Paper
Nº 048

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


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Francisco Arrebola, MS.

Francisco Arrebola, Francisco O'Valle, Asunción Olmo, Mariano Aguilar, Marco Masseroli, Mª Eugenia Reguero, Francisco Revelles, Rafael Carvia, Marina Higueras, Belén Espigares, Raimundo G. Del Moral.

Department of Pathology, School of Medicine and University Hospital, University of Granada, Granada, Spain

favargas@goliat.ugr.es

Dpto. de Anatomía Patológica, Facultad de Medicina, Avda. de Madrid 11 - 18012 Granada, SPAIN

 

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.

Drugs

(IC70)

MDCK

(MISF)

LLC-PK1

(MISF)

7 days

15 days

7 days†

15 days

Control

22.68±2.14*

27.7±4.09§

35.44±8.4

27.98±5.16§

AZA

39.98±3.08

53.97±3.27

34.4±3.09

45.13±7.4

CsA

34.63±3.13

37.4±3.83

37.03±5.72

40.23±3.94

FK506

38.67±4.67

30.7±0.76

34.73±4.8

27.6±2.86

Values expressed as mean±s; *: Newman-Keuls p<0.01 of control vs all; §: Newman-Keuls p<0.01 of control vs CsA and AZA; †: ANOVA not statistically significant.

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.

Drugs

(30mM)

 

NIH-MDR-G185

(MFC)

Drugs

(30mM)

MDCK

(MFC)

LLC-PK1

(MFC)

Verapamil

120.4

7 days

15 days

7 days

15 days

Control

17.8

Control

155.2

114.5

134.5

148.6

AZA

21.2

AZA

181.8

136.2

217.9

127.9

CsA

155.9

CsA

81.5

124.0

165.3

144.8

FK506

153.9

FK506

99.5

137.5

164.3

130.2

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.

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Francisco Arrebola is at the Pathology Department of the School of Medicine, University of Granada, on a predoctoral fellowship from the Spanish Ministry of Education and Culture. His main research line is the study of drug nephrotoxicity and its relationship to drug multiresistance, using in vitro experimental models.

 

We look forward to seeing you at The Twenty-Second Annual Course on the Surgical Pathology of Neoplastic Diseases organized by the Memorial Sloan-Kettering Cancer Center in Granada (May 1999).

 F. Arrebola, MS.


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.
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