ISOLATION AND CHARACTERIZATION OF INFLAMMATORY LEUKOCYTES FROM GLOMERULI IN AN INSITU MODEL OF GLOMERULONEPHRITIS IN THE RAT

Abstract

Inflammatory cell populations in glomerulonephritis (GN) are not well characterized. A method is reported for isolating leukocytes from glomeruli. GN was induced in rats by perfusing left kidneys (LKs) with cationized human IgG followed by intravenous rat anti-human IgG serum. Actue GN developed in LKs with proteinuria, deposition of human and rat IgG and C3, leukocyte infiltration, and capillary wall electron-dense deposits. Glomeruli (GL) isolated at 24 hours were digested with colagenase, trypsin, and DNase, and the resulting cells were as follows (mean .+-. SEM): LK, 354 .+-. 25/GL, RK, 214 .+-. 32/GL. Cells were labeled with monoclonal antibody MRCOX1 (anti-rat leukocyte common [LC] antigen) followed by FITC F(ab'')2 rabbit anti-mouse Ig: LK, 170 .+-. 11 leukocytes/GL; RK, 8 .+-. 2 leukocytes/GL (P < 0.001). Isolated cells were sorted by flow cytometry to 98% pure LC+ cells with > 80% viability (Giemsa staining: 86% mononuclear cells, 14% neutrophils); the ultrastructure was that of maturing macrophages and neutrophils. This method quantitates leukocyte infilatration and provides leukocytes from nephritic glomeruli suitable forin vitro studies.