| Summary: | 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. === Pharmaceutical Sciences, Faculty of === Graduate
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