THE BIOSYNTHESIS OF SULFATED AND NON-SULFATED GLYCOSAMINOGLYCANS (GAG) AND GLYCOPEPTIDES BY RAT KIDNEY AND THEIR INCORPORATION INTO THE GLOMERULAR BASEMENT MEMBRANE (GBM)

The synthesis and incorporation of glycosaminoglycans (GAG) and glycopeptides into rat glomeruli and glomerular basement membrane (GBM) was investigated both in vivo and in vitro. This has provided a comprehensive inventory of newly synthesized glycoconjugates in GBM.;The results demonstrate the incorporation of {('35)S}sulfate and {('3)H}glucosamine into both sulfated and non-sulfated GAG and glycopeptides both in vivo and in vitro. In both cases, heparan sulfate was found to be the major GAG synthesized by glomeruli. Smaller amounts of newly synthesized hyaluronic acid and chrondroitin sulfate were also present. There was relatively more chondroitin sulfate and less heparan sulfate synthesized and incorporated into GBM in vitro than in vivo. The in vitro data suggest that different (two or more) species of heparan sulfate are synthesized by glomeruli and incorporated into GBM, which differ in the proportion of O-linked and N-linked sulfate present.;Electron microscopic autoradiography was used to study the incorporation of {('35)S}sulfate into glomeruli and GBM. This analysis confirmed the biochemical findings that newly sulfated macromolecules are incorporated into glomeruli and GBM.;The autoradiographic studies demonstrated that (TURN)36% of the ('35)S-label is found in the region of the GBM (over the GBM and within 2 HD or 4300(ANGSTROM) of the GBM) after 4 hr of continuous labeling, while the biochemical analyses demonstrated that (TURN)7% of the ('35)S-label incorporated into glomeruli is recovered in isolated GBM. The autoradiographic data, in combination with the biochemical findings, suggest that many of the newly synthesized sulfated macromolecules in glomeruli are either loosely associated with GBM and, therefore, extracted during the isolation of GBM, or are incorporated into regions of the cells or cell surfaces in proximity to the GBM.;For the in vivo studies, rats were given four intraperitoneal injections of {('35)S}sulfate and {('3)H}glucosamine (over 10 hr) and sacrificed 14 hr after the last injection. For in vitro studies, kidneys were labeled with both {('35)S}sulfate and {('3)H}glucosamine continuously for 4 hr in an isolated perfused kidney system. Fractions of glomeruli and GBM were prepared from the labeled kidneys. The percent of label incorporated into specific GAG and glycopeptides was determined by selective degradative techniques in conjunction wth gel filtration chromatography.