Expression of ATP-binding cassette membrane transporters in a HIV-1 transgenic rat model

https://doi.org/10.1016/j.bbrc.2014.01.092Get rights and content

Highlights

  • We report mRNA expression of ABC drug transporters in a HIV transgenic rat model.

  • Transgenic rats transporter expression is significantly different from controls.

  • The HIV transgene regulates transporter expression in age & tissue specific manner.

Abstract

P-glycoprotein (P-gp, product of Mdr1a and Mdr1b genes), multidrug resistance associated proteins (Mrps), and breast cancer resistance protein (Bcrp), all members of the ATP-binding cassette (ABC) membrane-associated drug transporters superfamily, can significantly restrict the entry of antiretroviral drugs (ARVs) into organs which exhibit a barrier function such as the central nervous system (CNS) and the male genital tract (MGT). In vitro, HIV-1 viral proteins such as glycoprotein-120 (gp120) and transcriptional transactivator (tat) have been shown to alter the expression of these transporters and ARVs permeability. The objective of this study was to compare mRNA expression of these transporters, in vivo, in several tissues obtained from HIV-1 transgenic rats (Tg-rat) (8 and 24 weeks) with those of age-matched wild-type rats. At 24 weeks, significant changes in several drug transporter mRNA expressions were observed, in particular, in brain, kidney, liver and testes. These findings suggest that HIV-1 viral proteins can alter the expression of ABC drug transporters, in vivo, in the context of HIV-1 and further regulate ARVs permeability in several organs including the CNS and MGT, two sites which have been reported to display very low ARVs permeability in the clinic.

Introduction

Successful treatment of human immunodeficiency virus type-1 (HIV-1) infection requires antiretroviral drugs (ARVs) to reach effective therapeutic concentrations within the host’s plasma and tissues. Despite the implementation of highly active antiretroviral therapy for the treatment of HIV infection and its success in eradicating viral load in the periphery, several tissues have been identified as viral sanctuary sites including the central nervous system (CNS), the male genital tract (MGT), gut-associated lymphatic tissues and renal epithelium [1], [2]. These sites allow HIV replication to occur even in the presence of therapeutic concentrations of ARVs in plasma [3]. Furthermore, clinical studies have documented limited penetration of ARVs into several of these sites protected by unique blood–tissue barriers such as the blood–brain barrier (BBB) of the CNS [4] and the blood–testis barrier of the MGT [5].

One potential factor which may contribute to reduced tissue ARVs permeability is the expression of ATP-binding cassette (ABC) membrane-associated drug efflux transporters i.e., P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp) and multidrug resistance associated proteins (Mrp), which can efflux extracellularly a wide range of substrates including many ARVs [6]. P-gp is encoded by ABCB1 gene in humans and abcb1a and abcb1b genes in rodents and is expressed in many tissues including the brain (BBB), the MGT (blood–testis barrier), heart, liver, kidney and gastrointestinal tract [7]. P-gp is capable of transporting many ARVs i.e., protease inhibitors (PI), nucleoside/nucleotide reverse transcriptase inhibitors, (NRTI), integrase-strand transfer inhibitors and the CCR5 antagonist, maraviroc [6]. BCRP/Bcrp, encoded by ABCG2 and Abcg2 in humans and rodents, respectively, is also expressed at the BBB, the blood-testis barrier, kidney, liver and gastrointestinal tract [8] and can transport several NRTIs [9]. PIs have also been shown to inhibit both P-gp and Bcrp [10], [11]. Encoded by ABCC1 in humans and Abcc1 in rodents, MRP1/Mrp1 is expressed in many tissues including lung, testis, kidney, skeletal and cardiac muscles, placenta and macrophages [12], [13], and similar to P-gp, is capable of transporting many of the PIs [14]. MRP4/Mrp4 and MRP5/Mrp5 encoded by ABCC4/Abcc4 and ABCC5/Abcc5 respectively are predominantly cyclic nucleoside/nucleotide monophosphate transporters that are also capable of exporting several NRTIs such as abacavir, tenofovir and zidovudine [15], [16]. Both transporters are known to be expressed in several tissues and cell-types including blood–brain and blood–testis barriers, kidney, hepatocytes and platelets [17]. The expression of P-gp, Bcrp, Mrp1, 4 and 5 could contribute to the reduced tissue concentrations of ARVs observed clinically in HIV infected patients [3].

