Deletion of Rac1GTPase in the Myeloid Lineage Protects against Inflammation-Mediated Kidney Injury in Mice

Abstract

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.

Fig 1. Validation of myeloid-specific Rac1 knockout mice.

(A and B) Immunoblotting (A) and qPCR (B) analyses of Rac1 in the bone marrow-derived macrophages (BMDM) from myeloid-selective Rac1 knockout (M-Rac1 KO) and flox control (M-Rac1 FC) mice. β actin was used as a loading control. Data are means ± s.e.m. Statistical analysis was performed using unpaired t-test. **P < 0.01. n = 3 per each group. (C) Expression of cre mRNA in the BMDM and kidneys from M-Rac1 FC and KO mice. Statistical analysis was performed by two-way ANOVA, P < 0.01 genotype effect, P < 0.01 organ effect, P < 0.01 interaction effect. **P < 0.01 by Bonferroni's post hoc test. n = 3 per each group.

Fig 2. Lipopolysaccharide (LPS)-evoked renal functional and histological changes in M-Rac1 FC and KO mice.

Vehicle or LPS (5 mg/kg) was injected intraperitoneally in M-Rac1 FC and KO mice, and animals were sacrificed 48 h later. (A and B) Blood urea nitrogen (BUN) and serum creatinine. Data are expressed as means ± s.e.m. Statistical analysis was performed by two-way ANOVA, P < 0.01 genotype effect, P < 0.01 treatment effect, P < 0.01 interaction effect. **P < 0.01 by Bonferroni's post hoc test. n = 8 per each group. (C) The mRNA expression of kidney injury biomarkers, Kim-1 and Ngal, in the kidney homogenates of Vehicle- or LPS-injected M-Rac1 FC and KO mice. The mRNA levels were compared using real-time quantitative RT-PCR and expressed relative to M-Rac1 FC Vehicle group. Statistical analysis was performed by two-way ANOVA, P < 0.01 genotype effect, P < 0.01 treatment effect, P < 0.01 interaction effect. **P < 0.01 by Bonferroni's post hoc test. n = 8 per each group. (D) Typical images of hematoxylin-eosin stained kidney sections. Original magnification x 400. (E) Semiquantitative analysis of tubular injury. Data were analyzed using nonparametric analysis with Kruskal-Wallis test. **P < 0.01 by Mann-Whitney U test. n = 5 per each group.

Fig 3. Effects of LPS injection on Rac1 activity, macrophage-related cytokine mRNA expression, NADPH oxidase activity, and ROS production in M-Rac1 FC and KO mice.

(A) Expression of GTP-bound active Rac1 in the kidneys from Vehicle- or LPS-injected M-Rac1 FC and KO mice, as evaluated by GST pull-down assay. Left panel, representative blots. Right panel, result of densitometric analysis. Data are means ± s.e.m. Statistical analysis was performed by two-way ANOVA, P < 0.01 genotype effect, P < 0.01 treatment effect, P < 0.01 interaction effect. **P < 0.01 by Bonferroni's post hoc test. n = 3 per each group. (B) The mRNA levels of macrophage M1 cytokines, IL-6 and TNFα, in the kidney homogenates. Statistical analysis was performed by two-way ANOVA, P < 0.01 genotype effect, P < 0.01 treatment effect, P < 0.01 interaction effect. **P < 0.01 by Bonferroni's post hoc test. n = 8 per each group. (C) NADPH oxidase activity in the kidney was expressed as ROS generation in the presence of NADPH by the lucigenin chemiluminescence method. Statistical analysis was performed by two-way ANOVA, P < 0.01 genotype effect, P < 0.01 treatment effect, P < 0.01 interaction effect. **P < 0.01 by Bonferroni's post hoc test. n = 5 per each group. (D) Representative immunoblot of p47phox in the membrane fraction of the kidney. AIF was used as a loading control. (E) ROS in the kidney homogenates were measured as malonyldialdehyde using TBARS Assay Kit. Statistical analysis was performed by two-way ANOVA, P < 0.01 genotype effect, P < 0.05 treatment effect, P < 0.01 interaction effect. **P < 0.01 by Bonferroni's post hoc test. n = 4 per each group. (F) Expression of cre mRNA in the kidneys from Vehicle- or LPS-injected M-Rac1 FC and KO mice. Statistical analysis was performed by two-way ANOVA, P < 0.01 genotype effect, P < 0.01 treatment effect, P < 0.01 interaction effect. **P < 0.01 by Bonferroni's post hoc test. n = 5 per each group.

Fig 4. Immunostainings of macrophages and neutrophils in the kidneys of M-Rac1 FC and KO mice.

(A) Representative micrographs of F4/80 immunostaining in the kidneys of Vehicle- or LPS-injected M-Rac1 FC and KO mice. Original magnification x 200. (B) Quantitative analysis of F4/80-positive macrophages. Data are means ± s.e.m. Statistical analysis was performed by two-way ANOVA, n.s. genotype effect, P < 0.01 treatment effect, n.s. interaction effect. **P < 0.01 by Bonferroni's post hoc test. n = 5 per each group. (C) Representative micrographs of Ly-6B.2 (mAb 7/4) immunostaining. (D) Quantitative analysis of Ly-6B.2-positive neutrophils. Statistical analysis was performed by two-way ANOVA, n.s. genotype effect, P < 0.01 treatment effect, n.s. interaction effect. **P < 0.01 by Bonferroni's post hoc test. n = 6 per group.

Fig 5. LPS-triggered cytokine mRNA induction and its underlying mechanisms in cultured macrophages.

(A) Dose dependency of IL-6 and TNFα mRNA induction in response to LPS (1, 3, 10, 100, and 1000 ng/ml) for 3 h in the cultured macrophage cell line RAW264.7. Data are means ± s.e.m. Statistical analysis was performed by one-way ANOVA. **P < 0.01 vs. Vehicle. n = 4 per each group. (B) Effects of pretreatment with the Rac1 inhibitor EHT1864 (10, 50, and 100 μM) on LPS (100 ng/ml)-evoked mRNA induction of IL-6 and TNFα. Statistical analysis was performed by one-way ANOVA. ^##P < 0.01 vs. Vehicle; **P < 0.01 vs. LPS without inhibitors. n = 4 per group. (C) The mRNA expression of NADPH oxidase components, NOX1, NOX2, NOX4, p22phox, p47phox, and p67phox, in cultured macrophage RAW264.7. (D) Effects of preincubation with the NADPH oxidase inhibitor diphenyleneiodonium (DPI: 2.5, 10, and 25 μM). Statistical analysis was performed by one-way ANOVA. ^##P < 0.01 vs. Vehicle; **P < 0.01 vs. LPS without inhibitors. n = 4 per group. (E) Representative immunoblots of NF-κB p65 in the nuclear fraction. Histone H3 was used as a loading control. (F) Effects of preincubation with the NF-κB inhibitor BAY11-7082 (2, 5, and 20 μM). Statistical analysis was performed by one-way ANOVA. ^##P < 0.01 vs. Vehicle; **P < 0.01 vs. LPS without inhibitors. n = 4 per group.