Luciana Barros de Moura Neiva

Polymyxin B Nephrotoxicity: From Organ to Cell Damage

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.

Proteção funcional da enzima heme-oxigenase-1 na lesão renal aguda isquêmica e tóxica

OBJETIVOS: Verificar la proteccion funcional de la heme-oxigenasa (HO-1), por medio del uso de su inductor (Hemin) y su inhibidor quimico (protoporfirina de zinc-ZnPP) en la lesion renal aguda isquemica y toxica 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. METODOS: Jaffe (clearance de creatinina, Clcr) y FOX-2 (peroxidos urinarios). RESULTADOS: La isquemia (30´) de los pediculos reales y la administracion de PmxB redujo el Clcr con manutencion del flujo urinario. Los peroxidos urinarios se elevaron en ambas lesiones. La administracion del Inductor de HO-1 determino mejora de la funcion renal y reduccion de los niveles de peroxidos urinarios. CONCLUSION: Los resultados de este estudio demuestran que la isquemia y la PmxB inducen AKL por la elevacion de los peroxidos urinarios. El inductor de HO-1 atenuo la lesion en ambos modelos por atenuacion del mecanismo redox.ABSTRACT Objective : To investigate the functional protection of heme-oxygenase-1 enzyme (HO-1) when using its inducer (Hemin) and inhibitor(zinc protoporphyrin-ZnPP) in ischemic and toxic acute kidney injury by Polymixin B in mice. Materials : Adult male Wistar mice dividedinto 8 groups were used: SHAM (control), Ischemic (Isq), Isq+Hemin (Inducer of H0-1), Isq+ZnPP (inhibitor of H0-1), SALINA(control), Polimyxin B (PmxB), PmxB+Hemin, PmxB+ZnPP. Method : Analysis consists of JaffO (creatinine clearance [crCl]) and FOX-2(urinary peroxides [UP]). Results : Thirty minutes renal ischemia and its treatment with PmxB reduced the crCl and maintained urinaryoutput. Urinary peroxide levels increased in both injuries. The administration of the inducer of H0-1 resulted in improvement in renalfunction and reduction in the levels of urinary peroxide. Conclusions : Findings indicated that ischemia and PmxB induce LAR (acutekidney injury [AKI]) by elevating the levels of urinary peroxide. The HO-1 inducer ameliorated the injury in both animal models throughredox mechanism.

Polimixina B: efeito dose e tempo dependente na nefrotoxicidade in vitro

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.

Functional protection of heme-oxygenase-1 enzyme in ischemic and toxic acute kidney injury

OBJETIVOS: Verificar la proteccion funcional de la heme-oxigenasa (HO-1), por medio del uso de su inductor (Hemin) y su inhibidor quimico (protoporfirina de zinc-ZnPP) en la lesion renal aguda isquemica y toxica 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. METODOS: Jaffe (clearance de creatinina, Clcr) y FOX-2 (peroxidos urinarios). RESULTADOS: La isquemia (30´) de los pediculos reales y la administracion de PmxB redujo el Clcr con manutencion del flujo urinario. Los peroxidos urinarios se elevaron en ambas lesiones. La administracion del Inductor de HO-1 determino mejora de la funcion renal y reduccion de los niveles de peroxidos urinarios. CONCLUSION: Los resultados de este estudio demuestran que la isquemia y la PmxB inducen AKL por la elevacion de los peroxidos urinarios. El inductor de HO-1 atenuo la lesion en ambos modelos por atenuacion del mecanismo redox.ABSTRACT Objective : To investigate the functional protection of heme-oxygenase-1 enzyme (HO-1) when using its inducer (Hemin) and inhibitor(zinc protoporphyrin-ZnPP) in ischemic and toxic acute kidney injury by Polymixin B in mice. Materials : Adult male Wistar mice dividedinto 8 groups were used: SHAM (control), Ischemic (Isq), Isq+Hemin (Inducer of H0-1), Isq+ZnPP (inhibitor of H0-1), SALINA(control), Polimyxin B (PmxB), PmxB+Hemin, PmxB+ZnPP. Method : Analysis consists of JaffO (creatinine clearance [crCl]) and FOX-2(urinary peroxides [UP]). Results : Thirty minutes renal ischemia and its treatment with PmxB reduced the crCl and maintained urinaryoutput. Urinary peroxide levels increased in both injuries. The administration of the inducer of H0-1 resulted in improvement in renalfunction and reduction in the levels of urinary peroxide. Conclusions : Findings indicated that ischemia and PmxB induce LAR (acutekidney injury [AKI]) by elevating the levels of urinary peroxide. The HO-1 inducer ameliorated the injury in both animal models throughredox mechanism.

[Nephrotoxicity of polymyxin B: experimental study in cells and implications for nursing practice].

