PUROMYCIN AMINONUCLEOSIDE AND ADRIAMYCIN DISTURB CYTOSKELETAL AND EXTRACELLULAR-MATRIX PROTEIN ORGANIZATION, BUT NOT PROTEIN-SYNTHESIS OF CULTURED GLOMERULAR EPITHELIAL-CELLS

Puromycin aminonucleoside (PA) and Adriamycin (ADR) cause glomerular p roteinuria associated with degenerative alterations of glomenular visc eral epithelial cells (GVEC) and detachment from the glomenular baseme nt membrane when administered to rats. This in vitro study was perform ed to define, in detail, the quantitative and qualitative changes of a number of adhesion-associated proteins (cytoskeletal, extracellular m atrix and integrin proteins) upon exposure to PA and ADR. By immunoflu orescence we observed: (1) dose- and incubation-time-dependent filamen t pattern changes and decreased staining of the cytoskeletal proteins actin, vimentin, keratin, and beta-tubulin; (2) an altered distributio n, and decreased expression of the extracellular matrix proteins lamin in and heparan sulfate and (3) a loss of the beta(1)-integrin focal ad hesions upon exposure to PA and ADR. Using an ELISA, a concentration-d ependent decrease was found (a 50% reduction with 50 mu g/ml PA for 48 h and with 2 mu g/ml ADR for 24 h) in the production of cytoskeletal and extracellular matrix proteins per cell. These general effects were suggestive of a disturbance of protein synthesis but, by metabolic la belling studies, no reduction in overall protein synthesis was found. Using two-dimensional PAGE on S-35-methionine steady-state labeled cel ls, no changes were found in intracellular protein patterns of PA- and ADR-treated cells (pH 5-7.5, MW 110-20 kD). We hypothesize that expos ure of GVEC in vitro to PA and ADR might result, directly or indirectl y, in perturbation of the macromolecular organization of cytoskeletal and extracellular matrix proteins with loss of GVEC adhesion.