about the nurses interventions to identify at risk patients and consider prevention or treatment of nephrotoxicity acute kidney injury. This is a quantitative experimental in vitro study, in which the cells were exposed to 375μM polymyxin B sulfate concentration. Cell viability was determined by exclusion of fluorescent dyes and morphological method with visualization of apoptotic bodies for fluorescence microscopy. Cells exposed to polymyxin B showed reduced viability, increased number of apoptotic cells and a higher concentration of the enzyme lactate dehydrogenase. The administration of polymyxin B in vitro showed the need for actions to minimize adverse effects such as nephrotoxicity. .
El objetivo del estudio fue caracterizar los mecanismos de daño celular implicado en la fisiopatología de la citotoxicidad de la polimixina B en las células tubulares proximales (LLC-PK1) y discutir las propuestas de intervención de enfermería para identificar a los pacientes de riesgo y considerar la prevención o el tratamiento de la lesión renal aguda nefrotóxica. Corresponde a un estudio experimental cuantitativo in vitro, en el cual las células fueron expuestas a sulfato de polimixina B. La viabilidad celular se determinó por exclusión de los colorantes fluorescentes y el método morfológico con la visualización de cuerpos apoptóticos a la microscopía de fluorescencia. Las células expuestas a polimixina B demostraron reducción de la viabilidad, aumento de células apoptóticas y mayor concentración de la enzima lactato deshidrogenasa. La administración de polimixina B in vitro demostró la necesidad de realizar acciones en la práctica clínica para minimizar los efectos adversos como la nefrotoxicidad.
O objetivo do estudo foi caracterizar os mecanismos de lesão celular envolvidos na fisiopatologia da citotoxicidade da polimixina B em células tubulares proximais (LLC-PK1) e discutir as proposições de intervenção do enfermeiro para identificar os pacientes de risco e considerar a prevenção ou o tratamento para lesão renal nefrotóxica. Estudo experimental in vitro , onde as células foram expostas ao sulfato de polimixina B. A viabilidade celular foi determinada pela exclusão dos corantes fluorescentes e o método morfológico com visualização de corpos apoptóticos à microscopia de fluorescência. As células expostas à polimixina B apresentaram redução de viabilidade, aumento do número de células em apoptose e maior concentração da enzima desidrogenase láctea. A administração de polimixina B in vitro demonstrou a necessidade de ações na prática clínica para minimizar os efeitos adversos como a nefrotoxicidade. .
Assuntos
Animais , Antibacterianos/efeitos adversos , Nefropatias/induzido quimicamente , Polimixina B/efeitos adversos , Nefropatias/enfermagem , Nefropatias/prevenção & controle , Células LLC-PK1 , SuínosRESUMO
Cross-sectional study, carried out at the outpatient clinic of an oncology hospital. Data were collected from 88 caregivers of cancer patients using the Caregiver General Comfort Questionnaire (GCQ) to assess the caregivers’ comfort. The caregivers’ GCQ score mean was 203.9; better comfort scores was associated with age, care time and current occupation; positive aspects of comfort were related to the fact that caregivers felt loved, to patients’ physical and environmental comfort and to caregivers’ spirituality. 203.9; better comfort scores were associated with age of the caregiver and current occupation; positive aspects of comfort were related to the fact that caregivers felt loved, to patients’ physical and environmental comfort and to caregivers’ spirituality. Caregivers, who didn’t have a paid job or leisure’s activities showed a worse GCQ. The GCQ scale can help to identify factors that interfere in caregivers’ comfort, as well as needs that can be modified through health professionals’ interventions. .
Estudo transversal cujo objetivo foi avaliar o conforto de cuidadores de pacientes com câncer. Envolveu 88 cuidadores de pacientes em atendimento ambulatorial de um hospital especializado em oncologia. Utilizou-se o General Comfort Questionnaire (GCQ) validado para o português. Verificou-se que o escore médio do GCQ dos cuidadores foi de 203,9. Os melhores escores de conforto estiveram relacionados à idade e ocupação do cuidador; os aspectos positivos do conforto envolveram sentir-se amado, o conforto ambiental e físico do paciente e a espiritualidade do cuidador. Cuidadores que não exerciam atividade remunerada ou lazer apresentaram piores escores de GCQ. Concluiu-se que escala de GCQ pode ajudar a identificar fatores que interferem no conforto dos cuidadores de pacientes com câncer, assim como necessidades que permitam a intervenção dos profissionais de saúde. .
