Tissue Culture of Normal Rat Glomeruli

Abstract

The biosynthesis of glycosaminoglycans (GAG) by cultivated rat glomerular epithelial and mesangial cells was studied by incorporation of [<sup>35</sup>S] sulfate or [<sup>14</sup>C] glucosamine for 48 h. After dialysis, the incubation medium was subjected to digestion with papain. Labeled GAG were isolated from the digests by precipitation with cetylpyridinium chloride and ethanol. Results of cellulose acetate electrophoresis of the isolated ‘epithelial’ GAG fraction revealed the presence of two [<sup>14</sup>C] spots and one [<sup>35</sup>S] spot. The [<sup>35</sup>S] spot was identified as heparan sulfate, because it comigrated with the heparan sulfate standard and it was insensitive to testicular hyaluronidase. One [<sup>14</sup>C] spot comigrated with the [<sup>35</sup>S] spot and with the heparan sulfate standard. This GAG fraction did not contain galactosamine. The second [<sup>14</sup>C] spot was identified as hyaluronic acid, since it comigrated with the hyaluronic acid standard and since it was sensitive to testicular hyaluronidase. Results of cellulose acetate electrophoresis of the isolated ‘mesangial’ GAG fraction revealed the presence of one [<sup>14</sup>C] spot only. No [<sup>35</sup>S] spot was detectable. The [<sup>14</sup>C] spot comigrated with the hyaluronic acid standard and was sensitive to hyaluronidase. The data therefore suggest that the glomerular epithelial cells synthesize and secrete both sulfated GAG (heparan sulfate) and nonsulfated GAG (hyaluoronic acid) into the culture medium, whereas the glomerular mesangial cells synthesize and secrete nonsulfated GAG (hyaluronic acid) only into the culture medium.