Experimental data suggesting that inflammation mediated rat liver mitochondrial dysfunction results from secondary hypoxia rather than from direct effects of inflammatory mediators

Abstract

Systemic inflammatory response (SIR) comprises both direct effects of inflammatory mediators (IM) and indirect effects, such as secondary circulatory failure which results in tissue hypoxia (HOX). These two key components, SIR and HOX, cause multiple organ failure (MOF). Since HOX and IM occur and interact simultaneously in vivo, it is difficult to clarify their individual pathological impact. To eliminate this interaction, precision cut liver slices (PCLS) were used in this study aiming to dissect the effects of HOX and IM on mitochondrial function, integrity of cellular membrane, and the expression of genes associated with inflammation. HOX was induced by incubating PCLS or rat liver mitochondria at pO2 < 1% followed by reoxygenation (HOX/ROX model). Inflammatory injury was stimulated by incubating PCLS with IM (IM model). We found upregulation of inducible nitric oxide synthase (iNOS) expression only in the IM model, while heme oxygenase 1 (HO-1) expression was upregulated only in the HOX/ROX model. Elevated expression of interleukin 6 (IL-6) was found in both models reflecting converging pathways regulating the expression of this gene. Both models caused damage to hepatocytes resulting in the release of alanine aminotransferase (ALT). The leakage of aspartate aminotransferase (AST) was observed only during the hypoxic phase in the HOX/ROX model. The ROX phase of HOX, but not IM, drastically impaired mitochondrial electron supply via complex I and II. Additional experiments performed with isolated mitochondria showed that free iron, released during HOX, is likely a key prerequisite of mitochondrial dysfunction induced during the ROX phase. Our data suggests that mitochondrial dysfunction, previously observed in in vivo SIR-models, is the result of secondary circulatory failure inducing HOX rather than the result of a direct interaction of IM with liver cells.

The cytoprotective effect of nitrite is based on the formation of dinitrosyl iron complexes

“…For efficient reoxygenation, the mitochondrial suspension was strongly diluted in incubation buffer equilibrated with air oxygen. The iron concentration is in line with our recently published data showing in an in vitro model in the liver that 1 h hypoxia results in the release of 20 mM ferrous ions [27]. The concentrations of ferrous ions and nitrite during hypoxia were much higher than those occurring/used for treatment in vivo (Supplemental Table 1), but reached these levels after dilution during reoxygenation (Supplemental Fig.…”

“…30 mmol/kg tissue [45]. The data of Weidinger et al, reporting 20 mmol/kg tissue, lie in a similar range [27]. We used a high nitrite concentration, because in our model only mitochondria reduced nitrite to NO, while in the tissue this process is accelerated by Fig.…”

“…5B), quantified as TBARS formation. A dose of 20 mM iron ions, which lies in the (patho)physiological range [27], induced significant but not total mitochondrial dysfunction and was therefore chosen for further experiments.…”

“…In fact, transition metal ions such as iron and copper ions execute the most toxic reactions, causing oxidative damage. Elevated levels of intracellular iron ions have already been associated with tissue damage at the mitochondrial level during reperfusion or reoxygenation [25], inflammation [26,27], and other conditions. Based on these data, we aimed to prove that the protective effect of nitrite upon hypoxia is based on a mechanism controlling ironmediated impairment of mitochondrial function.…”

Mitochondria: the cell powerhouse and nexus of stress

“…For example, it has been shown that mitochondria dysfunction plays also an important role in developing of inflammation. Experiments using rat liver slices and isolated mitochondria demonstrated that inflammation induced by inflammatory mediators resulted in mitochondrial dysfunction due to secondary hypoxia (Weidinger et al, 2013). This study revealed hypoxiainduced attenuation of complexes I and II function in response to inflammation.…”