Cyclosporine Stimulates the Renal Epithelial Sodium Channel

We used A6 distal nephron cells as a model to test a hypothesis that cyclosporine A (CsA) causes hypertension by stimulating the renal epithelial sodium channel (ENaC). Patch‐clamp experiments showed that addition of 10 μM CsA to the basolateral bath significantly increased ENaC open time from 7 ± 2 (before) to 33 ± 9 sec (n = 5; P < 0.05). Since CsA is a potent inhibitor of the ATP‐binding cassette A1 transporter that is responsible for a constant cholesterol efflux , whether CsA elevates intracellular cholesterol was determined by using a fluorescent NBD‐cholesterol. Confocal microscopy experiments showed that CsA did cause cholesterol accumulation in A6 cells. Addition of 50 μg/ml cholesterol mimicked the effect of CsA on ENaC activity by also increasing ENaC open time from 5 ± 2 (before) to 27 ± 5 sec (n = 6; P < 0.01). Moreover, a longer exposure of the cells to CsA elevated ENaC cell surface expression; the chance for a cell‐attached patch to contain more than one ENaC channel in A6 cells treated with 1 μM CsA for 24 h was increased to 83 ± 14% (n = 15, P < 0.001), compared to that in untreated cells (17 ± 14%, n = 17). Since phophatidylinositol‐4,5‐bisphosphate (PIP 2 ) can affect protein trafficking, whether CsA regulates membrane PIP 2 distribution was also examined by expression of a GFP‐tagged pleckstrin homology domain of phospholipase C‐δ1. We found that PIP 2 formed relatively large domains (macrodomains) and that CsA treatment dramatically changed PIP 2 macrodomains from a granule‐like to a net‐like configuration. These data together suggest that CsA stimulates ENaC activity and trafficking, probably by elevating intracellular cholesterol and reconfiguring PIP 2 macrodomains.