A polyclonal antibody was raised against a proteoglycan fraction purified from extracts of isolated rat renal glomeruli. The antibody was characterized by column chromatography and by NaDodSO4/PAGE analysis of immunoprecipitates and was shown to recognize specifically the core protein of a population of heparan sulfate proteoglycans (Mr 130,000) found in the glomerular basement membrane and in other renal basement membranes. The antibody was used to localize the core proteins of this population of heparan sulfate proteoglycans (HSPG) in the glomerulus by immunoelectron microscopic techniques. These HSPG were found to be concentrated in the lamina rarae interna and externa of the glomerular basement membrane (GBM) and precursor forms were detected in the endoplasmic reticulum and Golgi cisternae of glomerular visceral epithelial cells (podocytes).Heparan sulfate proteoglycans (HSPG) are now recognized to be widely distributed if not ubiquitous components of basement membranes (basal laminae) (1, 2). They were originally discovered in the glomerular basement membrane (GBM) (3, 4), where they represent important structural components required for the maintenance of its selective permeability properties (5, 6). Previous work from this laboratory (7) established that HSPG isolated from the rat GBM are relatively small (mean Mr 130,000) and possess approximately four heparan sulfate side chains (Mr 26,000). By cytochemical techniques the heparan sulfate side chains of the HSPG were found to be concentrated in regularly spaced 60-nm anionic sites in the inner and outermost layers (lamina rara interna and externa) of the GBM (3,8). The location of the core protein of these HSPG has not been resolved because to date no specific antibodies have been generated against HSPG from the GBM, and immunocytochemical studies utilizing antibodies raised against HSPG derived from other basement membrane sources have provided conflicting evidence on the localization of HSPG in the GBM (9, 10). HSPG from various sources, including different sources of basement membranes (11)(12)(13) (totaling 5 mCi) in 120-g rats over a 12-hr period (15).Isolation of Glomerular Proteoglycans. Glomeruli were isolated from the radiolabeled kidneys (7) and extracted in 10 vol of 4 M guanidine HCI (pH 6.0) containing 0.5% Chaps and protease inhibitors (1 mM PMSF/10 mM N-ethylmaleimide/10 mM EDTA/10 mM 6-aminohexanoic acid/5 mM benzamidine-HCI) for 16 hr at 4°C with shaking. The unextracted residue was pelleted by centrifugation (1000 x g for 10 min) and proteoglycans were purified according to Yanagishita and Hascall (16) by passing the clarified extract over a Sephadex G-50 column, elution with 8 M urea/50 mM sodium acetate/0.15 M NaCl, pH 6, followed by application of the excluded volume onto a DEAE-Sephacel column and elution with a continuous NaCl gradient (0. 15-1.5 M). Fractions corresponding to the peaks of radioactivity that eluted in the starting buffer (peak 1) and at 0.45-1.0 M NaCl (peak 2) were collected, dialyzed overnight aga...