Original Contribution Exposure to low- vs iso-osmolar contrast agents reduces NADPH-dependent reactive oxygen species generation in a cellular model of renal injury Giuseppe Stefano Netti a , Clelia Prattichizzo a , Eustacchio Montemurno b , Simona Simone c , Cesira Cafiero c , Federica Rascio b , Giovanni Stallone b , Elena Ranieri a , Giuseppe Grandaliano b , Loreto Gesualdo c,n a Department of Medical and Surgical Sciences, Section of Clinical Pathology, University of Foggia, Foggia 71122, Italy b Department of Medical and Surgical Sciences, Section of Nephrology, University of Foggia, Foggia 71122, Italy c Department of Emergency and Organ Transplantation, Section of Nephrology, University of Bari "Aldo Moro," Bari 70124, Italy article info Article history: Received 5 August 2013 Received in revised form 13 November 2013 Accepted 15 November 2013 Available online 1 December 2013 Keywords: Contrast-induced nephropathy Low-osmolar contrast medium Iso-osmolar contrast medium Reactive oxygen species NADPH oxidase Free radicals abstract Contrast-induced nephropathy represents the third cause of hospital-acquired acute renal failure. This study investigated the effects of low- vs iso-osmolar contrast medium (CM) exposure on NADPH- dependent reactive oxygen species (ROS) generation by tubular cells. X-ray attenuation of iohexol, iopamidol, and iodixanol was assessed at equimolar iodine concentrations and their effects on human renal proximal tubular cells (PTCs) were evaluated with equally attenuating solutions of each CM. Cytotoxicity, apoptosis, and necrosis were investigated by trypan blue exclusion, MTTassay, and annexin V/propidium iodide assay, respectively. ROS production was assessed by DCF assay, NADPH oxidase activity by the lucigenin-enhanced chemiluminescence method, and Nox4 expression by immunoblot. Yielding the same X-ray attenuation, CM cytotoxicity was assessed in PTCs at equimolar iodine concentrations. More necrosis was present after incubation with iohexol and iopamidol than after incubation with equal concentrations of iodixanol. Iohexol and iodixanol at low iodine concentrations induced less cytotoxicity than iopamidol. Moreover, both iohexol and iopamidol induced more apoptosis than iodixanol, with a dose-dependent effect. ROS generation was significantly higher with iopamidol and iohexol compared to iodixanol. NADPH oxidase activity and Nox4 protein expression significantly increased after exposure to iopamidol and iohexol, with a dose-dependent effect, compared with iodixanol. CM-induced Nox4 expression and activity depended upon Src activation. In conclusion, at angiographic concentrations, iodixanol induces fewer cytotoxic effects on cultured tubular cells than iohexol and iopamidol along with a lower induction of Nox4-dependent ROS generation. This enzyme may, thus, represent a potential therapeutic target to prevent iodinated CM-related oxidative stress. & 2013 Elsevier Inc. All rights reserved. Acute kidney injury (AKI) is a frequent and devastating pro- blem in hospitalized adults with increasing rates of mortality and morbidity [1]. Several studies in large adult cohorts have shown that contrast-induced AKI (CI-AKI), formerly known as contrast- induced nephropathy, is the third most common cause of hospital- acquired AKI, accounting for 4–10% of cases [2]. The incidence of CI-AKI in the general population does not exceed 2%; however, in high-risk patients it can rise to more than 50% [3]. CI-AKI is defined as a sudden decrease in renal function after iv administration of contrast medium (CM) in the absence of any other cause [4] and is significantly associate with increased cost, hospital stay, and in-hospital and long-term morbidity and mor- tality [5–7]. The pathogenesis of CI-AKI is poorly understood. CM osmolarity has been suggested to play a key role. Low-osmolar nonionic CMs have fewer nephrotoxic effects than high-osmolar ionic contrast media [8]. In addition, in high-risk patients the iso-osmolar dimer iodixanol has been recently shown to be associated with fewer nephrotoxic effects than low-osmolar monomeric CMs [9]. However, these results are still debated [10], and even iso-osmolar contrast agents have been reported to cause nephrotoxicity [11]. Renal medullary hypoxia and direct tubular toxic effects of CM are the major pathogenic events of CI-AKI [12,13]. A direct toxic effect of CM on renal tubules has been shown in both clinical trials and experimental models [14–16]. Studies in humans suggested that reactive oxygen species (ROS) may contribute to CI-AKI [17–19]. Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine 0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2013.11.016 Abbreviations: AKI, acute kidney injury; CM, contrast medium; DPI, diphenylene iodonium; DCFH-DA, 2′,7′-dichlorodihydrofluorescein diacetate; NAC, N-acetylcys- teine; ROS, reactive oxygen species n Corresponding author. Fax: 0039 080 5575710. E-mail address: loreto.gesualdo@uniba.it (L. Gesualdo). Free Radical Biology and Medicine 68 (2014) 35–42