bay-11-7082 and diphenyleneiodonium

Macrophage-mediated inflammation has been implicated in various kidney diseases. We previously reported that Rac1, a Rho family small GTP-binding protein, was overactivated in several chronic kidney disease models, and that Rac1 inhibitors ameliorated renal injury, in part via inhibition of inflammation, but the detailed mechanisms have not been clarified. In the present study, we examined whether Rac1 in macrophages effects cytokine production and the inflammatory mechanisms contributing to kidney derangement. Myeloid-selective Rac1 flox control (M-Rac1 FC) and knockout (M-Rac1 KO) mice were generated using the cre-loxP system. Renal function under basal conditions did not differ between M-Rac1 FC and KO mice. Accordingly, lipopolysaccharide (LPS)-evoked kidney injury model was created. LPS elevated blood urea nitrogen and serum creatinine, enhanced expressions of kidney injury biomarkers, Kim-1 and Ngal, and promoted tubular injury in M-Rac1 FC mice. By contrast, deletion of myeloid Rac1 almost completely prevented the LPS-mediated renal impairment. LPS triggered a marked induction of macrophage-derived inflammatory cytokines, IL-6 and TNFα, in M-Rac1 FC mice, which was accompanied by Rac1 activation, stimulation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and reactive oxygen species overproduction. These changes were inhibited in M-Rac1 KO mice. LPS evoked F4/80-positive macrophages accumulation in the kidney, which was not affected by myeloid Rac1 deficiency. We further tested the role of Rac1 signaling in cytokine production using macrophage cell line, RAW264.7. Exposure to LPS increased IL-6 and TNFα mRNA expression. The LPS-driven cytokine induction was dose-dependently blocked by the Rac1 inhibitor EHT1864, NADPH oxidase inhibitor diphenyleneiodonium, and NF-κB inhibitor BAY11-7082. In conclusion, genetic ablation of Rac1 in the myeloid lineage protected against LPS-induced renal inflammation and injury, by suppressing macrophage-derived cytokines, IL-6 and TNFα, without blocking recruitment. Our data suggest that Rac1 in macrophage is a novel target for the treatment of kidney disease through inhibition of cytokine production.

Topics: Animals; Biomarkers; Cell Line; Cell Lineage; Chemokine CCL2; Chemokine CXCL1; Chemokine CXCL2; Culture Media, Conditioned; Cytokines; Gene Deletion; Gene Expression Regulation; Inflammation; Interleukin-6; Kidney; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Knockout; Monocytes; Myeloid Cells; NADPH Oxidases; Neuropeptides; Nitriles; Onium Compounds; Pyrones; Quinolines; rac1 GTP-Binding Protein; Reactive Oxygen Species; Sulfones; Tumor Necrosis Factor-alpha

Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotide (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative IκB-α plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-κB, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-κB pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages.

Topics: Acetylcysteine; Animals; Cell Line, Tumor; Ethanol; Female; I-kappa B Proteins; Macrophages; Matrix Metalloproteinase 12; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; NADPH Oxidase 2; NADPH Oxidases; NF-kappa B; NF-KappaB Inhibitor alpha; Nitriles; Onium Compounds; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Sulfones