Xenobiotica, 2009; 39(10): 738–748
RESEARCH ARTICLE
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Tissue-specific changes in mRNA expression of Abc and Slc transporters in murine pulmonary tuberculosis S. H. Lee1,2, T. Oh3, B.-Y. Jeon3, E.-Y. Kwak1,2, W.-S Shim1,2, S.-N Cho3, D.-D Kim2, S.-J Chung2, and C.-K Shim1,2 National Research Laboratory for Transporters Targeted Drug Design and, 2Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, Korea, and 3Department of Microbiology and the Brain Korea 21 Project for the Medical Sciences, Yonsei University College of Medicine, Seoul, Korea 1
Abstract 1. A pulmonary tuberculosis mouse model was used to assess the pharmacodynamic and pharmacokinetic characteristics of tuberculosis therapeutics. While membrane transporters play important roles in drug disposition and physiological homeostasis, their expressional changes and contribution have never been analysed in a tuberculosis animal model. 2. The mRNA expression level of 20 Abc family transporters and 32 Slc family transporters in tuberculosisinfected mice were compared with those in naïve uninfected mice using real-time polymerase chain reaction (PCR). Mycobacterium tuberculosis infection induced many dramatic expression changes of families of both Abc transporters and Slc transporters at 4 and 8 weeks, as observed in the livers, kidneys, and intestines of test mice — and in a different mode, in the lungs and spleens as well. These changes were dependent on the tuberculosis progression with the tissue-specific manner, that is, in the lungs, the number of transporters of which the expression level changed due to M. tuberculosis infection had increased, and the magnitude of change also greater at 8 weeks, while in the spleen, the transcription of most transporters except Mrps had not changed or had recovered back to the same level of naïve transcription at 8 weeks. 3. Understanding the expression changes of transporters will assist in setting up rational preclinical dosing plans through the ability to predict the pharmacokinetics of new anti-tuberculosis chemotherapeutics and, furthermore, will assist in the design of safer and more efficient drug regimens. Keywords: Tuberculosis; transporter; mouse infection; real-time polymerase chain reaction (PCR)
Introduction Membrane transporters, expressed in various tissues such as intestine, kidney, liver, lung, and brain, have primarily been known to facilitate the transport of substrates through the cells for nutrient absorption, protection from endogenous and exogenous toxins, and ion balance. Many studies have shown these transporters to be generally involved in the absorption, distribution, and excretion of various drugs, thereby affecting their therapeutic efficacy and toxicity (Shitara et al. 2006; Tsuji 2006). Transporters can also transport endogenous substrates, such
as inflammatory mediators, hormones, bile acids, nucleotides, and glutathione, and might have a role in modulating cellular signalling, such as adenosinemonophosphate kinase (AMPK) and cyclic-AMP (cAMP)-dependent signalling pathways (Loffler et al. 2007; Russel et al. 2008). Membrane transporters impact not only drug disposition, but also the maintenance of cellular homeostasis. Thus, knowledge of the alteration of transporter expression in diseases will facilitate a deeper understanding of the pharmacokinetic characteristics of a drug and of the pathophysiological change in the disease state (Cheng et al. 2008; Morgan et al. 2008).
Address for Correspondence: C.-K. Shim, National Research Laboratory for Transporters Targeted Drug Design, College of Pharmacy, Seoul National University, 599 Gwanangno, Gwanak-gu, Seoul 151-742, Korea. Tel: 82-2-880-7873. Fax: 82-2-885-8429. E-mail:
[email protected] (Received 17 April 2009; revised 02 June 2009; accepted 03 June 2009) ISSN 0049-8254 print/ISSN 1366-5928 online © 2009 Informa UK Ltd DOI: 10.1080/00498250903089829
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Transporters expression in pulmonary tuberculosis 739 Tuberculosis (TB) is a typical infectious disease that should be studied using a live pathogen infection model, because Mycobacterium tuberculosis (Mtb) resides intracellularly and interacts with the host for its lifetime (North & Jung 2004; Kaufmann et al. 2005). The current regimens of TB treatment use a combination of three or four drugs including rifampicin and isoniazid (Nunn et al. 2008). They are effective for drug-susceptible TB but ineffective to latent TB infection. The emergence of multi-drug-resistant/extensively drug-resistant TB together with human immunodeficiency virus (HIV) coinfection has made it necessary for research to improve drug efficacy and to optimize current therapeutic regimens (Sacks & Behrman 2008). Novel chemotherapeutic agents and new drug regimens are actively being developed along with in vitro and in vivo evaluation systems (Lenaerts et al. 2008; Nathan et al. 2008). In the present study, we analysed the expression changes of membrane transporters that would affect drug disposition and cellular homeostasis using TB-infected mice as a model. This mouse model was utilized in predicting the potential efficacy of new anti-TB compounds and drug combinations, and in characterizing their pharmacokinetics and toxicity (Basaraba 2008; Lenaerts et al. 2008). Understanding the behaviour of transporters will assist in setting up more rational preclinical dosing plans through the prediction of the pharmacokinetics of new anti-TB chemotherapeutics. Furthermore, knowledge of the pathogenesis mechanism will facilitate the design of safer and more efficient drug regimens.
