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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-H(2)DCF-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-N(6)-(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.

Previous evidence suggests that both oxygen radicals and nitric oxide (NO) are important mediators of injury during renal ischemia-reperfusion (I-R) injury. However, the generation of reactive nitrogen species (RNS) has not been evaluated in this model at early time points. The purpose of these studies was to examine the development of oxidant stress and the formation of RNS during I-R injury. Male Sprague-Dawley rats were anesthetized and subjected to 40 min of bilateral renal ischemia followed by 0, 3, or 6 h of reperfusion. Control animals received a sham operation. Plasma urea nitrogen and creatinine levels were monitored as markers of renal injury. Glutathione (GSH) oxidation and 4-hydroxynonenal (4-HNE)-protein adducts were used as markers of oxidant stress. 3-Nitrotyrosine (3-NT) was used as a biomarker of RNS formation. Significant increases in plasma creatinine concentrations and urea nitrogen levels were found following both 3 and 6 h of reperfusion. Increases in GSH oxidation, 4-HNE-protein adduct levels, and 3-NT levels were observed following 40 min of ischemia with no reperfusion. Since these results suggested RNS generation during the 40 min of ischemia, a time course of RNS generation following 0, 5, 10, 20, and 40 min of ischemia was evaluated. Significant increases in 3-NT generation was detected as early as 10 min of ischemia and rose to values nearly 10-fold higher than Control at 40 min of ischemia. No additional increase was observed following reperfusion. The data clearly demonstrate that oxidative stress and RNS generation occur in the kidney during ischemia.

In chickens, oviposition is correlated with increased plasma levels of the neurohypophysial hormone vasotocin, and vasotocin stimulates contraction of uterine strips in vitro. A gene encoding a vasotocin receptor subtype that we have designated the VT1 receptor was cloned from the domestic chicken. The open reading frame encodes a 370-amino acid polypeptide that displays seven segments of hydrophobic amino acids, typical of guanine nucleotide-protein-coupled receptors. Other structural features of the VT1 receptor include two potential N-linked glycosylation sites in the extracellular N-terminal region, a conserved aspartic acid in transmembrane domain 2 that is found in nearly all guanine nucleotide-protein-coupled receptors, and two potential protein kinase C phosphorylation sites in the third intracellular loop and C-terminal tail. Expressed VT1 receptors in COS7 cells bind neurohypophysial hormones with the following rank order of potency: vasotocin ≅ vasopressin > oxytocin ≅ mesotocin > isot...