Estudio transversal que tuvo como objetivo evaluar la comodidad de los cuidadores de pacientes con cáncer. Participaron 88 cuidadores de pacientes en atención ambulatoria de un hospital oncológico. Para la recolección de los datos, se utilizó el General Comfort Questionnaire (GCQ) validado para el portugués. La puntuación media del GCQ de los cuidadores fue de 203,9. Las mejores puntuaciones de comodidad estaban relacionadas con la edad y la ocupación del cuidador; los aspectos positivos fueron sentirse amado, comodidad física del paciente y de su ambiente y la espiritualidad del cuidador. Las peores puntuaciones fueron observadas en los cuidadores que no tienen trabajo remunerado o descanso. Se concluye que la escala GCQ puede ayudar a identificar factores que interfieren en la comodidad de los cuidadores de pacientes con cáncer, así como identificar las necesidades que permitan la intervención de los profesionales de la salud. .
Assuntos
Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Cuidadores/psicologia , Emoções , Neoplasias/enfermagem , Estudos Transversais , Qualidade de Vida , Inquéritos e QuestionáriosRESUMO
Amphotericin B is one of the most effective antifungal agents; however, its use is often limited owing to adverse effects, especially nephrotoxicity. The purpose of this study was to evaluate the effect of inhibiting the PKA signaling pathway in nephrotoxicity using Amphotericin B from the assessment of cell viability, pro-inflammatory cytokines and nitric oxide (NO) production in LLC-PK1 and MDCK cell lines. Amphotericin B proved to be cytotoxic for both cell lines, as assessed by the mitochondrial enzyme activity (MTT) assay; caused DNA fragmentation, determined by flow cytometry using the propidium iodide (PI) dye; and activated the PKA pathway (western blot assay). In MDCK cells, the inhibition of the PKA signaling pathway (using the H89 inhibitor) caused a significant reduction in DNA fragmentation. In both cells lines the production of interleukin-6 (IL)-6 proved to be a dependent PKA pathway, whereas tumor necrosis factor-alpha (TNF-α) was not influenced by the inhibition of the PKA pathway. The NO production was increased when cells were pre-incubated with H89 followed by Amphotericin B, and this production produced a dependent PKA pathway in LLC-PK1 and MDCK cells lines. Therefore, considering the present study's results as a whole, it can be concluded that the inhibition of the PKA signaling pathway can aid in reducing the degree of nephrotoxicity caused by Amphotericin B.
Assuntos
Anfotericina B/toxicidade , Antifúngicos/toxicidade , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citocinas/biossíntese , Rim/efeitos dos fármacos , Óxido Nítrico/biossíntese , Animais , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Fragmentação do DNA/efeitos dos fármacos , Cães , Interleucina-6/biossíntese , Rim/enzimologia , Rim/imunologia , Rim/patologia , Células LLC-PK1 , Células Madin Darby de Rim Canino , Transdução de Sinais , Suínos , Fator de Necrose Tumoral alfa/biossínteseRESUMO
OBJETIVO: Caracterizar a toxicidade da polimixina B (PmxB) em células renais em dosagem e tempos diferentes. MÉTODOS: Células LLC-PK1, cultivadas em placas multiwell de 12 poços, foram divididas nos seguintes grupos: Controle (CTL) - células mantidas em meio DMEM suplementado a 5%; G1 - células expostas à concentração de 75mM de PmxB; G2 - células expostas à concentração de 375mM de PmxB. Cada grupo foi avaliado nos tempos de 24, 48 e 72 horas quanto à viabilidade celular (Acridine Orange/Brometo de Etídio) e apoptose (Hoechst 33342). RESULTADOS: Os dados demonstraram a viabilidade celular e a apoptose à exposição de três doses de PmxB em três intervalos de tempo, com um aumento significativo da toxicidade à elevação das doses e ao maior tempo de permanência no antibiótico para apoptose. CONCLUSÃO: A citotoxicidade pela PmxB, no modelo de cultivo celular, se mostrou tempo e dose dependente, aumentando com a maior exposição e maior dose de antibiótico.
