Endothelin release by rabbit proximal tubule cells: Modulatory effects of cyclosporine A, tacrolimus, HGF and EGF

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 renal failure. In addition, endothelin seems to play an important role in mediating the nephrotoxic side effects of cyclosporine A (CsA) and tacrolimus. The present study investi- gated the production of endothelin-1 (ET-1) and endothelin-3 (ET-3) by bipolar differentiated rabbit proximal tubule cells (PT-1 cells), and the modulatory effect of CsA, tacrolimus, hepatocyte growth factor (HGF) and epidermal growth factor (EGF) on ET-1 and ET-3 release. Methods. ET-1 and ET-3 mRNA was detected by RT-PCR, immunoreactive endothelin was localized to PT-1 cells by immu- nofluorescence staining with antibodies against ET-1 and ET-3. ET-1 and ET-3 release into the culture medium was determined by specific radioimmunoassays after solid phase extraction. Results. PT-1 cells exhibited a time-dependent increase of ET-1 release up to an incubation period of 36 hours, whereas ET-3 release already reached a steady state level after four hours. PT-1 cells, cultured on filter membranes, released a significantly higher amount of immunoreactive ET-1 into the basolateral compart- ment than into the apical compartment. ET-3 release did not differ significantly between the basolateral and the apical com- partment. Supplementation of the cell culture medium with 10% fetal calf serum induced a marked increase of the basolateral and apical ET-1 release, whereas ET-3 release was only slightly increased. CsA and tacrolimus (0.5 to 5000 mg/liter) induced a significant, dose-dependent increase of ET-1 and ET-3 release by PT-1 cells with a maximum stimulation at a CsA concentration of 500 mg/liter (P , 0.001) and a tacrolimus concentration of 50 mg/liter (P , 0.001). HGF and EGF (10 210 to 10 28 mol/liter) exerted a significant (P , 0.001) dose-dependent inhibitory effect on ET-1 release, whereas ET-3 release was not significantly reduced. Coincubation of PT-1 cells with CsA or tacrolimus and HGF or EGF also resulted in a marked reduction of ET-1 release. Conclusions. 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 of CsA and tacrolimus on ET-1 release is antagonized by HGF and EGF. The endothelins are a family of peptides with potent vasoactive and mitogenic properties. Endothelin-1 was originally isolated from the supernatant of cultured porcine aortic endothelial cells (1). To date, three isoforms have been identified, endothelin-1, -2 and -3 (ET-1, ET-2, ET-3), and they are encoded by three separate genes (2). Endo- thelial cells seem to represent the main site of endothelin production, however, endothelin synthesis has also been localized to other cell types, including renal mesangial cells (3), proximal tubule cells and collecting duct cells (4 - 6). Endothelial cells have been shown to secrete ET-1 predom- inantly into the basolateral compartment (7), and recently published data suggest that renal proximal tubule and collecting duct cells also exhibit a polar ET-1 secretion (8, 9). In the present study, we investigated the basolateral and apical ET-1 and ET-3 release by rabbit proximal tubule cells (PT-1 cells).