Mirian Watanabe

Role of Heme Oxygenase-1 in Polymyxin B-Induced Nephrotoxicity in Rats

ABSTRACT Polymyxin B (PMB) is a cationic polypeptide antibiotic with activity against multidrug-resistant Gram-negative bacteria. PMB-induced nephrotoxicity consists of direct toxicity to the renal tubules and the release of reactive oxygen species (ROS) with oxidative damage. This study evaluated the nephroprotective effect of heme oxygenase-1 (HO-1) against PMB-induced nephrotoxicity in rats. Adult male Wistar rats, weighing 286 ± 12 g, were treated intraperitoneally once a day for 5 days with saline, hemin (HO-1 inducer; 10 mg/kg), zinc protoporphyrin (ZnPP) (HO-1 inhibitor; 50 μmol/kg, administered before PMB on day 5), PMB (4 mg/kg), PMB plus hemin, and PMB plus ZnPP. Renal function (creatinine clearance, Jaffe method), urinary peroxides (ferrous oxidation of xylenol orange version 2 [FOX-2]), urinary thiobarbituric acid-reactive substances (TBARS), renal tissue thiols, catalase activity, and renal tissue histology were analyzed. The results showed that PMB reduced creatinine clearance (P < 0.05), with an increase in urinary peroxides and TBARS. The PMB toxicity caused a reduction in catalase activity and thiols (P < 0.05). Hemin attenuated PMB nephrotoxicity by increasing the catalase antioxidant activity (P < 0.05). The combination of PMB and ZnPP incremented the fractional interstitial area of renal tissue (P < 0.05), and acute tubular necrosis in the cortex area was also observed. This is the first study demonstrating the protective effect of HO-1 against PMB-induced nephrotoxicity.

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.

A sepse como causa de lesão renal aguda: modelo experimental

Sepsis associated with multiple organ failure such as acute kidney injury (AKI) shows a high mortality rate in critically ill patients. This study investigated the sepsis induced AKI in experimental models. Adult, males, Wistar rats divided into the following groups: Control-surgical control and Sepsis-sepsis induction for the cecal ligation and puncture (CLP). Physiological parameters (rectal temperature, mean arterial pressure-MAP, serum glucose and urinary flow); renal function (creatinine clearance); oxidative stress (urinary peroxides and thiobarbituric acid reactive substances-TBARS) and kidney histological analysis were evaluated. That study concludes that sepsis induces AKI by endothelial injury with hemodynamic dysfunction, release of inflammatory mediators and reactive oxygen species (ROS) generation by tubular cells, in an association of renal vasoconstriction due to hemodynamic and inflammatory disturbances.A sepse associada a falencia de multiplos orgaos como a lesao renal aguda (LRA) demonstra alta taxa de mortalidade no paciente critico. Este estudo investigou a LRA induzida pela sepse em modelo experimental. Foram utilizados ratos da raca Wistar, adultos e machos divididos nos seguintes grupos: Controle - controle cirurgico e Sepse - inducao da sepse pela ligadura e puncao do cecon (LPC). Foram avaliados os parâmetros fisiologicos (temperatura retal, pressao arterial media - PAM, glicemia serica e fluxo urinario); a funcao renal (clearance de creatinina); o estresse oxidativo (peroxidos urinarios e substâncias reativas com acido tiobarbiturico - TBARS) e realizada a analise histologica renal. O estudo conclui que a LRA induzida pela sepse caracteriza-se por lesao endotelial com disfuncao hemodinâmica, liberacao de mediadores inflamatorios e geracao de especies reativas de oxigenio (EROs) por celulas tubulares, caracterizando-se como uma associacao de vasoconstricao renal de origem hemodinâmica e inflamatoria.

