Cyclosporine stimulates Na+-K+-Cl- cotransport activity in cultured mouse medullary thick ascending limb cells

Background. Cyclosporine (CsA) has been shown to alter the activity of plas ma membrane transporters in kidney epithelial cells. In this study, we have investigated the effects of CsA on Na+K+-ATPase and Na+-K+-Cl- cotransport activities in cultured cells derived from microdissected mouse medullary t hick ascending limb (mTAL) cells. Methods. Experiments were carried out on subcultured confluent mouse TAL ce lls. Reverse transcription-polymerase chain reaction experiments showed tha t they expressed the mNKCC2 electroneutral Na+-K+-Cl- cotransporter and ROM -K1 and ROMK2 potassium channel mRNA. Western blotting also revealed the pr esence of the 40 kD ROMK protein using an anti-ROMK antibody. The effect of CsA (100 ng/mL) on ion transport was assessed by measuring the influx and efflux of rubidium (Rb-86(+)) and Cl-36(-), used as tracers of K+ and Cl- m ovements, on cells grown on Petri dishes or permeable filters. Results. CsA inhibited by 38 % the ouabain-sensitive component of Rb-86(+) influx mediated by the Na+,K+-ATPase pumps. CsA also increased by 38% the o uabain-resistant furosemide-sensitive component (Or-Fs) of Rb-86(+) influx, reflecting the Na+-K+-Cl- cotransport activity and stimulated the basolate ral efflux of Cl-36(-) from mTAL cells grown on filters. The CsA-stimulated basal efflux of Cl- was prevented by the basal addition of the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoate (NPPB, 10(-4) mol/L). Api cal addition of the K+ channel blocking agent Ba2+ (10(-4) mol/L) partially prevented the CsA-stimulated basal efflux of Cl-. Adding Ba2+ to the lumin al side of cells grown on Petri dishes also prevented the rise in apical Rb -86(+) efflux and the increased Or-Fs component of Rb-86(+) influx caused b y CsA. Conclusion. These results indicated that CsA may stimulate the Na+-K+-Cl- c otransport activity and also suggested that this immunosuppressive agent ma y interfere in the recycling of apical K+ in this model of cultured mouse T AL cells.