OBJECTIVE: To characterize the toxicity of polymyxin B (PmxB) in renal cell in different dosage and times. METHODS: LLC-PK1 cells grown in 12 well multiwell plates were divided into the following groups: Control (CTL) - cells maintained in DMEM supplemented with 5%; G1 - cells exposed to concentration of 75µM PmxB G2 - cells exposed to concentration of 375µM PmxB. Each group was assessed at 24,48 and 72 hours as for cell viability (Acridine orange/ethidium bromide) and apoptosis (Hoechst 33342). RESULTS: The data demonstrate the cell viability and apoptosis exposure of three doses of PmxB in three time intervals, with a significant increase in toxicity to high doses and longer duration of stay in the antibiotic to apoptosis. CONCLUSION: Cytotoxicity by PmxB in cell culture model, showed to be time and dose dependent, increasing with increased exposure and higher dose of antibiotic.
Assuntos
Antibacterianos/administração & dosagem , Antibacterianos/toxicidade , Apoptose , Células LLC-PK1 , Técnicas In Vitro , Polimixina B/administração & dosagem , Polimixina B/toxicidade , Sobrevivência Celular , Estudos de Avaliação como AssuntoRESUMO
Trabalhos prévios têm indicado que soluções homeopáticas modificam os aspectos celulares e bioquímicos de células mantidas em cultura. No presente estudo, os efeitos de Natrum muriaticum, medicamento usado na clínica homeopática para o tratamento de hipertensão, foram avaliados em células renais das linhagens MDCK e LLC-PK1. Os seguintes parâmetros celulares foram analisados: viabilidade, morfologia e expressão da (Na+-K+)-ATPase e dos receptores AT1 e AT2 da angiotensina II. As linhagens celulares foram plaqueadas (5,0 x 104 células/mL) em DMEM suplementado com 10% de soro fetal bovino (SFB). Após 24 horas a 37°C, o meio DMEM foi substituído com a adição de 10% (v/v) e 1% (v/v) das seguintes amostras: Natrum muriaticum 30CH, água dinamizada 30CH e água destilada estéril, para a medida de viabilidade celular por MTT. As absorbâncias quantificadas em leitor de ELISA (490 nm) foram comparadas entre si e com os valores obtidos com o grupo controle (células isentas de tratamento) Os valores obtidos de quatro experimentos independentes realizados em quintuplicata foram tabulados e analisados estatisticamente pelo Software Sigma Plot v.11 (Jandel Scientific). A morfologia das células MDCK foi avaliada por microscópio óptico após coloração de Giemsa e a expressão da (Na+-K+)-ATPase e AT1/AT2 de células LLC-PK1 por Western Blot (WB). Para ambos experimentos, 5,0 x 104 células/mL foram incubadas em DMEM suplementado com 10% SFB sendo o meio de cultura diariamente substituído por um novo, contendo 1% de Natrum muriaticum 30CH e água dinamizada 30CH, por 5, 10 e 15 dias. A quantificação de proteínas totais foi realizada pelo método de Lowry, após lise celular. As amostras foram analisadas por eletroforese em SDS-PAGE (12%) e transferidos para membrana de nitrocelulose. Esta membrana foi incubada com anticorpos específicos primário (anti-(Na+-K+)-ATPase, anti-AT1 e AT2 ou anti-anti-beta-actina). A detecção foi realizada utilizando o sistema ECL e Hyperfilm. Os ensaios de MTT mostraram uma redução estatisticamente significativa na atividade mitocondrial celular (p <0,001), indicando um efeito osmótico decorrente da incubação com as soluções teste na concentração de 10% (v/v) Não foram detectadas alterações morfológicas significativas quando ambas as linhagens foram submetidas aos diferentes tratamentos . A análise por WB indicou um aumento proporcional da quantidade (Na+-K+)-ATPase expressa na linhagem LLC-PK1 em função do número de estímulos homeopáticos aplicado. Embora preliminares estes resultados evidenciam, pela primeira vez, que o estímulo homeopático parece modificar a expressão de importantes marcadores fisiológicos da linhagem LLC-PK1 que estão diretamente envolvidos na gênese da hipertensão arterial.(AU)
