Cristina García Caballero
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Melania Guerrero Hue
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Alejandra Palomino Antolín
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Matilde Cabanillas
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Cristina Vazquez Carballo
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...
Abstract
Background and Aims
Massive intravascular hemolysis is a common condition of several pathologies. It is associated with acute kidney injury (AKI) and progressive impairment of renal function. In this context, free hemoglobin (Hb) can exert harmful effects by accumulating in the kidney, where induces oxidative stress and it becomes cytotoxic. NADPH oxidase 4 (Nox4) is the principal source of reactive oxygen species (ROS) in the kidney. Nox4 is mostly expressed in proximal tubular cells with lower levels in glomerulus. The role of Nox4 in renal damage is not clear, with studies reporting beneficial or deleterious actions depending of the environmental conditions. For that reason we aimed to investigate the role of Nox4 in massive intravascular hemolysis-associated AKI.
Method
To study the role of Nox4 in AKI caused by massive intravascular hemolysis, we performed an experimental model of intravascular hemolysis by intraperitoneal injection of phenylhydrazine (200 mg/kg) in wild type (Nox4+/+) and Nox4 knockout mice (Nox4-/-). Mice were sacrificed 24 and 72 hours after intravascular hemolysis induction. We collected serum, urine and tissues sample. We analyzed renal function, oxidative stress, cell death and inflammation in these samples. In other experiments, wild type mice were treated with GKT137831 (10mg/kg/day), a potent Nox4 and Nox1 inhibitor, and mice were sacrificed 72h after induction of hemolysis. We also performed in vitro experiments in murine tubular epithelial cells (MCT) and murine podocytes cells to investigate the regulation of Nox4 in Hb-stimulated cells treated or not with GKT137831.
Results
Induction of intravascular hemolysis in Nox4+/+ mice increased creatinine and BUN levels and enhanced the expression of tubular injury markers, such as NGAL. These pathological effects were reduced in Nox4 knockout mice. Then, we analyzed oxidative stress in our experimental model thought determination of HO-1, ferritin, GSH and lipid peroxidation levels. All of these oxidative markers were reduced in Nox4-/- mice with intravascular hemolysis as compared with Nox4+/+ mice. We also observed that inflammatory markers such as IL-6, cell death and podocytes injury markers were reduced in Nox4-/- mice than in wild type mice, specially 72 hours after phenylhydrazine injection. In line with these results, GKT137831 administration ameliorated intravascular hemolysis-associated renal function impairment. Moreover, oxidative stress, tubular injury markers and podocyte injury were reduced in hemolytic mice treated with GKT137831. GKT137831 also reduced Hb- and heme-mediated oxidative stress in MCT and podocytes.
Conclusion
Our results show the important role of Nox4 in renal injury associated to massive intravascular hemolysis. Moreover, the inhibition of Nox4 may be a potential therapeutic target to prevent renal damage associated to Hb accumulation. These findings provide new insights into novel aspects of Hb-toxicity and may have important pathogenic and therapeutic implications for intravascular hemolysis related diseases
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