Effect of Inflammation on Molecular Targets and Drug Transporters

Inflammation, the host’s response to infection and injury, is associated with altered expression of genes such as metabolizing enzymes, transporters, receptors and plasma proteins. The purpose of the present work was to characterize the effect of inflammation on selected molecular targets and transporters that affect drugs’ action and disposition. We have used rats with adjuvant arthritis (AA), an animal model of chronic inflammation. The AA group received 0.2 ml of 50 mg ml -1 Mycobacterium butyricum suspended in squalene into the tail base. On day 12, the rats were euthanized and their organs (heart, liver, kidneys and intestine) excised. Expression of Cav1.2, β 1-AR, β 2-AR, α 1A-AR, Nav1.2, Nav1.6, Kv1.5, Kv2.1, Kv3.1, oatp1a1, oatp1a5, oatp1b2, oatp2b1, oatp4a1, oat2, oat3, oct1, mdr1a, bsep, mrp1, mrp3, mrp6, IL-1 α , IFN- γ , iNOS, MCP-1, IL-10, Cox-1 and Cox-2 were determined by real time polymerase chain reaction (RT-PCR). Inflammation resulted in a significant reduction of oct1, oatp4a1 and mrp1 gene expression in the liver and oatp2b1, mrp6 and bsep gene expression in the kidney. Oatp4a1 and mdr1a were found to be significantly upregulated in rat heart. In conclusion, inflammation alters the gene expression of some mediators and drug transporters that can influence the behavior of drugs in the body and contribute to therapeutic failure. This article is open to POST-PUBLICATION REVIEW . Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.


INTRODUCTION
Inflammation is the host's response to infection, injury or irritation. The development of the inflammatory response is a complex process that involves increased expression of chemicals such as cytokines and chemokines in a paracrine, autocrine, and/or endocrine fashion. This enhances the host's defense through immune cell recruitment and activation, antibody production and complement system activation [1]. The process has been found to be associated with altered expression of other genes either as a part of the host's defense mechanism or because of shifting priorities in the cellular expression machinery. Examples of the affected genes are metabolizing enzymes [2][3][4], transporters [5], receptors [6][7][8][9][10] and plasma proteins [11][12][13]. Inflammation-induced alterations of gene expression may be responsible, at least in part, for disease pathogenesis. For example, atrial fibrillation developed in septic shock patients [14,15] has been attributed to inflammation-induced L-type calcium channel dysfunction [16]. In addition, animal and human studies have shown that inflammation is an important factor that may alter drug action and/or disposition [6,8,[17][18][19][20][21][22]. Recently, we have reported that inflammation significantly downregulated angiotensin-converting enzyme-2 in rat heart, an observation that may contribute to the cardiovascular complications seen in inflammatory diseases [23]. The purpose of the present work was to investigate whether the effect of inflammation on target proteins goes beyond what has been reported earlier. We investigated the effect of inflammation on selected molecular targets and transporters that are known to be involved in the pharmacokinetics and pharmacodynamics of some drugs (Table 1). We have used rats with adjuvant arthritis (AA), an animal model of chronic inflammation that is known to mimic human rheumatoid arthritis [24].

Adrenergic Receptors
Each receptor provides important physiological functions mediated by the sympathetic nervous system. They are considered the main site of action of many drugs that are clinically in use such as β1 adrenoceptor and α1-blockers.

OTHERS: COX-1 and 2
Cyclooxygenases are important enzymes involved in the production of essential prostaglandins in all parts of the body.

IL-1α
An in inflammatory mediator; Activates T-cells and macrophages; Implicated in the pathogenesis of RA, septic shock and atherosclerosis [82].

