Mesangial cells derived from isolated glomeruli of rat kidney were grown as homogeneous cell lines in culture. They released, into the culture medium, erythropoietin that had free terminal galactosyl residues and was therefore not active in vivo. The production of erythropoietin by these cells was significantly enhanced by either lowering the Po2 in the incubation atmosphere or by adding cobalt chloride to the culture medium. Therefore, mesangial cells in culture may be considered as an in vitro system in which the regulation of erythropoietin production can be studied under well-defined conditions. With this in mind, we sought to develop a cell culture system in which the biosynthesis of Ep can be studied under conditions that are much better defined than in the intact organ. We show here that renal glomerular cells, which were clearly identified to be of mesangial origin, in culture produce Ep that has free galactosyl residues. The Ep production of these cells could be enhanced by hypoxia and cobalt chloride which are well-known stimuli of Ep formation in vivo. Therefore, the present culture system can be regarded as a useful model for the study of the hypoxia-induced increase of Ep synthesis.
MATERIALS AND METHODSThe preparation of isolated glomeruli from rat kidneys and subculture of glomerular outgrowths were done as described (5).Cells subcultured 3 weeks after the first inoculation were used. The cells were grown in RPMI 1640 (Boehringer Mannheim) supplemented with 10% fetal bovine serum (Boehringer Mannheim), penicillin at 100 units/ml, streptomycin at 10 jig/ml, and bovine insulin (Sigma) at 0.66 unit/ml. Kalden et al. (11).Ep Assay. The Ep activity of the culture medium was determined with the fetal mouse liver cell test (12) and a serumfree incubation mixture (6). In some experiments, bone marrow cells from adult mice were used for the test. The total volume of one test was 600 ,ul including 60 ,Al of the sample to be tested for Ep activity. Standard Ep (human urinary Ep standardized against International Reference Preparation B) was kindly provided by the National Institutes of Health. All unknown samples from the culture medium were chromatographed on DEAE-cellulose at pH 4.5 (13)