In Vitro Model of Sepsis-Induced Renal Epithelial Reactive Nitrogen Species Generation

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Abstract
Sepsis-induced acute kidney injury (AKI) is a complex disease characterized by generation of inducible nitric oxide synthase (iNOS)–derived reactive nitrogen species (RNS) by the renal tubular epithelium. While most in vitro models of sepsis use combinations of lipopolysaccharide and cytokines to simulate exposure to inflammatory mediators thought to play a role in sepsis, the relevance of these models is limited. To address the need for a model that more closely mimics the tubular microenvironment during sepsis, we developed an in vitro model where mIMCD-3 (murine tubular epithelial) cells are treated with media containing 5% serum collected from mice at 4 h after cecal ligation and puncture (CLP) or sham surgery (no sepsis). After exposure to CLP serum, induction of iNOS messenger RNA occurred and NO generation was significantly increased compared to sham. This increase was accompanied by increased RNS as measured by oxidation of 5-(and-6)-carboxy-2,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCF-DA) and 2-(3,6-diamino-9H-xanthen-9-yl)-benzoic acid, methyl ester (dihydrorhodamine 123) and moderate cytotoxicity in cells treated with CLP serum, similar to what is observed in mice subjected to CLP. Since iNOS has been shown to play an important role in sepsis-induced AKI, the iNOS inhibitor L-N6-(1-iminoethyl)-lysine (L-NIL) was tested in this in vitro model. L-NIL completely blocked NO generation, RNS generation, and cytotoxicity, similar to its effects in vivo. Therefore, this new in vitro model exhibits many of the characteristics observed in vivo, suggesting that it is a relevant model for studying the mechanism of sepsis-induced renal epithelial RNS generation and injury.