HIV-1 Alters Intestinal Expression of Drug Transporters and Metabolic Enzymes: Implications for Antiretroviral Drug Disposition

Abstract

This study investigated the effects of HIV-1 infection and antiretroviral therapy (ART) on the expression of intestinal drug efflux transporters, i.e., P-glycoprotein (Pgp), multidrug resistance-associated proteins (MRPs), and breast cancer resistance protein (BCRP), and metabolic enzymes, such as cytochrome P450s (CYPs), in the human upper intestinal tract. Intestinal biopsy specimens were obtained from HIV-negative healthy volunteers, ART-naive HIV-positive (HIV(+)) subjects, and HIV(+) subjects receiving ART (10 in each group). Intestinal tissue expression of drug transporters and metabolic enzymes was examined by microarray, real-time quantitative reverse transcription-PCR (qPCR), and immunohistochemistry analyses. Microarray analysis demonstrated significantly lower expression of CYP3A4 and ABCC2/MRP2 in the HIV(+) ART-naive group than in uninfected subjects. qPCR analysis confirmed significantly lower expression of ABCC2/MRP2 in ART-naive subjects than in the control group, while CYP3A4 and ABCG2/BCRP showed a trend toward decreased expression. Protein expression of MRP2 and BCRP was also significantly lower in the HIV(+) naive group than in the control group and was partially restored to baseline levels in HIV(+) subjects receiving ART. In contrast, gene and protein expression of ABCB1/Pgp was significantly increased in HIV(+) subjects on ART relative to HIV(+) ART-naive subjects. These data demonstrate that the expression of drug-metabolizing enzymes and efflux transporters is significantly altered in therapy-naive HIV(+) subjects and in those receiving ART. Since CYP3A4, Pgp, MRPs, and BCRP metabolize or transport many antiretroviral drugs, their altered expression with HIV infection may negatively impact drug pharmacokinetics in HIV(+) subjects. This has clinical implications when using data from healthy volunteers to guide ART.

FIG 1

DNA microarray analysis of mucosal gene expression in treated and untreated HIV-1 infection. (A) Gene expression changes in the gut biopsy specimens of HIV-negative participants (n = 5), HIV⁺ ART-naive participants (n = 7), and HIV⁺ participants on ART (n = 6). The patients were arranged based on when the microarray sample was analyzed (see Materials and Methods for details). (B) Differentially expressed genes with important pharmacological and physiological functions, such as genes for metabolic enzymes, drug efflux and uptake transporters (i.e., ABC and SLC drug transporters), nuclear receptors, and tight junctional proteins, and genes associated with the intestinal immune response and inflammation, were identified through hierarchical cluster analysis. For clarity, the sequence of patients was organized by patient identification number matching the arrangement that was used in panel A. Abbreviations: CYP, cytochrome P450 enzyme; UGT, UDP-glucuronyl transferase; Pgp, P-glycoprotein; MRP, multidrug resistance-associated protein; BCRP, breast cancer resistance protein; OATP, organic anion transporting polypeptide; OAT, organic anion transporter; OCT, organic cation transporter; CNT, concentrative nucleoside transporter; ENT, equilibrative nucleoside transporter; PXR, pregnane X receptor; CAR, constitutive androstane receptor; FXR, farnesoid X receptor; LXR, liver X receptor; PPAR, peroxisome proliferator-activated receptor; IL, interleukin; IFN, interferon; MCP, monocyte chemoattractant protein; COX, cyclooxygenase; MAPK, mitogen-activated protein kinase; NF, nuclear factor; TGF, transforming growth factor.

FIG 2

Real-time qPCR analysis of gene expression of selected intestinal metabolic enzymes and drug efflux transporters. Total RNA extracted from jejunal tissue biopsy specimen obtained from each subject was used for analysis of mRNA expression levels of each target gene using TaqMan gene expression assays specific for human CYP3A4 (A), ABCB1 (B), ABCC2 (C), or ABCG2 (D), obtained from Life Technologies Inc., Burlington, Ontario, Canada (assay Hs00604506_m1, Hs00184500_m1, Hs00166123_m1, or Hs01053790_m1, respectively). In each sample, the expression of the target gene was normalized to an internal control, GAPDH, by the ΔC_T method. Statistically significant differences in mRNA expression between groups (negative versus HIV⁺ naïve, negative versus HIV⁺ on ART, and HIV⁺ naive versus HIV⁺ on ART) were determined using GraphPad Prism (version 5.01; Graph Pad Software, San Diego, CA) by applying the nonparametric two-tailed Mann-Whitney test with significance defined by a P value of <0.05.

FIG 3

Immunohistochemistry analysis of protein expression of selected intestinal metabolic enzymes and drug efflux transporters. To detect protein expression and localization, paraffin-embedded jejunal tissue biopsy specimens from each subject, fixed in 4% paraformaldehyde and mounted onto glass slides, were immunostained with anti-CYP3A4 mouse polyclonal antibody from Sigma-Aldrich (Oakville, Ontario, Canada) at a 1:600 dilution (A), anti-Pgp mouse monoclonal D-11 antibody from Santa Cruz Biotechnology, Inc. (Dallas, TX) at a 1:100 dilution (B), anti-MRP2 mouse monoclonal M2III-6 antibody from Kamiya Biomedical Company (Seattle, WA) at a 1:250 dilution (C), or anti-BCRP rat monoclonal BXP-21 antibody from Abcam Inc. (Toronto, Ontario, Canada) at a 1:100 dilution (D). After washing, each tissue slice was immunostained with the corresponding Alexa-488-conjugated secondary antibodies, i.e., donkey anti-rat IgG for BCRP or donkey anti-mouse IgG for all other proteins (Life Technologies Inc., Burlington, Ontario, Canada). Standard DAPI staining was used to identify cell nuclei. For each gene of interest, the entire set of tissue slices was immunostained in a single experiment with all steps performed simultaneously, and images were obtained at constant exposure, zoom, and background settings using a Zeiss LSM700 confocal microscope.

FIG 4

Group comparison of protein expression levels estimated from relative fluorescence intensities of immunostained jejunal tissue slices. For each protein of interest, all fluorescent images obtained from tissue slices from each subject were analyzed using the Image Pro Premier software (Media Cybernetic, Rockville, MD). The fluorescence intensity (integrated optical density [OD], lumens/pixel²) for the protein of interest, i.e., CYP3A4 (A), Pgp (B), MRP2 (C), or BCRP (D), was normalized to the intensity of nuclear staining (i.e., the integrated OD measured for DAPI) within the same area. This analysis was performed in triplicate on three different areas within each tissue slice obtained from each study subject. The average normalized intensity ratio (gene of interest/DAPI) for each subject was used to estimate the mean ± standard error of the mean (SEM) for the study group. Statistically significant differences in protein expression between groups (negative versus HIV⁺ naïve, negative versus HIV⁺ on ART, and HIV⁺ naive versus HIV⁺ on ART) were determined using GraphPad Prism software, version 5.01 (Graph Pad Software, San Diego, CA) by applying the nonparametric two-tailed Mann-Whitney test with significance defined by a P value of <0.05.