Materials and methods Mice Specific pathogen-free female C57BL/6 mice at 5–6 weeks of age were purchased from Japan SLC, Inc. (Shijuoka, Japan) and maintained under barrier conditions in a BL-3 biohazard animal room at Yonsei University Medical Research Center. The mice were fed a sterile commercial mouse diet and given water ad libitum. Bacteria Mycobacterium tuberculosis H37Rv (ATCC27294) was used in the experiments and was grown for about 10 days at 37°C as a surface pellicle on Sauton medium enriched with 0.4% sodium glutamate and 3.0% glycerine. The surface pellicles were collected and disrupted with 6 mm glass beads by gentle vortexing. After the clumps settled, the upper suspension was collected and aliquots were stored at −70°C until use. After thawing, viable organisms were then enumerated by plating serial
dilutions on Middlebrook 7H11 agar (Difco, Detroit, MI, USA). For inoculation of M. tuberculosis into mice, bacterial suspension was sonicated briefly in a sonic bath and diluted with phosphate-buffered saline to reach the desired numbers. M. tuberculosis infection and bacterial counts Briefly, mice were exposed to a predetermined dose of Mtb for 30 min in the inhalation chamber of an airborne infection apparatus (Glas-Col, Terre Haute, IN, USA). Bacteria were counted 1 day after exposure to measure the initial load of viable Mtb that had been delivered to the lungs. Bacterial counts were taken from the lungs and spleens of mice at 4 and 8 weeks after the aerosol infection. The number of viable bacteria in the lungs and spleens were determined by plating serial dilutions of whole-organ homogenates on Middlebrook 7H11 agar (Difco). Colonies were counted after 3–4 weeks of incubation at 37°C. The resulting values were based on the experimental group of five mice and are presented as the mean values of log10CFU ± standard error of the mean (SEM) per organ, where CFU is colony-forming units. RNA isolation and quantitative real-time PCR Primers specific for each transporter and cytokine were designed using Primer3 software (Rozen & Skaletsky 2000). The sequences for each primer are listed in Table 1. The tissues for real-time PCR analysis were collected from three mice at 4 weeks, from five mice at 8 weeks, and from five naïve mice. Total RNA was extracted from the livers, kidneys, lungs, spleens, and from three parts of the intestine (duodenum, jejunum and ileum) of TB-infected, and naïve mice using Trizol™ reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), in which tissues were homogenized. Reverse transcription was performed using an RNA LA PCR kit Ver. 1.1 (Takara Bio Co., Shiga, Japan) under the following conditions: 42°C for 60 min, 99°C for 5 min, and 5°C for 5 min. mRNA expression levels for the transporters in the TB-infected mice were determined using either an Applied Biosystems 7300 or 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Quantitation of mRNA expression was performed using SYBR Premix Ex Taq™ (Takara Bio Co., Shiga, Japan). The cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 5 s, and extension at 72°C for 35s. To confirm the specific amplification product from non-specific products or primer dimers, a melting curve was constructed from the dissociation stage that was programmed using an ABI Real-Time PCR system.
