nox2-deficient (nox2 ko, h-2 b ; b6.129s- cybb tm1din /j, stock no. 002365) mice  (Jackson Laboratory)

Journal: Frontiers in Medicine

Article Title: Deletion of NADPH oxidase 2 attenuates cisplatin-induced acute kidney injury through reducing ROS-induced proximal tubular cell injury and inflammation

doi: 10.3389/fmed.2023.1097671

Figure Lengend Snippet: Increased NOX2 expression in cisplatin-induced AKI. Three days after cisplatin injection, western blot analysis was used to examine the main NOX family expression in cisplatin-induced AKI. (A) Western blots of NOX1, NOX2, and NOX4. (B–D) Quantitative analysis of NOX2 indicated overexpression compared to WT mice. However, NOX1 and NOX4 decreased after cisplatin injury. (E) Immunofluorescence staining suggests NOX2 expression in the epithelial cells along the renal proximal tubule in cisplatin-induced AKI. NOX2-KO mice did not show NOX2 expression in the normal saline and cisplatin groups. Data represent the mean ± standard error of four mice per group. Scale bar = 50 μM. WT-NS, wild type-normal saline; WT-CDDP, wild type-cisplatin; AQP1: aquaporin 1.

Article Snippet: Mice deficient in Cybb, the gene encoding the gp91phox subunit of NOX2, (B6.129S-Cybbtm1Din/J, No. 002365) were purchased from Jackson Laboratory.

Techniques: Expressing, Injection, Western Blot, Over Expression, Immunofluorescence, Staining, Saline

Journal: Frontiers in Medicine

Article Title: Deletion of NADPH oxidase 2 attenuates cisplatin-induced acute kidney injury through reducing ROS-induced proximal tubular cell injury and inflammation

doi: 10.3389/fmed.2023.1097671

Figure Lengend Snippet: NOX2-KO mice markedly attenuate the cisplatin-induced ROS increase in WT mice. The glutathione disulfide/glutathione ratio, an indicator of oxidative stress, was increased 3 days after cisplatin injection in the WT group. In contrast, ROS were a significantly blunt in the NOX2-KO CDDP group compared with the WT-CDDP group. Data represent the mean ± standard error of five mice per group. The experiment was repeated twice with similar results.

Article Snippet: Mice deficient in Cybb, the gene encoding the gp91phox subunit of NOX2, (B6.129S-Cybbtm1Din/J, No. 002365) were purchased from Jackson Laboratory.

Techniques: Injection

Journal: Frontiers in Medicine

Article Title: Deletion of NADPH oxidase 2 attenuates cisplatin-induced acute kidney injury through reducing ROS-induced proximal tubular cell injury and inflammation

doi: 10.3389/fmed.2023.1097671

Figure Lengend Snippet: Mice with NOX2 deletion had milder cisplatin-induced AKI. (A) Serum creatinine levels indicated that NOX2-KO suppressed the cisplatin-mediated decrease in renal function. (B) BUN levels had a lower trend in NOX2 KO-CDDP mice compared with WT CDDP mice. (C,D) PAS staining of renal tubules revealed cisplatin-induced cast formation, tubular dilatation (black arrow), and tubular epithelial cell injury in WT CDDP mice. Quantitative analysis of PAS staining indicated NOX2 deletion suppressed the tubular injury score 3 days after cisplatin injection. Data represent the mean ± standard error of five mice per group. Scale bar = 100 μM.

Article Snippet: Mice deficient in Cybb, the gene encoding the gp91phox subunit of NOX2, (B6.129S-Cybbtm1Din/J, No. 002365) were purchased from Jackson Laboratory.

Techniques: Staining, Injection

Journal: Frontiers in Medicine

Article Title: Deletion of NADPH oxidase 2 attenuates cisplatin-induced acute kidney injury through reducing ROS-induced proximal tubular cell injury and inflammation

doi: 10.3389/fmed.2023.1097671

Figure Lengend Snippet: Mice with NOX2 deletion had decreased Kim-1 levels in cisplatin-induced AKI. (A,B) Kim-1, a biomarker of renal tubular injury, was overexpressed and accumulated in the WT CDDP group’s peritubular area (black arrow). Immunohistochemical analysis indicated that the area of Kim-1 was greater after cisplatin and reduced in the NOX2 KO mice. (C) NOX2 deletion reduced Kim-1 protein levels in cisplatin-induced AKI, as indicated by the Luminex assay. Data represent the mean ± standard error of five mice per group. Scale bar = 100 μM. The experiment was repeated twice with similar results.

Article Snippet: Mice deficient in Cybb, the gene encoding the gp91phox subunit of NOX2, (B6.129S-Cybbtm1Din/J, No. 002365) were purchased from Jackson Laboratory.

