Contrast-induced acute kidney injury (CIAKI) is a leading cause of acute kidney injury following radiographic procedures. Intrarenal oxidative stress plays a critical role in CIAKI. Nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidases (Noxs) are important sources of reactive oxygen species (ROS). Among the various types of Noxs, Nox4 is expressed predominantly in the kidney in rodents. Here, we evaluated the role of Nox4 and benefit of Nox4 inhibition on CIAKI using in vivo and in vitro models. HK-2 cells were treated with iohexol, with or without Nox4 knockdown, or the most specific Nox1/4 inhibitor (GKT137831). Effects of Nox4 inhibition on CIAKI mice were examined. Expression of Nox4 in HK-2 cells was significantly increased following iohexol exposure. Silencing of Nox4 rescued the production of ROS, downregulated pro-inflammatory markers (particularly phospho-p38) implicated in CIAKI, and reduced Bax and caspase 3/7 activity, which resulted in increased cellular survival in iohexol-treated HK-2 cells. Pretreatment with GKT137831 replicated these effects by decreasing levels of phospho-p38. In a CIAKI mouse model, even though the improvement of plasma blood urea nitrogen was unclear, pretreatment with GKT137831 resulted in preserved structure, reduced expression of 8-hydroxy-2’-deoxyguanosine (8OHdG) and kidney injury molecule-1 (KIM-1), and reduced number of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive cells. These results suggest Nox4 as a key source of reactive oxygen species responsible for CIAKI and provide a novel potential option for prevention of CIAKI.

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Phenotype transition of mesothelial cells, such as epithelial‐to‐mesenchymal transition (EMT), is one of the early mechanisms of peritoneal fibrosis, which is mediated by oxidative stress and inflammation. Nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a multiprotein oligomer that promotes the maturation of IL‐lβ and IL‐18. Paricalcitol is reported to exert an antiinflammatory effect; however, there are no studies as to whether paricalcitol modulates the activation of NLRP3 inflammasome. We investigated the role of NLRP3 inflammasome in peritoneal EMT with an exploration of the effect of paricalcitol on oxidative stress, NLRP3 inflammasome, and EMT of mesothelial cells. TGF‐βl‐induced EMT in human peritoneal mesothelial cells (HPMCs) was associated with an up‐regulation of NLRP3, apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC), and ρrocasρase‐1, with an increased production of IL‐lβ and IL‐18, which was ameliorated by small interfering (si)NLRP3, siASC, caspase inhibitors, or neutralizing antibodies for IL‐lβ and IL‐18. TGF‐βl enhanced reactive oxygen species generation with an increase in NADPH oxidase (NOX) activity and mitochondrial NOX4 production. Paricalcitol alleviated TGF‐βl‐induced EMT and the NLRP3 inflammasome, which was associated with a down‐regulation of NOX activity by interfering with p47phox and p22phox interaction and mitochondrial NOX4 production in HPMCs. Taken together, paricalcitol ameliorated EMT of HPMCs via modulating an NOX‐dependent increase in the activity of NLRP3 inflammasome. Paricalcitol could be a novel approach to protect the peritoneum from the development of EMT and peritoneal fibrosis.—Ko, J., Kang, H.‐J., Kim, D.‐A., Ryu, E.‐S., Yu, M., Lee, H., Lee, H. K., Ryu, H.‐M., Park, S.‐H., Kim, Y.‐L., Kang, D.‐H. Paricalcitol attenuates TGF‐βl–induced phenotype transition of human peritoneal mesothelial cells (HPMCs) via modulation of oxidative stress and NLRP3 inflammasome. FASEB J. 33, 3035–3050 (2019). http://www.fasebj.org

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