Therapeutic concentrations of cyclosporine A, but not FK506, increase P-glycoprotein expression in endothelial and renal tubule cells  Ingeborg A. Hauser, Michael Koziolek, Ulrich Hopfer, Frank Thévenod  Kidney International  Volume 54, Issue 4, Pages 1139-1149 (October 1998) DOI: 10.1046/j.1523-1755.1998.00095.x Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 1 Expression of P-gp and Na+/K+-ATPase in lysates from human arterial endothelial cells (HAEC) treated for seven days with different concentrations of cyclosporine A (CsA) or FK506. (A) The immunoblot was probed with mAb C219 (5 μg/ml). HAEC were pretreated for seven days with either 0.1% (vol/vol) DMSO in controls (lane 1) and 0.1 to 1.6 μm CsA (upper immunoblot) or 0.01–0.2 μm FK506 (lower immunoblot). (B) Quantification of P-gp expression in HAEC treated with 0.1 to 1.6 μm CsA for seven days (left) or HAEC treated with 0.01 to 1.2 μm FK506 for seven days (right). Immunoreactive bands were analyzed by densitometry, and P-gp expression was expressed as a percentage of the total amount of immunoreactive protein present in HAEC that had been treated with 0.1% (vol/vol) DMSO for seven days. Values represent means ± sd of 5 different experiments. *P < 0.05 using unpaired Student’s t-test. (C) The immunoblot was probed with mAb Abα5 (0.5 μg/ml). HAEC were pretreated for seven days with either 0.1% (vol/vol) DMSO in controls, 0.8 and 1.6 μm CsA, or 0.6 and 1.2 μm FK506. The blot is typical for three different preparations. Kidney International 1998 54, 1139-1149DOI: (10.1046/j.1523-1755.1998.00095.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 2 Time course of the effect of cyclosporine A (CsA) on the expression of P-gp in lysates from human arterial endothelial cells (HAEC). HAEC were left untreated [0.1% (vol/vol) DMSO for 7days] or exposed to 0.8 μm CsA for one to seven days. The immunoblot was probed with mAb C219 (5 μg/ml). Typical for three different preparations. Kidney International 1998 54, 1139-1149DOI: (10.1046/j.1523-1755.1998.00095.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 3 Immunocytochemical staining of P-gp in the plasma membrane of human arterial endothelial cells (HAEC) incubated without and with cyclosporine A (CsA) or FK506 for seven days. Cytospins of HAEC were fixed in 4% paraformaldehyde, incubated with MRK-16mAb (1:100) and stained by immunoperoxidase reaction. In (A), control HAEC had been incubated with 0.1% (vol/vol) DMSO for seven days. In (B and C), HAEC had been exposed to 0.8 μm CsA or 0.1 μm FK506 for seven days, respectively. Typical for four different cytospin preparations. Original magnification ×400. Bar = 15 μm. Kidney International 1998 54, 1139-1149DOI: (10.1046/j.1523-1755.1998.00095.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 4 Fluorimetric time course of calcein accumulation in suspensions of human arterial endothelial cells (HAEC) incubated in the absence and presence of verapamil. Five × 105 cells/ml Tyrode solution were incubated in the presence of 0.25 μm calcein-AM at 37°C. The increase in fluorescence was monitored as the dye was hydrolyzed intracellularly. The plot shows calcein fluorescence (arbitrary units) against time. The inhibitor of P-gp verapamil (100 μm) was added where indicated. The different curves represent experiments with cells which had been exposed for seven days to either 0.1% (vol/vol) DMSO (control), 0.8 μm cyclosporine A (CsA), or 0.1 μm FK506 (FK506). Typical for three different experiments. Kidney International 1998 54, 1139-1149DOI: (10.1046/j.1523-1755.1998.00095.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 5 Expression of P-gp (A) and Na+/K+-ATPase (B) in lysates from rat proximal tubule cells (RPTC) treated for seven days with different concentrations of cyclosporine A (CsA) or FK506. (A) RPTC were exposed for seven days to either 0.1% (vol/vol) DMSO in controls (lane 1) and 0.1 to 1.6 μm CsA (upper immunoblot) or 0.01 to 0.2 μm FK506 (lower immunoblot) dissolved in 0.1% (vol/vol) DMSO. The immunoblot was probed with mAb C219 (5 μg/ml). One out of five similar experiments is shown. (B) The immunoblot was probed with mAb Abα5 (0.5 μg/ml) directed against the α-subunit of Na+/K+-ATPase. RPTC were pretreated for seven days with 0.1% (vol/vol) DMSO in controls (lane 1), 0.8 or 1.6 μm CsA, and 0.1 or 0.2 μm FK506. One out of three similar experiments is shown. Kidney International 1998 54, 1139-1149DOI: (10.1046/j.1523-1755.1998.00095.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Figure 6 Fluorimetric time course of calcein accumulation in suspensions of rat proximal tubule cells (RPTC) incubated in the absence and presence of verapamil. Five × 105 cells/ml Tyrode solution were incubated in the presence of 0.25 μm calcein-AM at 37°C. The plot shows calcein fluorescence (arbitrary units) against time. The inhibitor of P-gp verapamil (100 μm) was added where indicated. The different curves represent experiments with cells which had been exposed for seven days to either 0.1% (vol/vol) DMSO (control), 0.8 μm cyclosporine A (CsA), or 0.1 μm FK506 (FK506). Kidney International 1998 54, 1139-1149DOI: (10.1046/j.1523-1755.1998.00095.x) Copyright © 1998 International Society of Nephrology Terms and Conditions

Kidney International 1998 54, 1139-1149DOI: (10. 1046/j. 1523-1755 Kidney International 1998 54, 1139-1149DOI: (10.1046/j.1523-1755.1998.00095.x) Copyright © 1998 International Society of Nephrology Terms and Conditions