Nox2 Is a Mediator of Ischemia Reperfusion Injury

Abstract

Delayed graft function (DGF) results from ischemia-reperfusion injury (IRI) and the generation of reactive oxygen species. We hypothesized that NADPH oxidase 2 (Nox2) plays an important role in pathways leading to DGF. We tested this hypothesis in vitro, in an animal model of IRI using wild type and Nox2−/− mice, and in patients with DGF. Under hypoxic conditions, primary tubular epithelial cells from Nox2−/− mice had reduced expression of MMP2, vimentin and HSP27. BUN and creatinine levels were significantly increased in both Nox2−/− and WT mice at 4 weeks and 6 months after IRI, suggesting the development of acute and chronic kidney injury. At 4 weeks, kidney fibrosis (α-SMA, picrosirius) and oxidative stress (dihydroethidine, HNE) were significantly reduced in Nox2−/− mice, confirming the oxidative and pro-fibrotic effects of Nox2. The molecular signature of IRI using genomic analyses demonstrated a significant decline in hypoxia reponse, oxidative stress, fibrosis, and inflammation in Nox2−/− mice. Immunohistochemical analyses of pre-implanatation kidney allograft biopsies from patients with subsequent DGF showed significantly greater Nox2 levels and vascular injury compared with patients without DGF. These studies demonstrate that Nox2 is a modulator of IRI and its absence is associated with reduced inflammation, OS, and fibrosis.

Nox2 and Cyclosporine-Induced Renal Hypoxia

“…Our findings are also consistent with previous reports demonstrating that Nox2 inhibition attenuates renal fibrosis resulting from ischemia reperfusion injury, chronic rejection, and CsA 25,28,35 . This time, we used noninvasive molecular imaging to demonstrate that chronic CsA was associated with hypoxia in the cortex and the medulla without a decline in corticomedullary perfusion.…”

“…Immunohistochemical studies were performed as described previously 25,28,35 . H&E, PAS, trichrome, picrosirius red, Nox2, HSF, and α-SMA staining was performed using the concentrations outlined in the section below.…”

“…Depending on the size of the stained section, either the entire section or 5 random non-overlapping images were taken for each section. Automated quantification was performed using a custom macro written for ImageJ software (NIH, imagej.nih.gov/ij/) as described previously 35 .…”

“…WT and Nox2−/− treated with vehicle or CsA for 8 weeks (n=4 in each group) were studied using the Affymetrix Mouse ST 2.0 microarray chips (Affymetrix, cat # 901169) and an Affymetrix GeneChip Analysis System. Mouse specific gene lists were generated from the rat genome database (rgd.mcw.edu) as described previously 35 . We identified gene group classifiers associated with injury-repair, metabolism, and OS-hypoxia.…”

“…Student’s T test and Mann-Whitney rank sum test were utilized as described previously 25,35 to compare parametric and non-parametric continuous data when appropriate. Similarly, Chi-square or Fisher’s exact tests were utilized when appropriate to compare categorical data between groups.…”