The sepsis as cause of acute kidney injury: an experimental model

Sepsis associated with multiple organ failure such as acute kidney injury (AKI) shows a high mortality rate in critically ill patients. This study investigated the sepsis induced AKI in experimental models. Adult, males, Wistar rats divided into the following groups: Control-surgical control and Sepsis-sepsis induction for the cecal ligation and puncture (CLP). Physiological parameters (rectal temperature, mean arterial pressure-MAP, serum glucose and urinary flow); renal function (creatinine clearance); oxidative stress (urinary peroxides and thiobarbituric acid reactive substances-TBARS) and kidney histological analysis were evaluated. That study concludes that sepsis induces AKI by endothelial injury with hemodynamic dysfunction, release of inflammatory mediators and reactive oxygen species (ROS) generation by tubular cells, in an association of renal vasoconstriction due to hemodynamic and inflammatory disturbances.A sepse associada a falencia de multiplos orgaos como a lesao renal aguda (LRA) demonstra alta taxa de mortalidade no paciente critico. Este estudo investigou a LRA induzida pela sepse em modelo experimental. Foram utilizados ratos da raca Wistar, adultos e machos divididos nos seguintes grupos: Controle - controle cirurgico e Sepse - inducao da sepse pela ligadura e puncao do cecon (LPC). Foram avaliados os parâmetros fisiologicos (temperatura retal, pressao arterial media - PAM, glicemia serica e fluxo urinario); a funcao renal (clearance de creatinina); o estresse oxidativo (peroxidos urinarios e substâncias reativas com acido tiobarbiturico - TBARS) e realizada a analise histologica renal. O estudo conclui que a LRA induzida pela sepse caracteriza-se por lesao endotelial com disfuncao hemodinâmica, liberacao de mediadores inflamatorios e geracao de especies reativas de oxigenio (EROs) por celulas tubulares, caracterizando-se como uma associacao de vasoconstricao renal de origem hemodinâmica e inflamatoria.

Lesão renal aguda por glicerol: efeito antioxidante da Vitis vinifera L

BACKGROUND AND OBJECTIVES The Acute Kidney Injury (AKI) is the most serious complication of rhabdomyolysis. In this syndrome, the delivery of heme pigment induces an injury that distinguishes itself by glomerular vasoconstriction and direct cellular toxicity with oxidative component. The renoprotection with antioxidants has demonstrated satisfactory effect. The proanthocyanidins are natural antioxidants found in the grape seed extract. The aim of this study was to evaluate the antioxidant effect of Vitis vinifera (grape seed extract) on the renal function of rats submitted to the injury by rhabdomyolysis. METHODS Wistar rats, male, adults, weight ranging from 250-300g were used. The AKI was induced by intramuscular administration of glycerol 50%. The animals were distributed in 4 groups: Saline group (6 mL/kg of NaCl 0.9% intraperitoneal once a day), Glycerol group (6 mL/kg) of intramuscular glycerol each femoral region received 3 mL/kg of glycerol, once a day), Vitis vinifera group (3 mg/kg/day v.o by 5 days) and Glycerol + Vitis vinifera by 5 days before glycerol). RESULTS Renal function (RF-creatinine clearance) and oxidative profile (urinary peroxides-FOX-2 and MDA-TBARS) were evaluted. The Glycerol group treated with Vitis vinifera has shown improvements in RF and reduction levels of lipid peroxidation. CONCLUSION The results of this study have confirmed the antioxidant protection of Vitis vinifera in AKI induced by glycerol.BACKGROUND AND OBJECTIVES: The Acute Kidney Injury (AKI) is the most serious complication of rhabdomyolysis. In this syndrome, the delivery of heme pigment induces an injury that distinguishes itself by glomerular vasoconstriction and direct cellular toxicity with oxidative component. The renoprotection with antioxidants has demonstrated satisfactory effect. The proanthocyanidins are natural antioxidants found in the grape seed extract. The aim of this study was to evaluate the antioxidant effect of Vitis vinifera (grape seed extract) on the renal function of rats submitted to the injury by rhabdomyolysis. METHODS: Wistar rats, male, adults, weight ranging from 250-300g were used. The AKI was induced by intramuscular administration of glycerol 50%. The animals were distributed in 4 groups: Saline group (6 mL/kg of NaCl 0.9% intraperitoneal once a day), Glycerol group (6 mL/kg) of intramuscular glycerol each femoral region received 3 mL/kg of glycerol, once a day), Vitis vinifera group (3 mg/kg/day v.o by 5 days) and Glycerol + Vitis vinifera by 5 days before glycerol). RESULTS: Renal function (RF-creatinine clearance) and oxidative profile (urinary peroxides-FOX-2 and MDA-TBARS) were evaluted. The Glycerol group treated with Vitis vinifera has shown improvements in RF and reduction levels of lipid peroxidation. CONCLUSION: The results of this study have confirmed the antioxidant protection of Vitis vinifera in AKI induced by glycerol.

