Suitability of LLC-PK1 pig kidney cells for the study of drug action on renal cell cholesterol uptake: identification and characterization of low-density lipoprotein receptors.
ABSTRACT The purpose of this study was to identify and characterize the presence of low-density lipoprotein receptors (LDLr) in LLC-PK(1) cells.
LLC-PK(1) cells were assessed for the presence of LDLr by conducting dose-response, LDL specific binding and competitive studies with DiI-LDL, and Western blot and RT-polymerase chain reaction (PCR) analyses. Assay conditions with IgG-C7, a monoclonal antibody (mAb) to the LDLr, were optimized, including temperature, preincubation time, and concentration in LLC-PK(1) cells.
LLC-PK(1) cells express LDL receptors as determined by LDL specific and competitive binding studies and Western blot and RT-PCR analysis (specific binding 0.5 ng DiI-LDL/mug of cellular protein).
Taken together, these findings confirm the presence of LDL receptors on LLC-PK1 cells and support the appropriateness of using these cells in studies involving renal cell cholesterol uptake and metabolism.
Article: Modifications in low-density lipoprotein receptor expression affects Cyclosporin A cellular uptake and cytotoxicity.[show abstract] [hide abstract]
ABSTRACT: The purpose of this study was to test the effect of modulating the expression of the human low-density lipoprotein receptor (LDLr) in human embryonic kidney (293T) cells on Cyclosporin A (CsA) cellular uptake and CsA-mediated cytotoxicity. LDLr expression was modulated using RNA interference (RNAi) and an LDLr overexpression plasmid. One of the small-interfering RNA (siRNA) constructs, LDLr-792, showed a 60% decrease in LDLr protein expression. The downregulation effect was specific as transfection with an annexin V (AxV) siRNA construct did not decrease LDLr expression levels. AxV and ABCA1 expression levels were not affected in the cells transfected with LDLr-792 (LDLr(LOW) cells) compared to the controls. At a functional level, fluorescent low-density lipoprotein (LDL) (DiI-LDL) internalization in the LDLr(LOW) cells was decreased (30%) compared to control cells. We tested the dose-dependent cytotoxicity induced by CsA using a respiration assay. We found a decrease in CsA-mediated cytotoxicity in the range of CsA doses studied (1-10 microg/mL) in the LDLr(LOW) cells compared to the pSHAG-transfected cells, reaching a statistical significance at 10 microg/mL CsA. At higher CsA doses we found a significant decrease in LDLr expression. When the control and LDLr(LOW) cells were treated with another cytotoxic drug, gentamycin, there was no difference in the cell viability, suggesting that this effect is specific for CsA. We confirmed the association of LDLr expression levels with CsA uptake by overexpressing the LDLr. The LDLr overexpressing cells showed an enhanced uptake of radiolabelled CsA. Taken together these results suggest that CsA internalization and cytotoxicity are affected by the LDL receptor expression levels.Journal of Pharmaceutical Sciences 07/2008; 97(6):2350-61. · 3.06 Impact Factor