IL-18 is a proinflammatory cytokine produced by macrophages and other cell types present in the kidney during ischemia-reperfusion injury (IRI), but its role in this injury is unknown. Here, compared with wild-type mice, IL-18^−/− mice subjected to kidney IRI demonstrated better kidney function, less tubular damage, reduced accumulation of neutrophils and macrophages, and decreased expression of proinflammatory molecules that are downstream of IL-18. For determination of the relative contributions of leukocytes and parenchymal cells to IL-18 production and subsequent kidney damage during IRI, bone marrow–chimeric mice were generated. Wild-type mice engrafted with IL-18^−/− hemopoietic cells showed less kidney dysfunction and tubular damage than IL-18^−/− mice engrafted with wild-type bone marrow. In vitro, macrophages produced IL-18 mRNA and protein in response to ischemia. These data suggest bone marrow–derived cells are the key contributors to IL-18–mediated effects of renal IRI. Finally, similar to IL-18^−/− mice, pretreatment of wild-type mice with IL-18–binding protein was renoprotective in this model of IRI. In conclusion, IL-18, derived primarily from cells of bone marrow origin, contributes to the renal damage observed during IRI. IL-18–binding protein may have potential as a renoprotective therapy.

Extracellular and cell surface proteins are generally modified with N-linked glycans and glycopeptide enrichment is an attractive tool to analyze these proteins. The role of N-linked glycoproteins in cardiovascular disease, particularly ischemia and reperfusion injury, is poorly understood. Observation of glycopeptides by mass spectrometry is challenging due to the presence of abundant, nonglycosylated analytes, and robust methods for purification are essential. We employed digestion with multiple proteases to increase glycoproteome coverage coupled with parallel glycopeptide enrichments using hydrazide capture, titanium dioxide, and hydrophilic interaction liquid chromatography with and without an ion-pairing agent. Glycosylated peptides were treated with PNGase F and analyzed by liquid chromatography-MS/MS. This allowed the identification of 1556 nonredundant N-linked glycosylation sites, representing 972 protein groups from ex vivo rat left ventricular myocardium. False positive “glycosylations” were observed on 44 peptides containing a deamidated Asn-Asp in the N-linked sequon by analysis of samples without PNGase F treatment. We used quantitation via isobaric tags for relative and absolute quantitation (iTRAQ) and validation with dimethyl labeling to analyze changes in glycoproteins from tissue following prolonged ischemia and reperfusion (40 mins ischemia and 20 mins reperfusion) indicative of myocardial infarction. The iTRAQ approach revealed 80 of 437 glycopeptides with altered abundance, while dimethyl labeling confirmed 46 of these and revealed an additional 62 significant changes. These were mainly from predicted extracellular matrix and basement membrane proteins that are implicated in cardiac remodeling. Analysis of N-glycans released from myocardial proteins suggest that the observed changes were not due to significant alterations in N-glycan structures. Altered proteins included the collagen-laminin-integrin complexes and collagen assembly enzymes, cadherins, mast cell proteases, proliferation-associated secreted protein acidic and rich in cysteine, and microfibril-associated proteins. The data suggest that cardiac remodeling is initiated earlier during reperfusion than previously hypothesized.

Phalloidin was found to block nucleotide exchange in F‐actin, without interfering with nucleotide hydrolysis. This inhibition of nucleotide exchange occurs under conditions in which monomers are able to exchange. The distance separating a fluorescent chromophore attached to phalloidin from the nucleotide on actin was determined using fluorescence resonance energy‐transfer spectroscopy. They are separated by less than 1.0 nm. Added confirmation of the close proximity of phalloidin to nucleotide was obtained by extracting a small peptide‐ATP complex from an actin digest. The peptide comprises residues 114–118, which are from the same region as the residues that others have shown to crosslink to phalloidin [Vandekerckhove et al. (1985) EMBO J. 4, 2815–2818]. The results suggest that phalloidin has two major effects. It traps actin monomers in a conformation which appears to be distinct from G‐actin and it stabilizes the structure of F‐actin, an event accompanied by the trapping of ADP.