Calcium oxalate crystals and oxalate induce an epithelial-to-mesenchymal transition in the proximal tubular epithelial cells: Contribution to oxalate kidney injury.

TGF-ß1 is the main mediator of epithelial-to-mesenchymal transition (EMT). Hyperoxaluria induces crystalluria, interstitial fibrosis, and progressive renal failure. This study analyzed whether hyperoxaluria is associated with TGF-ß1 production and kidney fibrosis in mice and if oxalate or calcium oxalate (CaOx) could induce EMT in proximal tubule cells (HK2) and therefore contribute to the fibrotic process. Hyperoxaluria was induced by adding hydroxyproline and ethylene glycol to the mice's drinking water for up to 60 days. Renal function and oxalate and urinary crystals were evaluated. Kidney collagen production and TGF-ß1 expression were assessed. EMT was analyzed in vitro according to TGF-ß1 production, phenotypic characterization, invasion, cell migration, gene and protein expression of epithelial and mesenchymal markers. Hyperoxaluric mice showed a decrease in renal function and an increase in CaOx crystals and Ox urinary excretion. The deposition of collagen in the renal interstitium was observed. HK2 cells stimulated with Ox and CaOx exhibited a decreased expression of epithelial as well as increased expression mesenchymal markers; these cells presented mesenchymal phenotypic changes, migration, invasiveness capability and TGF-ß1 production, characterizing EMT. Treatment with BMP-7 or its overexpression in HK2 cells was effective at preventing it. This mechanism may contribute to the fibrosis observed in hyperoxaluria.

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.

Priming prevent nephrotoxic acute renal failure through stimulation of antioxidant defense mechanism. / Priming previne a insuficiência renal aguda nefrotóxica através da estimulação do mecanismo de defesa antioxidante.

INTRODUCTION: Priming is the mechanism of protection induced by a previous exposition of a cell or organ to low or equal concentrations of a toxic substance. OBJECTIVE: To analyze the mechanism of priming induced by the previous exposition to gentamicin in human proximal tubular cells and nephrotoxic acute renal failure (ARF). METHODS: Wistar rats and tubular cells were exposed to gentamicin 2mM during 24h or 40 mg/kg during 3 days and after one rest week were exposed to the same concentration during 24h in cells and additional ten days in rats. The primed animals were compared to control rats receiving vehicle and GENTA animals treated with the gentamicin during the same period. Biochemical parameters were analyzed. The oxidative stress was analyzed by urinary hydroperoxides and carbonylated protein while antioxidant defense was studied by antioxidant activity of the plasma (FRAP), catalase, superoxide dismutase, heme-oxygenase 1 (HO-1) immunostaining and enzymatic activity in kidney. Necrosis, apoptosis, proliferation, endothelin 1 (ET-1) and HO-1 expression were studied in cells. RESULTS: Priming of the animals inhibited the increase in creatinine, urea, sodium excretion and urinary protein induced by gentamicin. Bosentan, ET-1 receptor antagonist, and hemin, HO-1 inducer, potentiate the inhibition. The mechanism of protection was mediated by induction of the antioxidant enzymes HO-1, catalase and SOD activity and oxidative stress reduction. Priming inhibited cell death and induced proliferation through ET-1 production. CONCLUSION: Priming is a persistent and multifactorial mechanism, the stimulation of the antioxidant defense could mimics partially the priming process and prevent the ARF.

Priming previne a insuficiência renal aguda nefrotóxica através da estimulação do mecanismo de defesa antioxidante / Priming prevent nephrotoxic acute renal failure through stimulation of antioxidant defense mechanism

RESUMO Introdução: Priming é um mecanismo de proteção induzida pela exposição anterior de uma célula ou órgão a baixas ou mesmas concentrações de uma substância tóxica. Objetivo: analisar o mecanismo de priming induzido pela exposição a gentamicina em células tubulares proximais humanas e na insuficiência renal aguda (IRA). Métodos: Células tubulares foram expostos a 2 mM de gentamicina durante 24 horas, enquanto ratos Wistar foram expostas a 40 mg/kg durante 3 dias. Depois de uma semana, as células foram expostas à mesma concentração durante 24h e os ratos durante dez dias. Os animais condicionados foram comparados com ratos controle e tratados com gentamicina durante 10 dias. Foram analisados parâmetros bioquímicos, o estresse oxidativo foi analisado por hidroperóxidos e proteínas carboniladas urinárias, enquanto a defesa antioxidante foi estudada pela atividade antioxidante do plasma e imunomarcação e atividade da catalase, superóxido dismutase, heme oxigenase-1 (HO-1) nos rins. Necrose, apoptose, proliferação e expressão da endotelina-1 (ET-1) e HO-1 foram estudadas em células. Resultados: o condicionamento dos animais inibiu o aumento da creatinina, ureia, excreção urinária de sódio e de proteína induzida por gentamicina. Bosentana, antagonista do receptor ET-1, e hemin, indutor de HO-1, potencializaram a inibição. O mecanismo de proteção foi mediado pela indução de enzimas antioxidantes HO-1, catalase e SOD atividade e redução do estresse oxidativo. O condicionamento inibiu a morte celular e induziu a proliferação via produção de ET-1. Conclusão: o mecanismo de condicionamento é persistente e multifactorial, o estímulo da defesa antioxidante poderia mimetizar o processo de condicionamento e impedir a IRA.

ABSTRACT Introduction: Priming is the mechanism of protection induced by a previous exposition of a cell or organ to low or equal concentrations of a toxic substance. Objective: To analyze the mechanism of priming induced by the previous exposition to gentamicin in human proximal tubular cells and nephrotoxic acute renal failure (ARF). Methods: Wistar rats and tubular cells were exposed to gentamicin 2mM during 24h or 40 mg/kg during 3 days and after one rest week were exposed to the same concentration during 24h in cells and additional ten days in rats. The primed animals were compared to control rats receiving vehicle and GENTA animals treated with the gentamicin during the same period. Biochemical parameters were analyzed. The oxidative stress was analyzed by urinary hydroperoxides and carbonylated protein while antioxidant defense was studied by antioxidant activity of the plasma (FRAP), catalase, superoxide dismutase, heme-oxygenase 1 (HO-1) immunostaining and enzymatic activity in kidney. Necrosis, apoptosis, proliferation, endothelin 1 (ET-1) and HO-1 expression were studied in cells. Results: Priming of the animals inhibited the increase in creatinine, urea, sodium excretion and urinary protein induced by gentamicin. Bosentan, ET-1 receptor antagonist, and hemin, HO-1 inducer, potentiate the inhibition. The mechanism of protection was mediated by induction of the antioxidant enzymes HO-1, catalase and SOD activity and oxidative stress reduction. Priming inhibited cell death and induced proliferation through ET-1 production. Conclusion: Priming is a persistent and multifactorial mechanism, the stimulation of the antioxidant defense could mimics partially the priming process and prevent the ARF.