Transgenic animal models are useful to study the role of viral proteins in HIV pathogenesis in tissues that are not directly infected with the actively-replicating virus. About a decade ago, Reid et al. developed a HIV transgenic rat (Tg-rat) model in which affected animals express a modified HIV transgene which has a functional deletion of the gag and pol genes that renders the virus non-infectious [18]. Due to the expression of the modified HIV transgene and subsequent expression of viral proteins, Tg-rats progressively develop immune abnormalities, cognitive and motor deficits, muscle wasting, cataracts, nephropathy and skin lesions that are remarkably similar to HIV infection [18]. Furthermore, these rats also have circulating levels of several HIV viral proteins [18] such as glycoprotein-120 (gp120) and trans-activator of transcription (tat) which have been previously shown by our group and others to regulate the expression of ABC transporters i.e., P-gp and Mrp1, in rodent and human astrocytes and brain microvessel endothelial cells, independent of active viral replication [19], [20]. However, to the best of our knowledge, no studies have investigated the effect of the HIV transgene and viral proteins on ABC transporters mRNA expression in vivo. In this study, we examined the role of HIV viral proteins in the regulation of ABC transporter mRNA expression in several tissues obtained from two different ages of Tg-rats (8 and 24 weeks).

Section snippets

Animal model and tissue isolation

Male Sprague–Dawley Tg-rats and wild-type Sprague–Dawley rats (WT-rats) were purchased from Harlan Incorporated (Indianapolis, IN) at 6 weeks of age [18]. All rats were housed in pairs and provided with ad libitum food and water on a 12 h light/dark schedule. At 8 and 24 weeks of age, animals were anesthetized with 2-cc isoflurane and exsanguination was performed by cardiac puncture. Brain, heart, kidney, liver and testes were isolated and immediately frozen in liquid nitrogen. All animal

Comparison of HIV viral gp120 and tat mRNA expression between the 8 and 24 weeks old Tg-rat groups

Applying qPCR analysis, HIV gp120 mRNA expression was detected in all the tissues obtained from the Tg-rats. We observed a significant increase in HIV gp120 mRNA expression in 24 week Tg-rat kidneys (3.42 ± 1.72-fold) and liver (5.76 ± 3.45-fold) compared to 8 week Tg-rats (Fig. 1A). Similar to HIV gp120, mRNA expression of HIV tat was observed in all the tissues tested with a trend in an increase in the expression at 24 weeks compared to the younger Tg-rat group (Fig. 1B).

Comparison of ABC transporters mRNA expression between WT-rats and Tg-rats at 8 and 24 weeks of age

To determine the effect of

Discussion

The Tg-rat provides a unique animal model to investigate the effects of HIV-1 viral transgene on various pathologies related to HIV-1 infection including: chronic inflammation, increased oxidative stress and chronic immune dysfunction [18], [23], [24], [25]. In this study, we report for the first time that the expression of the HIV viral transgene can significantly alter, in vivo, in an age-dependent and tissue-specific manner, the relative mRNA expression of ABC drug transporters i.e., P-gp,

Acknowledgments

This research was funded by operating Grants from the Canadian Institutes of Health Research and the Ontario HIV Treatment Network (OHTN). Dr. Reina Bendayan is a Career Scientist of the OHTN, Ministry of Health of Ontario.

References (42)

  • B. McGee et al.

    HIV-1 pharmacology: barriers to the eradication of HIV from the CNS

    HIV Clinical Trials

    (2006)
  • D.M. Smith et al.

    Long-term persistence of transmitted HIV drug resistance in male genital tract secretions: implications for secondary transmission

    J. Infect. Dis.

    (2007)
  • I. Cascorbi

    P-glycoprotein: tissue distribution, substrates, and functional consequences of genetic variations

    Handb. Exp. Pharmacol.

    (2011)
  • M. Maliepaard et al.

    Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues

    Cancer Res.

    (2001)
  • X. Wang et al.

    The role of breast cancer resistance protein (BCRP/ABCG2) in cellular resistance to HIV-1 nucleoside reverse transcriptase inhibitors

    Antiviral Chem. Chemother.

    (2005)
  • A. Gupta et al.

    HIV protease inhibitors are inhibitors but not substrates of the human breast cancer resistance protein (BCRP/ABCG2)

    J. Pharmacol. Exp. Ther.

    (2004)
  • R.G. Deeley et al.

    Transmembrane transport of endo- and xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins

    Physiol. Rev.

    (2006)
  • E.R. Meaden et al.

    P-glycoprotein and MRP1 expression and reduced ritonavir and saquinavir accumulation in HIV-infected individuals

    J. Antimicrob. Chemother.

    (2002)
  • T. Imaoka et al.

    Functional involvement of multidrug resistance-associated protein 4 (MRP4/ABCC4) in the renal elimination of the antiviral drugs adefovir and tenofovir

    Mol. Pharmacol.

    (2007)
  • C.A. Ritter et al.

    Cellular export of drugs and signaling molecules by the ATP-binding cassette transporters MRP4 (ABCC4) and MRP5 (ABCC5)

    Drug Metab. Rev.

    (2005)
  • D. Keppler

    Multidrug resistance proteins (MRPs, ABCCs): importance for pathophysiology and drug therapy

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