Association of cyclophilin A with renal brush border membranes: redistribution by cyclosporine A.

BACKGROUND: Administration of the immunosuppressive agent cyclosporine A (CsA) is associated with nephrotoxicity. The main target for CsA, cyclophilin A (CypA), was found in high levels in epithelial cells of renal proximal tubules. In the present study, CypA was immunodetected and characterized following CsA treatment in subcellular fractions of renal cortex. METHOD: The renal content and distribution of CypA was evaluated in untreated rats and in rats treated with a subcutaneous injection of CsA (10 mg. kg-1. day-1) for 10 days. RESULTS: In untreated rats, membrane-bound CypA represents 0.25% of total brush border membrane (BBM) proteins, similar to the proportion found in the soluble fraction. High ionic strength treatment was unable to extract CypA from BBMs, whereas alkaline treatment (Na2CO2, pH 11) and detergent 3 - [(3 - cholamidopropyl) - dimethyl - ammonio] - 1 - propanesulfate (CHAPS) released it from BBMs. These results indicate that CypA is associated with renal BBMs, and that hydrophobic interactions are involved in this association. The CypA distribution was strongly modified in both BBMs and the soluble fraction after CsA treatment, but its affinity for CsA estimated by photoaffinity labeling was unaffected. The CypA expression level decreased by 45% in BBMs, while it increased by 33% in the soluble fraction, compared with control rats. CypA remained associated with the membranes following in vitro incubation of renal BBMs with CsA. However, incubation of CypA with one of its substrates released CypA from renal BBMs. CONCLUSIONS: These experiments suggest that renal BBMs contain a significant amount of CypA and chronic exposure to CsA, and acute exposure to one of CypA substrates may modify its subcellular distribution.

Inhibition of P-glycoprotein by cyclosporin A analogues and metabolites.

The interaction between P-glycoprotein (P-gp) from membranes isolated from multidrug-resistant Chinese hamster ovary cells and cyclosporin A (CsA) analogues and its metabolites was characterized. Screening of these latter as chemosensitizers was performed using three different assays: (i) vinblastine uptake, (ii) photoaffinity labeling by [125I]iodoaryl azidoprazosin, and (iii) P-gp ATPase activity. Oxidation of the hydroxyl group at position I of CsA (200-096), CsG (215-834), or CsD (PSC-833) increased their inhibition of P-gp. CsA analogues (208-032, 208-183) modified at position 11 retained their ability to inhibit P-gp while analogues modified at position 2 (CsC and CsD) lost their efficiency. The inhibitions induced by metabolites of CsA were also compared to those obtained with CsG metabolites. From all the molecules tested, PSC-833 and 280-446 peptolide were the strongest inhibitors. Our results indicate that modifications of CsA analogues at position 1 and 2 are critical for their interaction with P-gp and that CsA metabolites retain a portion of the inhibitory activity of the parent drug.