Paper Nº 048

Indice

Effect of Tacrolimus^® and Cyclosporin A on the Expression of P Glycoprotein in Renal Tubular Cells in Culture.

[Título] [Introducción] [Material y Métodos] [Resultados] [Discusión] [Iconografía] [Bibliografía]

favargas@goliat.ugr.es

INTRODUCTION: P glycoprotein (P-gp) is strongly expressed at the apical border of proximal convoluted tubules and is detected in their cell lines in culture. The precise physiological function of P-gp in the kidney has not been totally defined, but there is evidence that it plays an important role in renal detoxifying mechanisms when an aggression by xenobiotics is produced, acting as a "vacuum cleaner" of hydrophobic molecules. Our group has demonstrated in renal transplant biopsies a relationship between intrarenal deposits of Cyclosporin A (CsA) and an increase in the expression of P-gp. We have observed the same phenomenon with renal tubular cell lines. Alternatively, CsA may act by functionally blocking the P-gp. We present a comparative in vitro study of three immunosuppressors (IS) on the modulation of P-gp expression and its functional capacity.

MATERIAL AND METHODS: The in vitro assays were done with the renal tubular cell lines MDCK y LLC-PK1 maintained in culture medium of MEM plus 5% IFBS, and with the NIH-MDR-G185 cell line transfected with the human mdr1 gene and incubated with DMEM plus 10% of IFBS. The drug induction was done with sublethal doses (IC₇₀) of the IS: CsA (1.85µM and 10µM), Tacrolimus^® (FK506) (10µM), Azathioprine (AZA) (105µM y 207µM) and incubation with the drugs was for 7 and 15 days. Flow cytometry was used to evaluate the following parameters: P-gp expression (MoAb JSB-1); number of positive cells (%POS) and the mean incorporation of specific fluorescence (MISF); cell cycle and the growth fraction (GF). Induction or functional block of P-gp was assessed using fluorescent colorant Rhodamine123 (Rh123) as substrate and determining the mean fluorescent channel (MFC).

RESULTS AND DISCUSSION: In baseline conditions the %POS and intensity of expression of P-gp measured as MISF were similar for the renal tubular lines (%POS MDCK: 83.65±22.71 and LLC-PK1: 81.28±20.5) (MISF MDCK: 22.6±2.1 and LLC-PK1: 27.9±5.1) and much higher in the transfected control line NIH-MDR-G185 (%POS: 99±0.1% MISF: 122.8±15.4). The incubation with the IS modified the MISF in both lines (ANOVA, p<0.001), after 7 days for the MDCK line and after 15 days in the LLC-PK1 line. As shown in the following table, the greatest induction was with AZA, followed by CsA and FK506.

Thus, after 15 days of treatment P-gp induction was maintained in the two lines versus the control cultures (ANOVA, p<0.01) except for FK506, where statistical significance was lost. Comparing FK506 with CsA, the latter induced a significantly greater expression of P-gp (Newman-Keuls, p<0.05) in both renal tubular lines and at the two times measured. In the transfected line NIH-MDR-G185 the assays with Rh123 showed the modulating effect of the IS on the P-gp. FK506 and CsA produced a significantly greater intracellular accumulation of Rh123 versus the blocker Verapamil, and AZA produced the least accumulation. As shown in the following table, for the tubular lines the modulating effect of the IS on the P-gp was less evident.

CONCLUSIONS: 1) In vitro, drugs with nephrotoxic potential induce the overexpression of P-gp after prolonged incubation. 2) FK506 induces a comparatively smaller expression of P-gp and simultaneously induces the significant functional blocking of this molecule; this dual stimulating and blocking action may be of great utility in the management of transplant patients.

Key Words: Nephrotoxicity, Tissue culture, P-Glycoprotein, FK506, Cyclosporin A, Azathioprine, Rhodamine123.

• Introduction
• Materials and Methods
• Results
• Discussion
• Pictures
• Bibliography

Francisco Arrebola, MS.
Copyright © 1996, Departamento de Anatomía Patológica, Facultad de Medicina y Hospital Universitario, Universidad de Granada, 18012 Granada, España. Reservados todos los derechos.