Isolation and characterization of mouse glomerular basement membrane.
Glomerular basement membrane (GBM) antigens used for immunological studies have until now been isolated from human and rat glomeruli but not from mice. However, given the growing awareness of extracellular matrix species-specificity, the need for a purification method of mouse GBM exists. We now report the purification of GBM from isolated mouse glomeruli. 10 ml 0.01 M phosphate buffered saline (PBS) containing 1.25% Fe3O4 was perfused through the aortae of (C57BL10 x DBA/2)F1 mice over 1 min, kidneys were decapsulated and passed through a 0.075 mm mesh metal screen and collected. After pelletting and washing, the tube was placed against one pole of a magnet and the pelleted glomeruli were washed. This procedure was repeated three times. The glomerular suspension was examined by light microscopy, sonicated, and lyophilized. In suspension no free fragments of tubuli or Bowman's capsule were present as observed by light microscopy. Mouse GBM was prepared from the isolated glomeruli using a modification of earlier described methods for human and rat GBM by enzymatic digestion. Elisa studies showed the presence of laminin, type IV collagen, and fibronectin. Monoclonal antibody directed against tubular epithelial antigen gp160 did not react to either mouse or rat GBM. Sera and glomerular eluates from (C57BL10 x DBA/2)F1 mice suffering from chronic graft-versus-host autoimmune disease gave a higher signal against mouse GBM than against rat GBM. Normal rabbit serum, normal mouse serum and normal mouse eluate did not show significant activity against mouse GBM. Immunoblotting showed the presence of both laminin B1 and B2 chains as well as type IV collagen alpha 1 and alpha 2 subunits. In summary, we describe a method by which it is possible to extract mouse GBM with a high purity.
Baelde
H J
HJ
Department of Pathology, University of Leiden, The Netherlands.
Bergijk
E C
EC
Bruijn
J A
JA
D000818:Animals; D000906:Antibodies / Q000032:analysis; D001485:Basement Membrane / Q000737:chemistry* / Q000378:metabolism; D003094:Collagen / Q000032:analysis / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000032:analysis / Q000276:immunology; D015151:Immunoblotting; D007678:Kidney Glomerulus / Q000737:chemistry* / Q000378:metabolism; D007797:Laminin / Q000032:analysis / Q000276:immunology; D008722:Methods; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D003012:Microbial Collagenase / Q000378:metabolism
D000906:Antibodies; D005353:Fibronectins; D007797:Laminin; D003094:Collagen; D003012:Microbial Collagenase