MCP-1
A chemokine that Promotes monocytes and T cells chemotaxis to site of inflammation [82]

Experimental Protocol
The study protocol was approved by the Health Sciences Animal Policy and Welfare Committee of the University of Alberta. Experiments were carried out on male Sprague-Dawley rats (220-280 g). They were housed in a temperature-controlled room with a 12-h dark/light cycle. Two animal groups (n=4/group), inflamed (AA) and healthy (Control) were used. The inflamed group received 0.2 ml of 50 mg/ml Mycobacterium butyricum suspended in squalene into the tail base. Control animals received an equal volume of normal saline into the tail base. On day 12, when inflammation emerged, i.e., increased paw diameter and elevated proinflammatory mediators [19], rats were euthanized and their organs (heart, liver, kidneys and upper intestine) were excised and quickly frozen in liquid nitrogen. The excised organs were stored at -80 o .

Real Time Polymerase Chain Reaction (RT-PCR)
To determine the potential genetic changes, RT-PCR was carried out. Total RNA was isolated from the frozen organs using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. This was followed by spectrophotometric quantitation of the isolated RNA by measuring the absorbance at 260 nm. cDNA was synthesized from 1.5 μg total RNA samples with the random primers scheme using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Real-Time PCR was performed on an ABI 7500 Real-Time PCR system (Foster City, CA, USA).
Forward and reverse primers used for the experiments are depicted in Table 2. The housekeeping gene tested was the rat 18s rRNA. Melting curves were carried out to confirm amplification of single sequences and absence of primer dimers. Primers were purchased from Integrated DNA technologies (Coralville, IA, USA). PCR products were produced and detected quantitatively using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). PCR data were analyzed using the delta delta C T method (ΔΔC T ) as described by Livak and Schmittgen [25]. The data are expressed as fold change relative to a calibrator (Control) and normalized to the housekeeping gene.

Data Analysis
Data are expressed as mean ± SD. Statistical significance between the control and AA groups was analyzed using a two-tailed Student's t-test at p<0.05. Statistics were analyzed using Prism software v. 4.03 (GraphPad Software, Inc., San Diego, CA, USA).

Tested Genes in organs
The constitutive expression of drug transporters, adrenergic receptors, voltage-gated ion channels and COX 1 and 2 genes was found to be tissue specific (Figures 1 and 2). Tissue distribution of drug transporters in the liver, kidney and intestine was found to be similar to what has been previously reported (Table 3). In addition, MCP-1 was detected in the organs of Control rats.

Effect of Inflammation on the Tested Genes' mRNA
The influence of inflammation on the examined genes' mRNA in various organs is depicted in Figure 3.
Liver -Inflammation resulted in a significant reduction of oct1, oatp4a1 and mrp1 gene expression in the liver. There was also a general trend towards drug transporter downregulation in the liver except for mrp3 and mdr1a. The changes, however, were not statistically significant. There was no difference between AA and control animals with respect to COX-1, COX-2, ion channels and adrenergic receptors gene expression.

Figure 1.
Constitutive expression of drug transporter genes in different rat organs (n=4/group). Expression was determined using real time PCR in rat liver, kidney, heart and intestine. Gene expression was normalized to 18s rRNA. Relative expression was determined relative to a calibrator, the gene with the lowest expression in each organ. a, was not detected in the organ.
Heart -Out of the detected transporters in the hearts of control animals, oatp4a1 and mdr1a were found to be significantly increased by inflammation. Mrp3 was not detected in inflamed animals. Interestingly, despite being undetected in control animals, oatp1b2, oat2 and oat3 were detected in inflamed rats. There was no significant difference between AA and Control animals with respect to ion channels and adrenergic receptors gene expression. While not detectable in Control animals, COX-2 gene expression was detected in the AA group. On the other hand, inflammation significantly inhibited COX-1 gene expression.
Intestine -Due to the high variability observed in intestinal tissue, none of the tested genes were significantly different from the control. However, some transporters (such as oct1, mdr1a and mrp3) showed a strong trend towards downregulation.

