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The role of the low-density lipoprotein receptor family on Cyclosporine A uptake and toxicity in renal cells

University of British Columbia

Background: Cyclosporine A (CsA) is an effective immunosuppressant drug to treat patients who have undergone transplantation or to treat autoimmune diseases. However, the drug is limited by its narrow therapeutic index and usually becomes discontinued due to high nephrotoxicity. CsA is known to highly associate with lipoproteins, especially LDL and increased toxic effects of CsA have been reported in patients who are hypocholesterolemic. A significant reduction in [3H]CsA uptake and toxicity was observed when LLC-PK1 cells were treated with increased concentrations of LDL. Purpose: Based on the experimental and clinical evidence, it is hypothesized that when the LDL receptor family activity is decreased upon IgG-C7 treatment, both CsA uptake and toxicity are reduced in LLC-PK1 cells, a renal proximal tubule cell line. Methods: The appropriateness of LLC-PK1 cells as a cell model was assessed by conducting dose-response, LDL specific binding and competitive studies with Dil- LDL, and Western blot analysis of the LDL receptor. Assay conditions with IgG-C7, a monoclonal antibody to the LDL receptor, were optimized including temperature, preincubation time and concentration in LLC-PK1 cells. Finally, the effect of IgG-C7 on [3H]CsA uptake and toxicity with LDL was determined. Results: Significant results in both mean percent bound (2.6% ± 0.6% vs. 5.1% ± 1.3%) and mean percent toxicity (1.8% ± 0.5% vs. 3.2% ± 0.5%) were observed in the [3H]CsA alone group in the presence of IgG-C7 versus its absence (p<0.05 with unpaired t-test). However, no significant differences were observed in the [3H]CsA- LDL complex or [3H]CsA with LDL coaddition groups. In addition, LDL was not associated with a significant reduction in both [3H]CsA bound, uptake and toxicity. Conclusion: These results suggest that CsA may be binding directly to the LDL receptor family independent of its association with LDL and thus, eliciting its toxic effects at the membrane level. This study provides preliminary evidence of the family of LDL receptors playing a role in CsA binding and toxicity in LLC-PK1 cells.

Thesis/Dissertation

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2009-11-21

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http://hdl.handle.net/2429/15474

Pharmaceutical Sciences

University of British Columbia

UBCV

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