ORIGIN OF THE GLOMERULAR BASEMENT-MEMBRANE VISUALIZED AFTER INVIVO LABELING OF LAMININ IN NEWBORN RAT KIDNEYS

To examine the origin and assembly of glomerular basement membranes (GBM), affinity purified anti-laminin IgG was directly coupled to horseradish peroxidase (HRP) and i.v. injected into newborn rats. Kidneys were then processed for peroxidase histochemistry and microscopy. Within 1 h after injection, anti-laminin bound to basement membranes of nephrons in all developmental stages (vesicle, comma, S-shaped, developing capillary loop, and maturing glomeruli). In S-shaped and capillary loop glomeruli, anti-laminin-HRP labeled a double basal lamina between the endothelium and epithelium. Sections incubated with anti-laminin in vitro showed labeling within the rough endoplasmic reticulum of endothelium and epithelium, indicating that both cell types synthesized laminin for the double basement membrane. In maturing glomeruli, injected anti-laminin-HRP bound throughout the GBM, and double basement membranes were rarely observed. At this stage, however, numerous knobs or outpockets of basement membrane material extending far into the epithelial side of the capillary wall were identified and these were also labeled throughout their full thickenss. No such outpockets were found in the endothelial cell layer of newborn rats (and they normally are completely absent in fully mature, adult glomeruli). In kidneys fixed 4-6 d [days] after anti-laminin IgG-HRP injection, basement membranes of vesicle, comma and S-shaped nephrons were unlabeled, indicating that they were assembled after injection. GBM labeling was seen in maturing glomeruli, however. In addition, the outpockets of basement membrane extending into the epithelium were often completely unlabeled whereas GBMs lying immediately beneath them were labeled intensely, which indicates that the outpockets were probably assembled by the epithelium. Injections of sheep anti-laminin IgG followed 8 d later with injections of biotin-rabbit anti-laminin IgG and double-label immunofluorescence microscopy confirmed that GBM formation continued during individual capillary loop expansion. GBM assembly therefore occurs by at least 2 different processes at separate times in development: fusion of endothelial and epithelial basement membranes followed by addition of new basement membrane from the epithelium into existing GBM.