Biochemical and Biophysical Research Communications
Expression of ATP-binding cassette membrane transporters in a HIV-1 transgenic rat model
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.
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