Effects of hypertonic stress on transforming growth factor-β activity in normal rat kidney cells. Hypertonicity is known to modulate the expression of some genes and the action of several cytokines. We evaluated whether hypertonicity would increase the expression and/or activity of transforming growth factor-β (TGF-β) in normal rat kidney (NRK) cells. The bioassay for TGF-β showed that mature TGF-β activity was significantly increased when the cells were cultured in a hypertonic medium (500 mOsm/kg). Comparing to the isotonic medium, hypertonicity accelerated the increase in TGF-β activity during the initial 24 hours after changing the medium. The activity was increased as the medium osmolality increased from 300 to 500 mOsm/kg. Raffinose was found to be the most effective in increasing TGF-β activity. NaCl, glucose, and mannitol also increased TGF-β activity. In contrast, total TGF-β (mature and latent) activity and TGF-β mRNA abundance did not change significantly, suggesting that hypertonicity activated TGF-β without affecting the synthesis of TGF-β. To determine whether collagen synthesis was increased by hypertonicity, we examined [³H] proline incorporation into NRK cells cultured in hypertonic medium. Proline incorporation increased in an osmolality-dependent manner. Raffinose was also the most effective solute at increasing the proline incorporation. Furthermore, anti-TGF-β antibody prevented the increase in proline incorporation induced by hypertonicity. These results suggest that hypertonicity promotes the processing of latent TGF-β to the biologically active form, resulting in the stimulation of collagen synthesis in NRK cells.