Previous papers have indicated that homeopathic solutions modify the cellular and biochemical aspects of cells maintained in culture. In this study, the effects of Natrum muriaticum, a medicine used in the homeopathic clinic for the treatment of hypertension, were evaluated in kidney MDCK and LLC-PK1 cell lines. The following cellular parameters were analyzed: viability, morphology and expression of the (Na+-K+)-ATPase and the angiotensin II receptors AT1 and AT2. The cell lines were plated (5.0 x 104 cells/mL) in DMEM supplemented with 10% fetal calf serum (FCS). After 24 hours at 37°C, DMEM was re-fed with the addition of 10% (V/V) and 1% (V/V) of the following samples: Natrum muriaticum 30CH, water 30CH and non-dynamized sterile distilled water to do the MTT assay. The results obtained from these groups were compared to those obtained by incubation of the cells in culture medium free of these solutions (Control). Cell viability was assessed by a colorimetric MTT ELISA assay (490nm). The values from four independent experiments performed in quintuplicate were plotted and statistically analyzed by Sigma Plot v.11 (Jandel Scientific). The morphology of MDCK cells was evaluated by optical microscope after Giemsa?s staining. The expression of the (Na++K+)-ATPase and AT1/AT2 of LLC-PK1 cells was evaluated by Western Blot (WB) analysis. For this experimental set, 5.0x104 cells/mL were incubated in DMEM supplemented with 10% FCS and daily culture medium was replaced by a new one, containing: Natrum muriaticum 30CH and water 30CH. Additionally, cells were treated for 5, 10 and 15 days with 1% of specific solutions and the total protein was measured by the Lowry method, after cell lysis. The samples were analyzed by electrophoresis in SDS-PAGE (12% gel) and transferred to nitrocellulose membrane. This membrane was incubated with specific primary antibodies (anti-(Na++K+)-ATPase, anti-AT1 and AT2 or anti-anti-beta-actin). The detection was performed using ECL system and Hyperfilm. MTT assays showed a statistically significant reduction in cellular mitochondrial activity (p<0.001) probably attributed to an osmotic effect due to the use of 10% (V/V) concentration rather than 1% (V/V). The optical microscopy analysis revealed no significant morphological changes in MDCK and LLC-PK1 cells submitted to the different treatments when compared to controls groups. The WB analysis indicated a proportional increase in (Na++K+)-ATPase content according to an increase in the homeopathic stimuli. Although preliminary, these results show for the first time, that the homeopathic medicine is able to modify the expression of important physiological markers for LLC-PK1 cells, which are directly involved in the genesis of hypertension.(AU)
Assuntos
Natrium Muriaticum , HipertensãoRESUMO
Nephrotoxicity is the main side effect of antibiotics such as gentamicin. Preconditioning has been reported to protect against injuries as ischemia/reperfusion. The objective of the present study was to determine the effect of preconditioning with gentamicin on LLC-PK1 cells. Preconditioning was induced in LLC-PK1 cells by 24-h exposure to 2.0 mM gentamicin (G/IU). After 4 or 15 days of preconditioning, cells were again exposed to gentamicin (2.0 mM) and compared to untreated control or G/IU cells. Necrosis and apoptosis were assessed by acridine orange and HOESCHT 33346. Nitric oxide (NO) and endothelin-1 were assessed by the Griess method and available kit. Heat shock proteins were analyzed by Western blotting. After 15 days of preconditioning, LLC-PK1 cells exhibited a significant decrease in necrosis (23.5 ± 4.3 to 6.5 ± 0.3%) and apoptosis (23.5 ± 4.3 to 6.5 ± 2.1%) and an increase in cell proliferation compared to G/IU. NO (0.177 ± 0.05 to 0.368 ± 0.073 ìg/mg protein) and endothelin-1 (1.88 ± 0.47 to 2.75 ± 0.53 pg/mL) production significantly increased after 15 days of preconditioning compared toG/IU. No difference in inducible HSP 70, constitutive HSC 70 or HSP 90 synthesis in tubular cells was observed afterpreconditioning with gentamicin. The present data suggest that preconditioning with gentamicin has protective effects on proximal tubular cells, that involved NO synthesis but not reduction of endothelin-1 or production of HSP 70, HSC 70, or HSP 90. We conclude that preconditioning could be a useful tool to prevent the nephrotoxicity induced by gentamicin.
