Polymyxins have a long history of dose-limiting toxicity, but the underlying mechanism of polymyxin B-induced nephrotoxicity is unclear. This study investigated the link between the nephrotoxic effects of polymyxin B on renal metabolic functions and mitochondrial morphology in rats and on the structural integrity of LLC-PK1 cells. Fifteen Wistar rats were divided into two groups: Saline group, rats received 3 mL/kg of 0.9% NaCl intraperitoneally (i.p.) once a day for 5 days; Polymyxin B group, rats received 4 mg/kg/day of polymyxin B i.p. once a day for 5 days. Renal function, renal hemodynamics, oxidative stress, mitochondrial injury and histological characteristics were assessed. Cell membrane damage was evaluated via lactate dehydrogenase and nitric oxide levels, cell viability, and apoptosis in cells exposed to 12.5 μM, 75 μM and 375 μM polymyxin B. Polymyxin B was immunolocated using Lissamine rhodamine-polymyxin B in LLC-PK1 cells. Polymyxin B administration in rats reduced creatinine clearance and increased renal vascular resistance and oxidative damage. Mitochondrial damage was confirmed by electron microscopy and cytosolic localization of cytochrome c. Histological analysis revealed tubular dilatation and necrosis in the renal cortex. The reduction in cell viability and the increase in apoptosis, lactate dehydrogenase levels and nitric oxide levels confirmed the cytotoxicity of polymyxin B. The incubation of LLC-PK1 cells resulted in mitochondrial localization of polymyxin B. This study demonstrates that polymyxin B nephrotoxicity is characterized by mitochondrial dysfunction and free radical generation in both LLC-PK1 cells and rat kidneys. These data also provide support for clinical studies on the side effects of polymyxin B.
Original ArticleProteção funcional da enzima heme-oxigenase-1 na lesão renal aguda isquêmica e tóxica Protección funcional de la enzima heme-oxigenasa-1 en la lesión renal aguda isquémica y tóxica RESUMOObjetivos: Verificar a proteção funcional da heme-oxigenase (HO-1), por meio do uso do seu indutor (Hemin) e seu inibidor químico (protoporfirina de zinco-ZnPP) na lesão renal aguda isquêmica e tóxica pela Polimixina B (PmxB) em ratos. Material: Foram utilizados ratos Wistar, adultos e machos divididos em 8 grupos: SHAM (controle), Isquemia (Isq), Isq+Hemin (indutor de HO-1), Isq+ZnPP (inibidor de HO-1), SALINA (controle), Polimixina B (PmxB), PmxB+Hemin, PmxB+ZnPP. Métodos: Jaffé (clearance de creatinina, Clcr) e FOX-2 (peróxidos urinários). Resultados: A isquemia (30´) dos pedículos reais e a administração de PmxB reduziu o Clcr com manutenção do fluxo urinário. Os peróxidos urinários se elevaram em ambas as lesões. A administração do Indutor de HO-1 determinou melhora da função renal e redução dos níveis de peróxidos urinários. Conclusão: Os resultados deste estudo demonstraram que a isquemia e a PmxB induzem LRA oxidativa. O indutor de HO-1 atenuou a lesão em ambos os modelos por atenuação do mecanismo redox. Descritores: Rim/lesões; Heme-oxigenase-1 RESUMEN Objetivos: Verificar la protección funcional de la heme-oxigenasa (HO-1), por medio del uso de su inductor (Hemin) y su inhibidor químico (protoporfirina de zinc-ZnPP) en la lesión renal aguda isquémica y tóxica producida por la Polimixina B (PmxB) en ratas. Material: Fueron utilizadas ratas Wistar, adultas y machos divididos en 8 grupos: SHAM (control), Isquemia (Isq), Isq+Hemin (indutor de HO-1), Isq+ZnPP (inibidor de HO-1), SALINA (control), Polimixina B (PmxB), PmxB+Hemin, PmxB+ZnPP. Métodos: Jaffé (clearance de creatinina, Clcr) y FOX-2 (peróxidos urinarios). Resultados: La isquemia (30´) de los pedículos reales y la administración de PmxB redujo el Clcr con manutención del flujo urinario. Los peróxidos urinarios se elevaron en ambas lesiones. La administración del Inductor de HO-1 determinó mejora de la función renal y reducción de los niveles de peróxidos urinarios. Conclusión: Los resultados de este estudio demuestran que la isquemia y la PmxB inducen AKL por la elevación de los peróxidos urinarios. El inductor de HO-1 atenuó la lesión en ambos modelos por atenuación del mecanismo redox. Descriptores: Riñón/lesión; Hemo-oxigenase-1
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. ResumoObjetivo: Caracterizar a toxicidade da polimixina B (PmxB) em células renais em dosagem e tempos diferentes. Métodos: Células LLC-PK 1 , 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.
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