Nox4 is involved in acute kidney injury associated to intravascular hemolysis

Massive intravascular hemolysis occurs not unfrequently in many clinical conditions. Breakdown of erythrocytes promotes the accumulation of heme-derivates in the kidney, increasing oxidative stress and cell death, thus promoting acute kidney injury (AKI). NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species (ROS) in the kidney, however it is unknown the role of Nox4 in hemolysis and whether inhibition of this enzyme may protect from heme-mediated injury. To answer these questions, we elicited intravascular hemolysis in wild type and Nox4 knockout mice. We also evaluated whether nephrotoxic effects of heme may be reduced by using Nox4 siRNA and pharmacologic inhibition with GKT137831, a Nox4 inhibitor, both in vivo and in cultured renal cells. Our results showed that induction of massive hemolysis elicited AKI characterized by loss of renal function, morphological alterations of the tubular epithelium and podocytes, oxidative stress, inflammation, mitochondrial dysfunction, blockade of autophagy and cell death. These pathological effects were significantly prevented in Nox4-deficient mice and in animals treated with GKT137831. In vitro studies showed that Nox4 disruption by specific siRNAs or Nox4 inhibitors declined heme-mediated ROS production and cell death. Our data identify Nox4 as a key enzyme involved in intravascular hemolysis-induced AKI. Thus, Nox4 inhibition may be a potential therapeutic approach to prevent renal damage in patients with severe hemolytic crisis.