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740 S. H. Lee et al. Table 1. Primers used for real-time polymerase chain reaction (PCR) analysis. mRNA (GeneBank No.) Other names Forward primer (position) Abca1 (NM_013454) Abc1 5´-ccagacagttgtggatgtgg-3´ (7034) Abca3 (NM_013855) Abc-c 5´-ctgggccaaggtatttggta-3´ (5511) Abca7 (NM_013850) 5´-tccctcaattggtacgaagc-3´ (700) Abca8a (NM_153145) 5´-cagaaagcgaagggaaactg-3´ (2219) Abca8b (NM_013851) 5´-tggatccatccaacatctca-3´ (4702) Bsep (NM_021022) Abcb11 5´-ctgccaaggatgctaatgca-3´ (1719) Mdr1a (NM_011076) Mdr3, P-gp, Abcb1a 5´-agggtctaggcttgctgtga-3´ (2611) Mdr1b (NM_011075) Mdr1, Abcb1b 5´-gtgggggacagaaacagaga-3´ (1701) Mdr2 (NM_008830) Abcb4 5´-aggtggtggagttcttgtgg-3´ (553) Mrp1 (NM_008576) Abcc1 5´-gccctctttgcagtcatctc-3´ (3670) Mrp2 (NM_013806) Abcc2 5´-gcactgtaggctctgggaag-3´ (2102) Mrp3 (NM_029600) Abcc3 5´-gcagagacaggcaatgtgaa-3´ (2891) Mrp4 (NM_001033336) Moat-B, Abcc4 5´-caaagacatcggacacatgg-3´ (2572) Mrp5 (NM_013790) Abcc5 5´-cccaggagggagaagtaacc-3´ (3759) Mrp6 (NM_023732) Abcc6 5´-gaagtcctccctgctgtctg-3´ (2003) Mrp7 (NM_145140) Abcc10 5´-ctgcacgtctaccgagcata-3´ (2686) Bcrp (NM_011920) Abcg2 5´-gccttggagtactttgcatca-3´ (1270) Abcg1 (NM_009593) 5´-ggagaatgcgaagctgtacc-3´ (2014) Abcg5 (NM_031884) 5´-tcaggaccccaaggtcatgat-3´ (677) Abcg8 (NM_026180) 5´-gacagcttcacagcccacaa-3´ (834) 5´-tacagcttcaccaccacagc-3´ (671) -Actin (NM_007393) IL-10 (NM_010548) 5´-ccagaaatcaaggagcatt-3´ (381) IL-12 (NM_008352 ) 5´-ctcacatctgctgctccac-3´ (458) 5´-caggcaggcagtatcactc-3´ (247) IL-1 (NM_008361) IL-6 (NM_031168) 5´-gtccggagaggagacttca-3´ (118) 5´-aaatcctgcagagccagat-3´ (298) INF- (NM_008337) 5´-caaagggatgagaagttcc-3´ (330) TNF- (NM_013693) Oat1 (NM_008766) Slc22a6 5´-tgatggctgggtctatgaca-3´ (568) Oat2 (NM_144856) Slc22a7 5´-gggacagctcaccaagtagc-3´ (1665) Oat3 (NM_031194) Slc22a8 5´-ccagaagcagaaaaggcatc-3´ (1844) Oat5 (NM_144785) Slc22a9 5´-gaggagcgctgtctcctcta-3´ (1519) Oct1 (NM_009202) Slc22a1 5´-ccaatagcggcatcaaatct-3´ (1395) Oct2 (NM_013667) Slc22a2 5´-aaatggtctgcctggtcaac-3´ (1435) Oct3 (NM_011395) Slc22a3 5´-aatatcctgtttcggcgttg-3´ (923) Octn1 (NM_019687) Slc22a4 5´-cctctctggcctgattgaag-3´ (1245) Octn2 (NM_011396) Slc22a5 5´-gggaagtctgaccatcctga-3´ (1606) Octn3 (NM_019723) Slc22a21 5´-ggattgttgcaccttccact-3´ (1084) Urat1 (NM_009203) Slc22a12 5´-gagggagacacgttgaccat-3´ (1185) Oatp1a1 (NM_013797) Oatp1, Slc21a1 5´-tcaaggggagttgtgtttcc-3´ (2431) Oatp1a4 (NM_030687) Oatp2, Slc21a5 5´-aatgccaaagaggagaagca-3´ (1139) Oatp1a5 (NM_130861) Oatp3, Slc21a7 5´-tgtgtggagacaatggccta-3´ (1451) Oatp1a6 (NM_023718) Oatp5, Slc21a13 5´-gggaacaatggcctagcata-3´ (1394) Oatp1b2 (NM_020495) Oatp4, Slc21a10 5´-caaactcagcatccaagcaa-3´ (204) Oatp2a1 (NM_033314) Pgt, Slc21a2 5´-tctccctttcctttcccact-3´ (2664) Oatp2b1 (NM_175316) OATP-B, Slc21a9 5´-tgcaggttcctgtgagtcag-3´ (1706) Oatp3a1 (NM_023908) OATP-D, Slc21a11 5´-ctcaccagctccatttccat-3´ (2992) Oatp4c1 (NM_172658) OATP-H, Slc21a20 5´-tgccattggatatgtgttgg-3´ (881) Pept1 (NM_053079) Slc15a1 5´-agctctgatcgcagactcgt-3´ (242) Pept2 (NM_021301) Slc15a2 5´-atcattggttggcctgagtc-3´ (638) Cnt1 (NM_001114184) Slc28a1 5´-gctgtttgagtggatcagca-3´ (335) Cnt2 (NM_172980) Slc28a2 5´-atttgtggcctaccagcaac-3´ (2810) Cnt3 (NM_022317) Slc28a3 5´-attggttctggctgaaatgg-3´ (621)