O objetivo do estudo foi caracterizar os mecanismos de lesao celular envolvidos na fisiopatologia da citotoxicidade da polimixina B em celulas tubulares proximais (LLC-PK1) e discutir as proposicoes de intervencao do enfermeiro para identificar os pacientes de risco e considerar a prevencao ou o tratamento para lesao renal nefrotoxica. Estudo experimental in vitro , onde as celulas foram expostas ao sulfato de polimixina B. A viabilidade celular foi determinada pela exclusao dos corantes fluorescentes e o metodo morfologico com visualizacao de corpos apoptoticos a microscopia de fluorescencia. As celulas expostas a polimixina B apresentaram reducao de viabilidade, aumento do numero de celulas em apoptose e maior concentracao da enzima desidrogenase lactea. A administracao de polimixina B in vitro demonstrou a necessidade de acoes na pratica clinica para minimizar os efeitos adversos como a nefrotoxicidade.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.

Nefrotoxicidade da polimixina B: estudo experimental em células e implicações para a prática de enfermagem

O objetivo do estudo foi caracterizar os mecanismos de lesao celular envolvidos na fisiopatologia da citotoxicidade da polimixina B em celulas tubulares proximais (LLC-PK1) e discutir as proposicoes de intervencao do enfermeiro para identificar os pacientes de risco e considerar a prevencao ou o tratamento para lesao renal nefrotoxica. Estudo experimental in vitro , onde as celulas foram expostas ao sulfato de polimixina B. A viabilidade celular foi determinada pela exclusao dos corantes fluorescentes e o metodo morfologico com visualizacao de corpos apoptoticos a microscopia de fluorescencia. As celulas expostas a polimixina B apresentaram reducao de viabilidade, aumento do numero de celulas em apoptose e maior concentracao da enzima desidrogenase lactea. A administracao de polimixina B in vitro demonstrou a necessidade de acoes na pratica clinica para minimizar os efeitos adversos como a nefrotoxicidade.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.

La Nefrotoxicidad De Polimixina B: Estudio Experimental En Las Células Y Implicaciones Para La Práctica De Enfermería

O objetivo do estudo foi caracterizar os mecanismos de lesao celular envolvidos na fisiopatologia da citotoxicidade da polimixina B em celulas tubulares proximais (LLC-PK1) e discutir as proposicoes de intervencao do enfermeiro para identificar os pacientes de risco e considerar a prevencao ou o tratamento para lesao renal nefrotoxica. Estudo experimental in vitro , onde as celulas foram expostas ao sulfato de polimixina B. A viabilidade celular foi determinada pela exclusao dos corantes fluorescentes e o metodo morfologico com visualizacao de corpos apoptoticos a microscopia de fluorescencia. As celulas expostas a polimixina B apresentaram reducao de viabilidade, aumento do numero de celulas em apoptose e maior concentracao da enzima desidrogenase lactea. A administracao de polimixina B in vitro demonstrou a necessidade de acoes na pratica clinica para minimizar os efeitos adversos como a nefrotoxicidade.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.

Protección funcional de la enzima heme-oxigenasa-1 en la lesión renal aguda isquémica y tóxica

OBJETIVOS: Verificar la proteccion funcional de la heme-oxigenasa (HO-1), por medio del uso de su inductor (Hemin) y su inhibidor quimico (protoporfirina de zinc-ZnPP) en la lesion renal aguda isquemica y toxica 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. METODOS: Jaffe (clearance de creatinina, Clcr) y FOX-2 (peroxidos urinarios). RESULTADOS: La isquemia (30´) de los pediculos reales y la administracion de PmxB redujo el Clcr con manutencion del flujo urinario. Los peroxidos urinarios se elevaron en ambas lesiones. La administracion del Inductor de HO-1 determino mejora de la funcion renal y reduccion de los niveles de peroxidos urinarios. CONCLUSION: Los resultados de este estudio demuestran que la isquemia y la PmxB inducen AKL por la elevacion de los peroxidos urinarios. El inductor de HO-1 atenuo la lesion en ambos modelos por atenuacion del mecanismo redox.ABSTRACT Objective : To investigate the functional protection of heme-oxygenase-1 enzyme (HO-1) when using its inducer (Hemin) and inhibitor(zinc protoporphyrin-ZnPP) in ischemic and toxic acute kidney injury by Polymixin B in mice. Materials : Adult male Wistar mice dividedinto 8 groups were used: SHAM (control), Ischemic (Isq), Isq+Hemin (Inducer of H0-1), Isq+ZnPP (inhibitor of H0-1), SALINA(control), Polimyxin B (PmxB), PmxB+Hemin, PmxB+ZnPP. Method : Analysis consists of JaffO (creatinine clearance [crCl]) and FOX-2(urinary peroxides [UP]). Results : Thirty minutes renal ischemia and its treatment with PmxB reduced the crCl and maintained urinaryoutput. Urinary peroxide levels increased in both injuries. The administration of the inducer of H0-1 resulted in improvement in renalfunction and reduction in the levels of urinary peroxide. Conclusions : Findings indicated that ischemia and PmxB induce LAR (acutekidney injury [AKI]) by elevating the levels of urinary peroxide. The HO-1 inducer ameliorated the injury in both animal models throughredox mechanism.