Techniques: Biomarker Discovery, Immunohistochemical staining, Luminex

Journal: Frontiers in Medicine

Article Title: Deletion of NADPH oxidase 2 attenuates cisplatin-induced acute kidney injury through reducing ROS-induced proximal tubular cell injury and inflammation

doi: 10.3389/fmed.2023.1097671

Figure Lengend Snippet: Cytokine expression with Luminex assay in cisplatin-induced AKI in the WT and NOX2-KO groups. (A,B) IL-6 and IL-1α proinflammatory factors were significantly reduced in the NOX2 KO-CDDP group compared to the WT-CDDP group. (C–G) MCP-1 and T-helper lymphocyte cytokines such as IFN-γ, IL-4, IL-5, and IL-17A did not show significant changes between the WT-CDDP and NOX2 KO-CDDP groups. (H) IL-10 was significantly reduced in the WT-CDDP group compared with the WT-NS group. IL-10 was mildly recovered in the NOX2 KO-CDDP group compared with the WT-CDDP group. Data represent the mean ± standard error of five mice per group. The experiment was repeated twice with similar results.

Article Snippet: Mice deficient in Cybb, the gene encoding the gp91phox subunit of NOX2, (B6.129S-Cybbtm1Din/J, No. 002365) were purchased from Jackson Laboratory.

Techniques: Expressing, Luminex

Journal: Frontiers in Medicine

Article Title: Deletion of NADPH oxidase 2 attenuates cisplatin-induced acute kidney injury through reducing ROS-induced proximal tubular cell injury and inflammation

doi: 10.3389/fmed.2023.1097671

Figure Lengend Snippet: Mice with NOX2 deletion had reduced neutrophil infiltration in cisplatin-induced AKI. (A) Whole kidneys were homogenized into a single-cell suspension for flow cytometric analysis of immune cells. Hierarchical gating was performed to identify Ly6G + , CD11b + neutrophils; F4/80 + , CD11C − macrophages; and F4/80 − , CD11c + dendritic cells. (B,D,E) Neutrophil infiltration was increased on day 3 after cisplatin injection in both WT and NOX2 KO groups. However, the neutrophil percentage of total CD45 + cells was significantly reduced in the NOX2-KO CDDP group on day 3 compared to that of the WT CDDP group. (C,F–I) The percentages of macrophage and dendritic cells among total CD45 + cells were significantly reduced in the WT CDDP group on day 3 compared to the WT on day 0. However, the numbers and percentages of total CD45 + macrophage and dendritic cells were not significantly different between the WT CDDP and NOX2-KO CDDP groups on day 3. Data represent the mean ± standard error of 3–5 mice per group.

Article Snippet: Mice deficient in Cybb, the gene encoding the gp91phox subunit of NOX2, (B6.129S-Cybbtm1Din/J, No. 002365) were purchased from Jackson Laboratory.

Techniques: Suspension, Injection

Journal: Frontiers in Medicine

Article Title: Deletion of NADPH oxidase 2 attenuates cisplatin-induced acute kidney injury through reducing ROS-induced proximal tubular cell injury and inflammation

doi: 10.3389/fmed.2023.1097671

Figure Lengend Snippet: Cisplatin-induced increase in ICAM-1 and CXCL1 was reduced in NOX2-KO mice. (A,G,H) sICAM expression with Luminex assay was decreased in the NOX2-KO CDDP group compared to the WT CDDP group on day 3, and the area of ICAM-1 labeling (black arrow) through immunohistochemical staining was increased in WT-CDDP but not in NOX2 KO-CDDP mice. (B,C,F) sP-selectin, VEGF, and GM-CSF levels with Luminex assay were not significantly different between the WT CDDP and NOX2-KO CDDP groups on day 3. (D,E) The level of chemokine CXCL1 was significantly lower in the NOX2-KO CDDP group on day 3. However, another chemokine, CXCL2, showed no change between the WT CDDP and NOX2-KO groups. Data represent the mean ± standard error of five mice per group. Scale bar = 100 μM. The experiment was repeated twice with similar results.

Article Snippet: Mice deficient in Cybb, the gene encoding the gp91phox subunit of NOX2, (B6.129S-Cybbtm1Din/J, No. 002365) were purchased from Jackson Laboratory.

Techniques: Expressing, Luminex, Labeling, Immunohistochemical staining, Staining

Journal: Frontiers in Medicine

Article Title: Deletion of NADPH oxidase 2 attenuates cisplatin-induced acute kidney injury through reducing ROS-induced proximal tubular cell injury and inflammation

doi: 10.3389/fmed.2023.1097671

Figure Lengend Snippet: Schematic summarizing the mechanism of NOX2-induced ROS in the renal proximal tubular cells. NOX2 contributes to cisplatin-induced AKI and involves an increase Kim-1 and proinflammatory cytokines, IL6 and IL-1α which also increase neutrophil infiltration with higher endothelial adhesion molecule ICAM-1 and chemokine CXCL1. The box in the figure is NOX2 which has multiple membrane-bound subunits of NADPH oxidase including gp91 phox , p22 phox , p67 phox , p47 phox , and Rac.