Reverse primer (position) 5´-cctgtgtgaacgggattctt-3´ (7125) 5´-gggagatctggctcacagag-3´ (5590) 5´-cagggctgagactgttgtca-3´ (787) 5´-ccaggtctggacttctctcg-3´ (2314) 5´-gcctgaggatctccgtattg-3´ (4810) 5´-cgatggctaccctttgcttct-3´ (1835) 5´-cactccagctatcgcaatga-3´ (2743) 5´-tgcaccacagcttcactttc-3´ (1815) 5´-caaaccagcccatttcttgt-3´ (680) 5´-cagtctctccactgccacaa-3´ (3819) 5´-tgctgagggacgtaggctat-3´ (2210) 5´-ccaccatacaggaggcagat-3´ (2985) 5´-agcggaaccaatggtatgag-3´ (2706) 5´-aggacttccctgaccctgtt-3´ (3899) 5´-tgcctgaagcacacattctc-3´ (2143) 5´-cagctgagacagccagtgag-3´ (2817) 5´-aaatccgcagggttgttgta-3´ (1330) 5´-ggaggcggtttttacctctc-3´ (2133) 5´-aggctggtggatggtgacaat-3´ (809) 5´-gcctgaagatgtcagagcga-3´ (932) 5´-tctccagggaggaagaggat-3´ (791) 5´-cactcttcacctgctccac-3´ (509) 5´-tccggagtaatttggtgct-3´ (568) 5´-catgagtcacagaggatgg-3´ (389) 5´-tttccacgatttcccagag-3´ (225) 5´-ttgctgttgctgaagaagg-3´ (429) 5´-tccacttggtggtttgcta-3´ (462) 5´-gccaggtagccaaacatcat-3´ (717) 5´-agcaccagtagcagctccat-3´ (1758) 5´-tctctggaaggcaggaaaga-3´ (1924) 5´-gcagaggctgattcttggtc-3´ (1653) 5´-gaccatctgcaacacaatgg-3´ (1529) 5´-tccagccagatgtcagtgag-3´ (1573) 5´-tcacgatcacgaagcaagtc-3´ (1056) 5´-cacacctcctccccagaata-3´ (1353) 5´-tgcattcttgtctggctttg-3´ (1746) 5´-cataaatgtggtgcgactgg-3´ (1176) 5´-agcccaggtgtgtggagtag-3´ (1291) 5´-ttcaatgtgtcccaaccaga-3´ (2576) 5´-aaggcattgacctggatcac-3´ (1284) 5´-gcagctgcaattttgaaaca-3´ (1551) 5´-gcagctgcaattttgaaaca-3´ (1489) 5´-ggctgccaaaaatatcctga-3´ (290) 5´-gttccttcgttcctgctctg-3´ (2777) 5´-cacacctctcaggatgagca-3´ (1829) 5´-cacagcttcctgaggtcaca-3´ (3118) 5´-ggggatcatcctcagtcaga-3´ (972) 5´-cgtgtagacgatggatagtgaaa-3´ (309) 5´-gcctctgcatgttcctcttc-3´ (739) 5´-accacacaggtgatgacgaa-3´ (451) 5´-gcaaatccacagagggaaaa-3´ (2944) 5´-gatgagtccgccaaaggata-3´ (739) Table 1. continued on next page
Transporters expression in pulmonary tuberculosis 741 Table 1. Continued. mRNA (GeneBank No.) Ent1 (NM_022880) Ent2 (NM_007854) Ntcp (NM_011387) Asbt (NM_011388) Npc1L1 (NM_207242) Ost- (NM_145932) Ost- (NM_178933)
Other names Slc29a1 Slc29a2 Slc10a1 Slc10a2
Forward primer (position) 5´-ctgtgactgctgaggtggaa-3´ (1106) 5´-caggtgggattcgactgtct-3´ (1838) 5´-ggtgccctacaaaggcatta-3´ (499) 5´-tggaatgcagaacactcagc-3´ (835) 5´-atcgcactaccatccaggac-3´ (1877) 5´-gttgccatttttctggagga-3´ (550) 5´-ggaactgctggaagaaatgc-3´ (105)
The relative quantity of each mRNA to -actin mRNA in each tissue was calculated using the CT method of 7500 software for the 7500 Real-Time PCR System ver. 2.0.1 (Applied Biosystems). The ratio of target mRNA in each tissue of a TB-infected mouse to the same target mRNA in the same kind of tissue of a naïve mouse was calculated by the comparative CT (CT) method using the 7500 software, and the data are expressed as such. A 95% confidence interval was calculated using the 7500 software, and the data were analysed using two-tailed unpaired t-tests. A difference in mean values with a value of p
