ENDOTHELIN RELEASE BY RABBIT PROXIMAL TUBULE CELLS - MODULATORY EFFECTS OF CYCLOSPORINE-A, TACROLIMUS, HGF AND EGF

Background. Previous studies have suggested that endothelins, a family of 21 amino acid peptides with potent vasoconstrictive and mitogenic properties, are involved in the pathogenesis of acute and chronic rena l failure. In addition, endothelin seems to play an important role in mediating the nephrotoxic side effects of cyclosporine A (CsA) and tac rolimus. The present study investigated the production of endothelin-1 (ET-1) and endothelin-3 (ET-3) by bipolar differentiated rabbit proxi mal tubule cells (PT-1 cells), and the modulatory effect of CsA, tacro limus, hepatocyte growth factor (HGF) and epidermal growth factor (EGF ) on ET-1 and ET-3 release. Methods. ET-I and ET-3 mRNA was detected b y RT-PCR, immunoreactive endothelin was localized to PT-1 cells by imm unofluorescence staining with antibodies against ET-1 and ET-3. ET-I a nd ET-3 release into the culture medium was determined by specific rad ioimmunoassays after solid phase extraction. Results. PT-1 cells exhib ited a time-dependent increase of ET-1 release up to an incubation per iod of 36 hours, whereas ET-3 release already reached a steady state l evel after four hours. PT-1 cells, cultured on filter membranes, relea sed a significantly higher amount of immunoreactive ET-I into the baso lateral compartment than into the apical compartment. ET-3 release did not differ significantly between the basolateral and the apical compa rtment. Supplementation of the cell culture medium with 10% fetal calf serum induced a marked increase of the basolateral and apical ET-1 re lease, whereas ET-3 release was only slightly increased. CsA and tacro limus (0.5 to 5000 mu g/liter) induced a significant, dose-dependent i ncrease of ET-1 and ET-3 release by PT-1 cells with a maximum stimulat ion at a CsA concentration of 500 mu g/liter (P < 0.001) and a tacroli mus concentration of 50 mu g/liter (P < 0.001). HGF and EGF (10(-10) t o 10(-8) mol/liter) exerted a significant (P < 0.001) dose-dependent i nhibitory effect on ET-1 release, whereas ET-3 release was not signifi cantly reduced. Coincubation of PT-1 cells with CsA or tacrolimus and HGF or EGF also resulted in a marked reduction of ET-1 release. Conclu sions. The present data suggest that ET-1 and ET-3 release by cultured rabbit proximal tubule cells are regulated differently, and that the stimulatory effect BE CSA and tacrolimus on ET-1 release is antagonize d by I-IGF and EGF.