Article Snippet: Mice deficient in Cybb, the gene encoding the gp91phox subunit of NOX2, (B6.129S-Cybbtm1Din/J, No. 002365) were purchased from Jackson Laboratory.

Techniques: Membrane

Journal: Antioxidants

Article Title: Early Cytokine-Induced Transient NOX2 Activity Is ER Stress-Dependent and Impacts β-Cell Function and Survival

doi: 10.3390/antiox10081305

Figure Lengend Snippet: ROS production in pancreatic islets exposed to proinflammatory cytokines. ( A ) Superoxide production from wild-type islets in the absence (Ctrl) or presence of proinflammatory cytokines after different periods of exposure, as indicated. The fluorescence intensity was analyzed by flow cytometry using DHE dye. The results are expressed as the mean ± SD of 3–6 independent experiments. A.U.: arbitrary units. ( B ) Dynamic levels of the cytosolic/nuclear H 2 O 2 of islets from roGFP2-Orp1 mice incubated with proinflammatory cytokines. ( C ) Peak of the normalized ratio of ( B ) at 4 h. ( D ) Dynamic levels of mitochondrial H 2 O 2 of the islets from Mt-roGFP2-Orp1 mice incubated with proinflammatory cytokines. ( E ) Peak of the normalized ratio of ( D ) at 4 h. The fluorescence intensity was captured every 10 min for 22 h. The results are presented as the normalized ratio (405/488 nm) and mean ± SD of triplicates from 3 to 4 independent experiments. ( F , G ) Dynamic levels of NAD(P)H of the islets from roGFP2-Orp1 ( F ) and Mt-roGFP2-Orp1 ( G ) mice incubated with proinflammatory cytokines. The results are presented as the mean ± SD of triplicates from 3 to 4 independent experiments. * p < 0.05 and *** p < 0.001 when compared to the control using One-Way ANOVA + Dunnett’s ( A ) or a Student’s t -test ( C ). Proinflammatory cytokines mix: 10-U/mL IL-1β + 100-U/mL TNF + 14-U/mL IFN-γ.

Article Snippet: C57BL/6J wild-type (WT), NOX1 and NOX2-deficient (KO) mice were purchased from Jackson Laboratory (Bar Harbor, ME, EUA).

Techniques: Fluorescence, Flow Cytometry, Incubation, Control

Journal: Antioxidants

Article Title: Early Cytokine-Induced Transient NOX2 Activity Is ER Stress-Dependent and Impacts β-Cell Function and Survival

doi: 10.3390/antiox10081305

Figure Lengend Snippet: The role of NOX2 in the ROS production of pancreatic islets exposed to proinflammatory cytokines. ( A ) Superoxide production from wild-type (WT), NOX1 KO (NOX1) or NOX2 KO (NOX2) islets in the absence (Ctrl) or presence of proinflammatory cytokines after different periods of exposure, as indicated. The fluorescence intensity was analyzed by flow cytometry using DHE dye. The results are presented as the mean ± SD of 6–14 independent experiments. A.U.: arbitrary units. ( B ) Dynamic levels of cytosolic/nuclear H 2 O 2 of the islets from roGFP2-Orp1 mice incubated with proinflammatory cytokines in the absence or presence of 20 µM of the NOX2 inhibitor GSK2795039 (GSK). The fluorescence intensity was captured every 10 min for 18 h. The results are presented as the normalized ratio (405/488 nm) and mean ± SD of 3 independent experiments. ( C , D ) NOX2 KO mice and roGFP2-Orp1 mice were crossbred in order to obtain NOX2KO:roGFP2-Orp1. C57BL/6J WT mice and roGFP2-Orp1 mice were crossbred in order to obtain proper heterozygous controls. The islets were incubated in the absence (Ctrl) or presence of proinflammatory cytokines for 4 h and 30 min. ( C ) Representative images of the islets from the control heterozygous (WT) and NOX2KO:roGFP2-Orp1 (NOX2) mice. The color temperatures reflect the normalized 405/488 ratio, from black/purple (non-oxidized sensor) to yellow/white (completely oxidized sensor), thus reflecting the H 2 O 2 levels. ( D ) H 2 O 2 levels represented as the normalized ratio (405/488). The results are expressed as the mean ± SD of individual islets from 6–9 independent experiments. ( A ) * p < 0.05 when compared to the Ctrl. One-Way ANOVA + Sidak. ( D ) **** p < 0.0001 versus the respective controls in same genotypes; #### p < 0.0001 versus the WT cytokines. One-Way ANOVA + Tukey’s. Proinflammatory cytokines mix: 10-U/mL IL-1β + 100-U/mL TNF + 14-U/mL IFN-γ.

Article Snippet: C57BL/6J wild-type (WT), NOX1 and NOX2-deficient (KO) mice were purchased from Jackson Laboratory (Bar Harbor, ME, EUA).

Techniques: Fluorescence, Flow Cytometry, Incubation, Control