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.

Reactive oxygen species (ROS) generation during purine metabolism is associated with xanthine oxidase and uric acid. However, the direct effect of hypoxanthine on ROS generation and atherosclerosis has not been evaluated. Smoking and heavy drinking are associated with elevated levels of hypoxanthine. In this study, we investigated the role of hypoxanthine on cholesterol synthesis and atherosclerosis development, particularly in apolipoprotein E (APOE)‐deficient mice. The effect of hypoxanthine on the regulation of cholesterol synthesis and atherosclerosis were evaluated in Apoe knockout (KO) mice and cultured HepG2 cells. Hypoxanthine markedly increased serum cholesterol levels and the atherosclerotic plaque area in Apoe KO mice. In HepG2 cells, hypoxanthine increased intracellular ROS production. Hypoxanthine increased cholesterol accumulation and decreased APOE and ATP‐binding cassette transporter A1 (ABCA1) mRNA and protein expression in HepG2 cells. Furthermore, H2O2 also increased cholesterol accumulation and decreased APOE and ABCA1 expression. This effect was partially reversible by treatment with the antioxidant N‐acetyl cysteine and allopurinol. Hypoxanthine and APOE knockdown using APOE‐siRNA synergistically induced cholesterol accumulation and reduced APOE and ABCA1 expression. Hypoxanthine induces cholesterol accumulation in hepatic cells through alterations in enzymes that control lipid transport and induces atherosclerosis in APOE‐deficient cells and mice. These effects are partially mediated through ROS produced in response to hypoxanthine.

The protective effects of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) 1 inhibition against kidney ischemia-reperfusion injury (IRI) remain uncertain. The bilateral kidney pedicles of C57BL/6 mice were clamped for 30 min to induce IRI. Madin–Darby Canine Kidney (MDCK) cells were incubated with H2O2 (1.4 mM) for 1 h to induce oxidative stress. ML171, a selective NOX1 inhibitor, and siRNA against NOX1 were treated to inhibit NOX1. NOX expression, oxidative stress, apoptosis assay, and mitogen-activated protein kinase (MAPK) pathway were evaluated. The kidney function deteriorated and the production of reactive oxygen species (ROS), including intracellular H2O2 production, increased due to IRI, whereas IRI-mediated kidney dysfunction and ROS generation were significantly attenuated by ML171. H2O2 evoked the changes in oxidative stress enzymes such as SOD2 and GPX in MDCK cells, which was mitigated by ML171. Treatment with ML171 and transfection with siRNA against NOX1 decreased the upregulation of NOX1 and NOX4 induced by H2O2 in MDCK cells. ML171 decreased caspase-3 activity, the Bcl-2/Bax ratio, and TUNEL-positive tubule cells in IRI mice and H2O2-treated MDCK cells. Among the MAPK pathways, ML171 affected ERK signaling by ERK phosphorylation in kidney tissues and tubular cells. NOX1-selective inhibition attenuated kidney IRI via inhibition of ROS-mediated ERK signaling.

Although dipeptidyl peptidase-4 inhibitors, a class of antidiabetic drugs, have various pleiotropic effects, it remains undetermined whether gemigliptin has a beneficial effect on vascular calcification. Therefore, this study was performed to evaluate the effect of gemigliptin on vascular calcification in a rat model of adenine-induced chronic kidney disease and in cultured vascular smooth muscle cells. Gemigliptin attenuated calcification of abdominal aorta and expression of RUNX2 in adenine-induced chronic kidney disease rats. In cultured vascular smooth muscle cells, phosphate-induced increase in calcium content was reduced by gemigliptin. Gemigliptin reduced phosphate-induced PiT-1 mRNA expression, reactive oxygen species generation, and NADPH oxidase mRNA expression (p22phox and NOX4). The reduction of oxidative stress by gemigliptin was associated with the downregulation of phospho-PI3K/AKT expression. High phosphate increased the expression of frizzled-3 (FDZ3) and decreased the expression of dickkopf-related protein-1 (DKK-1) in the Wnt pathway. These changes were attenuated by gemigliptin treatment. Gemigliptin restored the decreased expression of vascular smooth muscle cells markers (α-SMA and SM22α) and increased expression of osteogenic makers (CBFA1, OSX, E11, and SOST) induced by phosphate. In conclusion, gemigliptin attenuated vascular calcification and osteogenic trans-differentiation in vascular smooth muscle cells via multiple steps including downregulation of PiT-1 expression and suppression of reactive oxygen species generation, phospho-PI3K/AKT, and the Wnt signaling pathway.