title: Cyclosporine stimulates Na+-K+-Cl- cotransport activity in cultured mouse medullary thick ascending limb cells.
abstract: BACKGROUND
  Cyclosporine (CsA) has been shown to alter the activity of plasma 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 thick ascending limb (mTAL) cells.
  
  
  METHODS
  Experiments were carried out on subcultured confluent mouse TAL cells. Reverse transcription-polymerase chain reaction experiments showed that they expressed the mNKCC2 electroneutral Na+-K+-Cl- cotransporter and ROM-K1 and ROMK2 potassium channel mRNA. Western blotting also revealed the presence 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 (86Rb+) and 36Cl-, used as tracers of K+ and Cl- movements, on cells grown on Petri dishes or permeable filters.
  
  
  RESULTS
  CsA inhibited by 38% the ouabain-sensitive component of 86Rb+ influx mediated by the Na+,K+-ATPase pumps. CsA also increased by 38% the ouabain-resistant furosemide-sensitive component (Or-Fs) of 86Rb+ influx, reflecting the Na+-K+-Cl- cotransport activity and stimulated the basolateral efflux of 36Cl- 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). Apical 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 luminal side of cells grown on Petri dishes also prevented the rise in apical 86Rb+ efflux and the increased Or-Fs component of 86Rb+ influx caused by CsA.
  
  
  CONCLUSIONS
  These results indicated that CsA may stimulate the Na+-K+-Cl- cotransport activity and also suggested that this immunosuppressive agent may interfere in the recycling of apical K+ in this model of cultured mouse TAL cells.
authors.affiliation: Division of Nephrology, Chang Gung Memorial Hospital, Taipei, Taiwan. maisuwu@ms9.hinet.net
authors.forename: M S
authors.initials: MS
authors.lastname: Wu
authors.forename: C W
authors.initials: CW
authors.lastname: Yang
authors.forename: M
authors.initials: M
authors.lastname: Bens
authors.forename: K C
authors.initials: KC
authors.lastname: Peng
authors.forename: H M
authors.initials: HM
authors.lastname: Yu
authors.forename: A
authors.initials: A
authors.lastname: Vandewalle
mesh_terms: D000818:Animals; D001464:Barium / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D006084:Graft Rejection / Q000188:drug therapy / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007679:Kidney Medulla / Q000166:cytology / Q000378:metabolism*; D016030:Kidney Transplantation / Q000502:physiology; D008138:Loop of Henle / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying; D012414:Rubidium Radioisotopes / Q000493:pharmacokinetics; D028021:Sodium-Potassium-Chloride Symporters; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D002712:Chlorides; D007166:Immunosuppressive Agents; C510693:Kcnj1 protein, mouse; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D012414:Rubidium Radioisotopes; D028021:Sodium-Potassium-Chloride Symporters; D001464:Barium; D016572:Cyclosporine; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium