Cyclosporin increases the density of angiotensin II subtype 1 (AT₁) receptors in mouse medullary thick ascending limb cells

Background. Cyclosporin A (CsA), a potent immunosuppressive agent, can be nephrotoxic. Because clinical studies have suggested that the intrarenal renin-angiotensin system may be involved in the mechanism responsible for CsA nephrotoxicity, we have analysed the effects of CsA on angiotensin II (Ang II) receptors in medullary thick ascending limb (mTAL) cells known to be sensitive to the action of CsA. Methods. Experiments were carried out on subcultured mouse mTAL cells. The expression of mRNA of Ang II subtype 1 and 2 (AT₁ and AT₂) receptors was investigated using reverse transcription-polymerase chain reaction (RT-PCR). [³H]Ang II was used for radioligand and binding studies. Fluorimetric recordings using the fluorescent dye fura-2/AM were performed to determine the effect of CsA on the intracellular calcium ([Ca²⁺]i) content of untreated and Ang II-treated mTAL cells. Results. Subcultured mTAL cells expressed AT₁ and AT₂ Ang II receptor mRNAs, and binding studies revealed that the AT₁ receptors were the predominant Ang II receptor subtype (∼90%) in mTAL cells. CsA (100 ng/ml, 24 h) increased (1.7-fold) the number of Ang II receptors (untreated, 315.8; +CsA, 543.6 fmol/mg protein) without altering the K_D (untreated, 7.16; +CsA, 7.06nM). CsA also significantly increased the level of [Ca²⁺]i measured in cultured mTAL cells both in the basal state (-CSA, 72.2; +CsA, 93.4 nM/10⁶ cells) and in the presence of Ang II (-CSA, 97.8; +CsA, 206.3 nM/10⁶ cells). Conclusions. These findings suggest that the increase in Ang II AT₁ receptors and [Ca²⁺]i caused by CsA may be involved in the mechanism(s) responsible for CsA nephrotoxicity.