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cyclosporine A; FK506; Na+/K+-ATPase; Na+/K+/2Cl(-) cotransporter; MDCK cells; kidney; distal nephron; toxicity; aldosterone
Abstract:
Background. Hyperkalemia and metabolic acidosis are common manifestations in patients receiving the immunosuppressive agent cyclosporine A (CsA) and the recently introduced FK506. We compared the acute toxic and antiproliferative effects as well as the effects on the transport activity of Na+/K+-ATPase and Na+/K+/2Cl(-) cotransporter of CsA and FK506 in an established cell line of distal/collecting tubule origin (MDCK cells). Methods: MDCK cells were exposed to various concentrations of CsA or FK506 and the effects on cell viability (MTT test and neutral red uptake), plasma membrane integrity (lactate dehydrogenase (LDH) release) and cell proliferation (bromodeoxyuridine (BrdU) incorporation) were compared. For transport studies, after confluence, MDCK cells were exposed to CsA or FK506 for 48 h in the presence and absence of aldosterone. Ouabain- and bumetanide-sensitive (86)Rubidium uptake measurements were used to study the activity of the Na+/K+-ATPase and Na+/K+/2Cl(-) cotransporter at the surface of intact cells. Results: After 24 h of exposure CsA reduced the number of viable cells to 50% at 30 muM, whereas for FK506 2-3 times higher concentrations had to be employed. Similarly, LDH release was stimulated tenfold by 30 muM CsA but only fourfold by 70 muM FK506. In contrast, DNA synthesis was affected at lower concentrations of FK506 than of CsA. In cells treated for 24 h BrdU incorporation was significantly inhibited by 3 muM FK506, whereas a similar inhibition required 10 muM CsA. The transport activity of Na+/K+-ATPase and of Na+/K+/2Cl(-) cotransporter were significantly decreased (37 and 63%, respectively) on CsA administration (8 muM). In CsA-treated cells the K+ channel blockers barium (1 mM), TEA (10 mM) and quinine (1 mM) did not further inhibit the transport activities suggesting that CsA might also act via inhibition of K+ channels. FK506 at 8 muM had no effect on Na+/K+-ATPase transport activity but stimulated Na+/K+/2Cl(-) cotransporter activity by 59%. The stimulatory effect was abolished by K+ channel blockers indicating that recycling of K+ might increase by FK506. The simultaneous presence of aldosterone (5 muM) protected the cells from the inhibitory effect of CsA on Na+/K+-ATPase and Na+/K+/2Cl(-) cotransporter activity. The stimulatory effect of FK506 on the Na+/K+/2Cl(-)cotransporter activity was completely abolished in the presence of aldosterone. Conclusions: Both CsA and FK506 showed acute toxicity in MDCK cells in vitro with the effects of FK506 being less pronounced. CsA and FK506 had different effects on the in vivo transport rates of the Na+/K+-ATPase and the Na+/K+/2Cl(-) cotransporter; CsA inhibited the activity of the Na+/K+-ATPase and the Na+/K+/2Cl(-) cotransporter whereas FK506 stimulated the activity of Na+/K+/2Cl(-) cotransporter. These effects were abolished by the application of aldosterone.
