CYCLOSPORINE INHIBITS NITRIC-OXIDE PRODUCTION IN MEDULLARY ASCENDING LIMB CULTURED-CELLS

Background. Nitric oxide (NO) has been shown to play a role in cyclosp orin (CsA) nephrotoxicity but its mechanism of action is still unclear . As inducible NO synthase (iNOS) mRNA has been found to be expressed in rat medullary thick ascending limb (mTAL) cells, we investigated th e effects of CsA on NO production in a model of mouse cultured mTAL ce lls. Materials and methods. The experiments were carried out on sub-cu ltured cells derived from isolated mTAL microdissected from the kidney of C57BL/6 mice. The identification of the iNOS mRNA in mTAL microdis sected segment and cultured cell was confirmed by RT-PCR and RsaI dige stion. Nitrite (NO2-) released by mTAL cells was determined using the modified Griess reagent method and taken as an index of nitric oxide p roduction. The cultured cells were treated with various concentrations of CsA and different signal transduction regulators to assess the eff ect and possible pathway(s) of action of CsA on NO production in mTAL cells. Results. The basal production of NO by mTAL cells increased by 1.8-fold following incubation with bacterial lipopolysaccaride (LPS). Both aminoguanidine and L-NAME inhibited NO production. CsA (10-300 ng /ml) also inhibited NO production in a dose-dependent manner and preve nted its increase induced by LPS. Phorbol 12-myristate 13-acetate (PMA ), a PKC stimulator, enhanced slightly the production of NO under basa l conditions and prevented the inhibitory action of CsA on NO producti on. These results suggest that the NO secreted by mouse cultured mTAL cells is dependent on the PKC pathway. Conclusion. These results show that CsA may down-regulate the production of NO by cultured mTAL cells expressing iNOS mRNA and that the PKC pathway is involved in this pro cess.