Expression Profile of Drug and Nutrient Absorption Related Genes in Madin-Darby Canine Kidney (MDCK) Cells Grown under Differentiation Conditions

Abstract

The expression levels of genes involved in drug and nutrient absorption were evaluated in the Madin-Darby Canine Kidney (MDCK) in vitro drug absorption model. MDCK cells were grown on plastic surfaces (for 3 days) or on Transwell® membranes (for 3, 5, 7, and 9 days). The expression profile of genes including ABC transporters, SLC transporters, and cytochrome P450 (CYP) enzymes was determined using the Affymetrix® Canine GeneChip®. Expression of genes whose probe sets passed a stringent confirmation process was examined. Expression of a few transporter (MDR1, PEPT1 and PEPT2) genes in MDCK cells was confirmed by RT-PCR. The overall gene expression profile was strongly influenced by the type of support the cells were grown on. After 3 days of growth, expression of 28% of the genes was statistically different (1.5-fold cutoff, p < 0.05) between the cells grown on plastic and Transwell® membranes. When cells were differentiated on Transwell® membranes, large changes in gene expression profile were observed during the early stages, which then stabilized after 5-7 days. Only a small number of genes encoding drug absorption related SLC, ABC, and CYP were detected in MDCK cells, and most of them exhibited low hybridization signals. Results from this study provide valuable reference information on endogenous gene expression in MDCK cells that could assist in design of drug-transporter and/or drug-enzyme interaction studies, and help interpret the contributions of various transporters and metabolic enzymes in studies with MDCK cells.

Figure 1

Principal Component Analysis (PCA) of gene expression profiles of MDCK cells in various culture conditions. Expression values from a total of 5554 confirmed probe sets that were considered present in at least one condition were used for analysis.

Figure 2

Expression profiles of ABC transporter genes involved in the drug absorption process.

Figure 3

Expression profiles of select SLC transporters involved in drug and nutrient absorption in MDCK cells grown under various conditions. Transporters are divided into 3 groups according to their expression patterns. (A) transporters whose Affymetrix signals were above 1000; (B) transporters whose Affymetrix signals were below 1000 and expressed in all conditions; and (C) transporters whose Affymetrix signals were below 1000 and not expressed on plastic surface.

Figure 4

Expression profiles of CYP enzymes in MDCK cells grown under various conditions.

Figure 5

qRT-PCR expression of SLC15A1 (PEPT1), SLC15A2 (PEPT2) and ABCB1 (MDR1) in MDCK cells carrying empty vector. The mRNA of PEPT1 was not detected. The data was expressed as relative mRNA abundance after normalization for GAPDH expression.