Co-immunostaining of ICAM-1, ICAM-2, and CD31 in Mouse Kidney Glomeruli

Glomerulonephritis (GN) is a common pathological condition in chronic kidney diseases that often leads to end stage renal failure. Mac-1 (CD11b/CD18)-mediated neutrophil, macrophage, and dendritic cell glomerular infiltration leading to cellular dysfunction and destruction is an important disease mechanism. The cellular distribution and dynamics of the expression of Mac-1 ligands ICAM-1 and ICAM-2 in GN have not been well studied because of the difficulties in tissue staining and colocalizing glomerular cells with surface antigens. To improve the visualization of cell surface marker and antigen expression in kidney compartments, we have devised an even but mild fixation procedure employing p-formaldehyde-lysine-periodate (PLP) perfusion. A large panel of antibodies (Ab) against cell surface markers was used to identify kidney cell types and adhesion molecules. When confocal microscopy was used in visualizing glomerular adhesion molecule staining, the endothelial cells were found to specifically express CD31, and these cells express ICAM-2 constitutively. Though ICAM-1 was not expressed by glomerular endothelial cells in homeostasis, it was highly upregulated in mice with chronic GN and severe proteinuria. VCAM-1, a ligand for VLA-4 important in leukocyte migration, was not expressed in the glomerulus. The results highlight the importance of ICAM-1 in the infiltration of macrophages and dendritic cells in cGN. This report will provide a widely applicable procedure for yielding high quality confocal images and for the identification and quantitation of receptors and other cellular antigens expressed in different kidney compartments and cell types.