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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 (?-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.

SUBMITTER: Karim AS

PROVIDER: S-EPMC4636908 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Nox2 is a mediator of ischemia reperfusion injury.

Karim A S AS Reese S R SR Wilson N A NA Jacobson L M LM Zhong W W Djamali A A

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons 20150623 11

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 ...[more]

PMID: 26104383

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Project description:BackgroundInhibiting SDH (succinate dehydrogenase), with the competitive inhibitor malonate, has shown promise in ameliorating ischemia/reperfusion injury. However, key for translation to the clinic is understanding the mechanism of malonate entry into cells to enable inhibition of SDH, its mitochondrial target, as malonate itself poorly permeates cellular membranes. The possibility of malonate selectively entering the at-risk heart tissue on reperfusion, however, remains unexplored.MethodsC57BL/6J mice, C2C12 and H9c2 myoblasts, and HeLa cells were used to elucidate the mechanism of selective malonate uptake into the ischemic heart upon reperfusion. Cells were treated with malonate while varying pH or together with transport inhibitors. Mouse hearts were either perfused ex vivo (Langendorff) or subjected to in vivo left anterior descending coronary artery ligation as models of ischemia/reperfusion injury. Succinate and malonate levels were assessed by liquid chromatography-tandem mass spectrometry LC-MS/MS, in vivo by mass spectrometry imaging, and infarct size by TTC (2,3,5-triphenyl-2H-tetrazolium chloride) staining.ResultsMalonate was robustly protective against cardiac ischemia/reperfusion injury, but only if administered at reperfusion and not when infused before ischemia. The extent of malonate uptake into the heart was proportional to the duration of ischemia. Malonate entry into cardiomyocytes in vivo and in vitro was dramatically increased at the low pH (≈6.5) associated with ischemia. This increased uptake of malonate was blocked by selective inhibition of MCT1 (monocarboxylate transporter 1). Reperfusion of the ischemic heart region with malonate led to selective SDH inhibition in the at-risk region. Acid-formulation greatly enhances the cardioprotective potency of malonate.ConclusionsCardioprotection by malonate is dependent on its entry into cardiomyocytes. This is facilitated by the local decrease in pH that occurs during ischemia, leading to its selective uptake upon reperfusion into the at-risk tissue, via MCT1. Thus, malonate's preferential uptake in reperfused tissue means it is an at-risk tissue-selective drug that protects against cardiac ischemia/reperfusion injury.

Project description:Timely reperfusion after a myocardial infarction is necessary to salvage the ischemic region; however, reperfusion itself is also a major contributor to the final tissue damage. Currently, there is no clinically relevant therapy available to reduce ischemia-reperfusion injury (IRI). While many drugs have shown promise in reducing IRI in preclinical studies, none of these drugs have demonstrated benefit in large clinical trials. Part of this failure to translate therapies can be attributed to the reliance on small animal models for preclinical studies. While animal models encapsulate the complexity of the systemic in vivo environment, they do not fully recapitulate human cardiac physiology. Furthermore, it is difficult to uncouple the various interacting pathways in vivo. In contrast, in vitro models using isolated cardiomyocytes allow studies of the direct effect of therapeutics on cardiomyocytes. External factors can be controlled in simulated ischemia-reperfusion to allow for better understanding of the mechanisms that drive IRI. In addition, the availability of cardiomyocytes derived from human induced pluripotent stem cells (hIPS-CMs) offers the opportunity to recapitulate human physiology in vitro. Unfortunately, hIPS-CMs are relatively fetal in phenotype, and are more resistant to hypoxia than the mature cells. Tissue engineering platforms can promote cardiomyocyte maturation for a more predictive physiologic response. These platforms can further be improved upon to account for the heterogenous patient populations seen in the clinical settings and facilitate the translation of therapies. Thereby, the current preclinical studies can be further developed using currently available tools to achieve better predictive drug testing and understanding of IRI. In this article, we discuss the state of the art of in vitro modeling of IRI, propose the roles for tissue engineering in studying IRI and testing the new therapeutic modalities, and how the human tissue models can facilitate translation into the clinic.

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Nox2 is a mediator of ischemia reperfusion injury.

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Nox2 is a mediator of ischemia reperfusion injury.

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