CULTURED HUMAN MESANGIAL CELLS PRODUCE BOTH TYPE-1 AND TYPE-2 PLASMINOGEN-ACTIVATOR INHIBITORS

Cultured human mesangial cells (HMC) derived from normal kidneys have been shown to synthesize tissue-type plasminogen activator (t-PA) and excess amounts of PA inhibitor type 1 (PAI-1). Conflicting results hav e been obtained concerning the production of urokinase-type PA (u-PA) and efforts to show PA inhibitor 2 (PAI-2) mel with failure, We evalua ted the fibrinolytic profile of cultured HMC lines obtained from 12 pa tients with renal carcinoma and one cadaveric kidney donor. Subconflue nt cells (third passage) were incubated overnight in serum-free medium . t-PA, u-PA, PAI-1 and PAI-2 antigens were assayed by ELISA methods a nd PA and PAI activities by amidolytic methods both in conditioned med ium (CM) and cell extracts (CE). Besides PAI-I, PAI-2 antigen was dete cted in all but one HMC lines. At variance with the former, which was largely released in the culture medium, PAI-2 was mainly cell-associat ed. t-PA antigen was found in all but two cell lines while u-PA antige n was detected in relatively high concentrations in 8 cell lines. PA a ctivity, identified as u-PA by functional and immunological criteria, was measured in CM of six of the eight u-PA producing cell lines, wher eas PAI activity was undetectable or very low in CM of all cell lines, suggesting that PAI-1 was largely inactive. Functional assays of cell extracts demonstrated the presence of PA activity, again identified a s u-PA, only in samples (five lines) containing u-PA antigen in excess over PAI-2. PAI activity was found instead in the extracts in which t he inhibitor was higher than the activator (six lines) and was identif ied as PAI-2, as it inhibited u-PA but not single-chain t-PA and was n eutralized by a polyclonal antl-PAI-2 antibody. The heterogeneous fibr inolytic pattern of HMC lines was confirmed by mRNA analysis of three representative lines. Results were similar when HMC lines at passage f ive were used except that the u-PA content was significantly reduced b oth in CM and CE. These findings indicate that the fibrinolytic profil e of cultured HMC is more complex than previously reported. The produc tion of large amounts of PAI-2 may represent an additional control mec hanism of proteinase activity.