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Reactive nitrogen species generation in a new in‐vitro model of sepsis‐induced renal epithelial injury
Author(s) -
Pathak Elina,
Mayeux Philip R
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.968.3
Subject(s) - sepsis , acute kidney injury , pharmacology , in vitro , lactate dehydrogenase , medicine , cytotoxicity , kidney , chemistry , immunology , biochemistry , enzyme
Our studies in a murine model of cecal ligation and puncture (CLP)‐induced sepsis show that iNOS‐derived reactive nitrogen species (RNS) generated by the tubular epithelium play a major role in acute kidney injury (AKI) associated with sepsis. Unfortunately, sepsis‐induced AKI is a complex disease and difficult to model in vitro . To address the need to study the mechanism of tubular oxidant generation and injury, we developed a new, more relevant in vitro model where mIMCD‐3 (murine tubular epithelial cells) are treated with media containing 5% serum collected from mice at 4h after induction of sepsis (CLP) or Sham (no sepsis). After exposure to Sham or CLP serum, induction of iNOS (by RT‐PCR) occurred and NO generation increased at 6h by 83% (P < .05 compared to Sham). This was accompanied by a 74% or 72% increase in RNS measured by oxidation of carboxy‐H2DCFDA or dihydrorhodamine 123, respectively, and a 30% increase in cytotoxicity (lactate dehydrogenase release) in cells treated with CLP serum (P < .05 compared to Sham). To analyze the source of the oxidants and injury, mIMCD‐3 cells were treated with iNOS inhibitor L‐N 6 ‐(1‐iminoethyl) lysine (L‐NIL). L‐NIL (1mM) completely blocked NO generation, RNS generation, and cytotoxicity (P < .05). Therefore, it appears this new cellular model is relevant for studying the mechanism of renal epithelial oxidant generation and injury induced by sepsis. Supported by NIH DK075991.