Expression of iNOS, eNOS, and peroxynitrite-modified proteins in experimental anti-myeloperoxidase associated crescentic glomerulonephritis

Abstract

Nitric oxide radicals are recognized as important mediators in various physiological and pathophysiological processes. During inflammation, increased amounts of nitric oxide (NO) are produced, but it is unclear whether NO radicals are either protective or harmful. To obtain more insight into the role of NO in glomerular inflammation, we studied the temporal expression of endothelial NO synthase (eNOS) and inducible NOS (iNOS) in conjunction with platelet aggregation, inflammatory cell influx, superoxide anion production cells, and nitrotyrosine formation in an experimental model of anti-myeloperoxidase (MPO) associated necrotizing crescentic glomerulonephritis (NCGN). Brown Norway rats were immunized with MPO in complete Freund's adjuvant (CFA) or CFA alone. After two weeks, the left kidney was perfused with a neutrophil lysosomal extract and H2O2. Rats were sacrificed at 24 hours, four days, and 10 days after perfusion. Kidney sections were stained by immunohistochemistry for eNOS, iNOS, platelets, nitrotyrosines, polymorphonuclear cells (PMN), monocytes, and T-cells. Superoxide anion producing cells were identified by enzyme cytochemistry using diaminobenzidine. Strong staining for eNOS was found in glomerular capillaries and interstitial tubular capillaries and larger vessels from non-perfused kidneys. At 24 hours after perfusion, glomerular and interstitial eNOS staining was greatly reduced, which was associated with massive platelet aggregation. At later time points, eNOS expression was absent in severely damaged glomeruli. Inducible NOS expression was found at all time points in infiltrating inflammatory cells, which by double labeling studies were identified as PMNs and monocytes. The peak in iNOS expression was observed at four days after perfusion but declined thereafter. Superoxide anion and nitrotyrosine generating cells were also found at all time points, but were most abundantly present at four days after perfusion, coinciding with the peak in iNOS expression. Double labeling experiments revealed that most nitrotyrosine generating cells also produced superoxide anions and expressed iNOS. In conclusion, these studies suggest that during the course of anti-MPO associated NCGN, loss of NO production by eNOS in conjunction with NO radical production by iNOS contribute to tissue injury. This is compatible with a protective role for eNOS contrasting with the possibly harmful effects of iNOS in anti-MPO associated NCGN.