CELL-DENSITY MODULATES GROWTH, EXTRACELLULAR-MATRIX, AND PROTEIN-SYNTHESIS OF CULTURED RAT MESANGIAL CELLS

Mesangial cell (MC) hyperplasia and accumulation of extracellular matr ix are hallmarks of chronic glomerular disease. The present in vitro s tudy examined the effects of cell density on growth, extracellular mat rix formation, and protein synthesis of cultured rat MCs. A negative l inear relationship was found between initial plating density and DNA s ynthesis per cell after 24 hours incubation in medium with 10% fetal c alf serum (range. 1 X 10(3) to 7 x 10(5) MCs/2cM2, r = 0.996, P < 0.00 1). Enzyme-linked immunosorbent assay of the amount of fibronectin in the conditioned medium after 72 hours showed a negative relationship w ith increasing cell density. In contrast, the amount of cell-associate d fibronectin increased to maximal values in confluent cultures, and x o further increase was seen at supraconfluency. ne relative collagen s ynthesis in the conditioned medium and cell layer-assessed by collagen ase digestion after 5 hours [H-3]proline pulse labeling-showed a simil ar pattern Secreted collagen decreased with increasing cell density fr om 3.4% to 0.2% of total protein synthesis. Ix contrast, cell-associat ed collagen increased from 1.1% to 11.8% of newly synthesized protein until confluency followed by a decrease to 4.2% at supraconfluency. Sp ecific immunoprecipitation of collagen types I, III, and IV revealed a significant (twofold) increase in collagen I synthesis per cell at co nfluency. Collagen III and IV synthesis was not affected by cell densi ty. Specific protein expression in both the medium and cell layer were analyzed by two-dimensional polyacrylamide gel electrophoresis (150 t o 20 kd, pl 5.0 to 70) after 20 hours steady-state metabolic labeling with [S-35]methionine. Supraconfluent MCs displayed overexpression of 10, underexpression of four, new expression of five, and changed mobil ity of three different intracellular proteins. Of interest was the ove rexpression of two proteins (89 kd, pI 5.31 and 72 kd, pI 5.32) that w ere identified by immunoblotting as the stress proteins beat-shock pro tein 90 and glucose-related protein 78, respectively. The progressive increase of cell-associated fibronectin and collagens, particularly co llagen type 1, in confluent MCs resembles extracellular matrix accumul ation in glomerular disease. The increased expression of stress protei ns in supraconfluent MCs is of interest in view of the analogy between glomerulosclerosis and atherosclerosis in which stress proteins are e xpressed in high concentrations.