The role of oxidative stress in streptozotocin-induced diabetic nephropathy in rats

Objective The objective of this study was to evaluate the role of oxidative stress in an experimental model of streptozotocin-induced diabetic nephropathy in rats. Materials and methods Wistar, adult, male rats were used in the study. Animals were divided in the following groups: Citrate (control, citrate buffer 0.01M, pH 4.2 was administrated intravenously - i.v - in the caudal vein), Uninephrectomy+Citrate (left uninephrectomy-20 days before the study), DM (streptozotocin, 65 mg/kg, i.v, on the 20th day of the study), Uninephrectomy+DM. Physiological parameters (water and food intake, body weight, blood glucose, kidney weight, and relative kidney weight); renal function (creatinine clearance), urine albumin (immunodiffusion method); oxidative metabolites (urinary peroxides, thiobarbituric acid reactive substances, and thiols in renal tissue), and kidney histology were evaluated. Results Polyphagia, polydipsia, hyperglycemia, and reduced body weight were observed in diabetic rats. Renal function was reduced in diabetic groups (creatinine clearance, p < 0.05). Uninephrectomy potentiated urine albumin and increased kidney weight and relative kidney weight in diabetic animals (p < 0.05). Urinary peroxides and thiobarbituric acid reactive substances were increased, and the reduction in thiol levels demonstrated endogenous substrate consumption in diabetic groups (p < 0.05). The histological analysis revealed moderate lesions of diabetic nephropathy. Conclusion This study confirms lipid peroxidation and intense consumption of the antioxidant defense system in diabetic rats. The association of hyperglycemia and uninephrectomy resulted in additional renal injury, demonstrating that the model is adequate for the study of diabetic nephropathy.

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.

Impact of Iodinated Contrast on Renal Function and Hemodynamics in Rats with Chronic Hyperglycemia and Chronic Kidney Disease

Iodinated contrast (IC) is clinically used in diagnostic and interventional procedures, but its use can result in contrast-induced acute kidney injury (CI-AKI). Chronic kidney disease (CKD) and chronic hyperglycemia (CH) are important predisposing factors to CI-AKI. The aim of this study was to investigate the impact of iodinated contrast on the renal function and hemodynamics in rats with chronic hyperglycemia and chronic kidney disease. A total of 30 rats were divided into six groups; Sham: control of chronic renal disease; Citrate: control of chronic hyperglycemia (CH); Nx5/6: rats with 5/6 nephrectomy; Chronic Hyperglycemia: rats receiving Streptozotocin 65 mg/kg; Nx5/6 + IC: rats Nx5/6 received 6 mL/kg of IC; CH + IC: Chronic hyperglycemia rats receiving 6 mL/kg of IC. Renal function (inulin clearance; urinary neutrophil gelatinase-associated lipocalin, NGAL) and hemodynamics (arterial blood pressure; renal blood flow; renal vascular resistance) were evaluated. Iodinated contrast significantly increased urinary NGAL and reduced inulin clearance, while the hemodynamics parameters showed changes in arterial blood pressure, renal blood flow, and renal vascular resistance in both CKD and CH groups. The results suggest that the iodinated contrast in risk factors models has important impact on renal function and hemodynamics. NGAL was confirmed to play a role of highlight in diagnosis of CI-AKI.

Renoprotective effect of the Echinodorus macrophyllus in induced renal injury

OBJECTIVE: Evaluating the renoprotective effect of Echinodorus macrophyllusin acute kidney injury induced by cyclophosphamide in rats.METHODS: Experimental research with Wistar rats, male adults, distributed into groups, namely: Control - administration of 1.5 ml sodium chloride 0.9% intraperitoneally; Echinodorous - administration of 2g/kg of Echinodorus macrophyllus by gavage for five days; Cyclophosphamide - administration of cyclophosphamide 150mg/kg intraperitoneally; and Cyclosphosphamide + Echinodorus - administration of Echinodorus macrophyllus and cyclophosphamide. Renal function (creatinine clearance) and the oxidative metabolites (peroxides and urinary substances reactive to thiobarbituric acid, thiols in kidney tissue) were evaluated.RESULTS: Preconditioning with Echinodorus macrophyllus elevated the creatinine clearance and reduced the levels of oxidative metabolites.CONCLUSION: The antioxidant action of Echinodorus macrophyllus has demonstrated renoprotective effects evidenced by the reduction of oxidative stress in acute renal injury induced by cyclophosphamide in rats.

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.