DISCUSSION
We used the AA model, which mimics human rheumatoid arthritis in terms of development of systemic inflammation and arthritis [24]. Six days following administration of the adjuvant, the inflammatory mediators start to rise [19] and in approximately 12 days, animals develop arthritis. This model allowed us to study the effect of chronic inflammation on the gene expression of drug transporters, adrenergic receptors, ion channels and select mediators of biological relevance (Table 1).
MCP-1 was present in all of the examined organs of healthy and AA rats and demonstrated a trend toward increased expression in the kidney and heart in response to inflammation. MCP-1 has previously been detected in rat interstitial fibroblasts [26] and smooth muscle cells [27], Figure 2. Constitutive expression of voltage-gated ion channels, adrenergic receptors, COX-1 and COX-2 in different rat organs (n=4/group). Expression was determined using real time PCR in rat liver, kidney, heart and intestine. Gene expression was normalized to 18s rRNA. Relative expression was determined relative to a calibrator, the gene with the lowest expression in each organ. a, was not detected in the organ.  which is suggestive of a basal housekeeping function of the chemokine.
With regard to other examined inflammatory mediators (iNOS, IL-1a, INF-γ, IL-10), their presence was organ-dependant. Other observed changes remained insignificant, except for IFN-γ in the kidney, which was significantly increased by AA. Pronounced alterations of these markers have previously been reported in the rat joints and lymphoid organs such as spleen and lymph nodes, the main sites ofinflammation in adjuvant arthritis [28]. Lack of statistically significant alterations of the examined expressions can, in part, be attributed to the variability which depends on the disease severity [20,118].
COX-2 is an important enzyme responsible for the development of inflammation in the AA model. COX-2 (but not COX-1) expression is increased in AA rat paws and inhibition of COX-2 reverses the inflammatory response [28]. Determination of COX-1 and COX-2 genes expression in other AA rat organs has not been reported. COX-1 expression was significantly and substantially (by 80%) reduced by inflammation only in the heart. There was a similar strong trend for intestine that did not reach significance. For the other examined organs, the present data do not suggest significant changes in either COX-1 or COX-2 gene expression in response to inflammation. Reduced expression of COX-1 has previously been observed in the hearts and lungs of rats treated with lipopolysaccharides [29,30]. The observed down-regulation of cardiac COX-1 may have therapeutic significance as prostaglandins are important regulatory mediators of cardiovascular function [29,30]. It has been found that COX-1-deficient mice have renal vasoconstriction and lower blood pressure reduction during sleep (non-dippers), a condition associated with increased risk of cardiovascular diseases in humans [31].
Oatp2b1 gene was detected in the rat heart. Cardiac oatp2b1 expression may imply its role in the cardiac uptake of digoxin, its substrate [32]. The expression of oatp2b1 was significantly reduced in the kidney of AA rats, an observation that may explain, at least in part, the previously reported reduced digoxin clearance in TNF-α-treated mice [33]. ]. This has been explained, in part, as being due to an inflammation-induced upregulation of thyroid hormone transporters leading to increased cellular uptake of thyroid hormones. For example, MCT8 in the liver and skeletal muscles has been found to be significantly upregulated in patients with critical illness [38]. These observations are in agreement with what we have found in the AA rat hearts and the upregulation trend in the kidney. In addition, oatp4a1 was significantly downregulated in the liver of AA rats, a process that if coupled with changes to other thyroid hormone transporters, may significantly alter the hepatic uptake of thyroid hormones.
The lack of effect of inflammation on the renal oat and oct may indicate that the renal excretion of drugs that are substrates of these transporters may not be affected by inflammation. This may explain, at least in part, the lack of influence of inflammatory conditions on the clearance of renally excreted drugs such as sotalol, valsartan and losartan [8,39,40]. In contrast to what was observed in the kidney, oct1 is significantly downregulated in the liver of AA rats suggesting the potential reduction of the hepatic uptake of its substrates. TNF-α and IL-6 have been implicated in inflammation-induced downregulation of hepatic OATs and OCTs in humans. Incubation of human hepatocytes with TNF-α or IL-6 has resulted in reduced expression of OCT1 and OAT2 [41]. On the other hand, lipopolysaccharide administration in rats does not seem to alter oat2 mRNA [42], which is suggestive of a model-dependency of the changes.
