Okadaic acid interferes with the maturation of human fetal kidney in serum-free culture

A chemically-defined culture model has been established in our laboratory, enabling the study of the respective effects of growth factors on the developing kidney. Since this system involves the use of defined medium unsupplemented with serum, hormones or glucose, these limited conditions induce a decrease in DNA synthesis. Addition of insulin and transferrin significantly restores this important cellular function. The objective of the present study was to verify the influence of okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, in order to determine whether the latter modulate insulin action on human fetal kidney. Okadaic acid was found to decrease H-3-thymidine incorporation below control (L-15) levels in a dose-dependent manner. The influence of 0.01 muM okadaic acid could be detected as early as the 4th h of culture, and was progressive until the 48th h; at 0.4 muM, the effect was more dramatic with maximum inhibition obtained between 8 and 12 h. Moreover, at both concentrations, the drug completely abolished the stimulating effect of insulin plus transferrin over a 5-day period. Following removal of the toxin after 24 h of culture, DNA synthesis after 5 days still remained at levels observed in the presence of okadaic acid and insulin plus transferrin on day 1. This result indicates that the inhibitory influence of okadaic acid remained at the same level once the drug was removed, and that this effect is irreversible. In addition, okadaic acid at 0.4 muM induced cell death and morphological changes possibly associated with apoptosis. These data suggest that insulin stimulation of DNA synthesis, at least in this 'in vitro' system, is mediated through specific protein phosphatases that may play a crucial role in growth regulation during kidney development.