Assuntos
Animais , Antibacterianos/farmacologia , Endotelina-1/biossíntese , Gentamicinas/farmacologia , Proteínas de Choque Térmico/biossíntese , Túbulos Renais Proximais/efeitos dos fármacos , Óxido Nítrico/biossíntese , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Células LLC-PK1 , Necrose/induzido quimicamente , SuínosRESUMO
A heme-oxigenase-1 (HO-1) é uma enzima induzível envolvida na degradação do grupo prostético heme, produzindo compostos com funções anti-oxidante, anti-inflamatória, anti-apoptótica e modulatória do sistema imune no rim. A importância de sua indução está associada à resposta adaptativa ao estresse oxidativo e à inflamação envolvidos na gênese da insuficiência renal aguda. O sulfato de polimixina B é um antibiótico usado no tratamento de infecções Gram-negativas e que apresenta um efeito nefrotóxico ainda não completamente elucidado. O objetivo deste estudo foi verificar a viabilidade e apoptose de células LLC-PK1 submetidas ao tratamento com polimixina B, com tempos de exposição diferentes, e pré-tratadas com hemin (indutor de heme oxigenase-1) ou protoporfirina de zinco (inibidor de heme oxigenase-1). Células renais de porco, LLC-PK1, foram cultivadas com polimixina B durante 24, 48 e 72 horas. A apoptose e viabilidade celular foram avaliadas usando diferentes doses do antibiótico: Controle (CTL, 0 µM); G1 (12,5µM); G2 (37,5µM); G3 (75µM); G4 (125µM) e G5 (375µM). O hemin (25µM) e a protoporfirina de zinco (10µM) foram administrados uma hora antes da polimixina B. Foram utilizados os métodos Acridine orange/ brometo de etídio (viabilidade) e Hoescht 33342 (apoptose). Os resultados demonstraram redução linear de viabilidade induzida pela polimixina B quando a dose e o tempo de exposição foram aumentados, isto foi confirmado pela variação inversa de apoptose. O hemin aumentou a viabilidade e reduziu apoptose na presença de polimixina B, sugerindo um efeito protetor da HO-1 neste modelo. O efeito observado para a protoporfirina de zinco foi semelhante ao descrito para o hemin. O estudo confirmou a citotoxicidade da polimixina B em células renais e constatou que esse efeito pode ser mediado pela HO-1 considerando o efeito obtido no tratamento com o indutor daquela enzima.
The heme oxygenase-1 is a inducible enzyme involved in degradation of the heme prosthetic group, producing compounds with antioxidant, anti-inflamatory, antiapoptotic and immune system modulatory actions. The importance of its induction is linked with the adaptative response to oxidative stress and inflammation involved in the genesis of the acute renal failure. Polymyxin B sulphate is an antibiotic used for the treatment of infections by Gram-negative bacteria and that can induce a nephrotoxic effect that is not completely elucidated yet. The objective of this study was to verify the viability and the apoptosis on LLC-PK1 cells submitted to treatment with polymyxin B and previously treated with hemin (heme oxygenase stimulator - Hm) or zinc protoporphyrin (heme oxygenase inhibitor - ZnPP). LLC-PK1 cells were cultivated during 24, 48 e 72 hours. The apoptosis and the viability were evaluated using different doses of antiobiotic: Control(0mM); G1(12,5mM); G2(37,5mM); G3(75mM); G4(125mM) and G5(375mM). The hemin (25mM) and ZnPP (10mM) were given 1 hour before the addition of polymyxin B. It was utilized the Acridine orange/ Ethydium bromide (viability) and Hoescht 33342 (apoptosis) methods. Results demonstrated the linear rise of apoptosis, as the dose and time exposition were increased. This was confirmed by the inverse variation of the cellular viability. The hemin increased the viability and reduced the apoptosis in the presence of polymyxin B, confirming the protector effect of the heme oxygenase-1 in this model. Similar data were obtained when zinc protoporphyrin was added to the culture. This study confirmed the polymyxin B cytotoxicity and that this effect can be mediated by HO-1.