Mdr1a and mdr1b are the rodent orthologs of human MDR1 (p-glycoprotein, P-gp). In the Sprague Dawley rats, mdr1a is the predominant transporter [43]. P-gp is the most studied efflux transporter owing to its ubiquitous distribution in most body tissues in humans and animals, its broad substrate specificity and its association with numerous clinically relevant drug interactions. Mdr1a is strategically located in different organs to facilitate specific functions. It is localized in the apical side of hepatocytes, renal tubular and intestinal cells and is responsible for the biliary secretion, tubular secretion and resisting absorption of drugs and their respective metabolites. The clinical significance of mdr1a has been investigated by the use of mdr1a inhibitors and mdr1a knockout animals. For example, injection of radiolabeled digoxin and cyclosporine in mdr1a knockout animals resulted in 20-to 50-fold increase of drug exposure [44]. The effect of inflammation on mdr1a expression has been found to be model, species and organ dependent. Acute inflammatory rats models (turpentine-induced [45] and endotoxin-induced [46]) are associated with reduced expression and activity of mdr1a in the rat liver, uppergastrointestinal tract and kidney. On the other hand, endotoxin-induced inflammation in mice has been associated with upregulation of renal mdr1a and increased doxorubicin renal clearance [47].
Other rodent models such as renal failure [48] and colitis [49] have been found to be associated with reduced expression of P-gp in intestinal tissues. However, no change has been found in experimental cholestasis [50]. The reason for these discrepancies has been explained, in part, by the variation in inflammatory mediator profiles in different diseases and models of inflammation [51]. For example, while in vitro incubation of IL-6 with hepatocytes results in mdr1a downregulation [52,53], incubation with IFN-γ yields increased mRNA and protein expression with no change in the activity in Caco-2 cells [54]. In the present study, mdr1a gene expression was not altered in the rat liver and kidney (the two major elimination organs), suggestive of unaltered biliary clearance and tubular secretion of mdr1a substrates. Interestingly, mdr1a mRNA was significantly increased more than 4-fold in the hearts of AA rats, in contrast to what has been previously reported in a rat model of acute inflammation [55]. This may, at least in part, explain the observed reduced response of cardiovascular drugs in AA rats [6,8,9,17,18,20]. Furthermore, altered cardiac P-gp function can have a crucial role in determining drug cardiotoxicities as in the case of idarubicin [57]. However, inflammation-induced pharmacodynamic alterations caused by target channel or receptor downregulation [9], seem to have higher impact than the reduced cardiac uptake. For example, the reduced verapamil response in rats with endotoxemia has been found to be independent of its reduced uptake [56].
Generally, bsep is exclusively expressed in the liver. However, tissue distribution studies have detected its presence in the rat gut [58]. Herein, we report the expression of bsep in the rat kidney as well. However, the biological function of bsep in the kidney and intestine is not known. Lipopolysaccharide administration in rats resulted in a decline in bsep expression, an effect that can explain LPS-induced intrahepatic cholestasis [42]. In the present study, bsep is downregulated in the liver and kidney by AA. This alteration may affect bile salt transport in AA rat liver and may lead to cholestasis similar to those produced in rats by estrogen and troglitazone, which are bsep inhibitors [59,60]. The consequences of the reduced function of bsep are species dependent. While bsep gene mutation or inhibition in humans can lead to progressive familial intrahepatic cholestasis or drug-induced cholestasis, respectively, bsep knockout mice experienced only mild cholestasis [61,62]. The latter has been explained by the compensatory effects of p-glycoprotein in bile salt transport in mice [61].
Changes at the gene level may not be reflected at the level of protein or protein function. Therefore, further confirmatory experiments may be instituted to test the significance of the changes reported herein. For example, in post-myocardial injury rats, despite its protein downregulation, Ca v 1.2 mRNA was not significantly different suggesting that inflammation-induced alterations are at the post-transcriptional (translational) level [63]. Similarly, the absence of a significant change in expression of Ka v 1.5 may not be extrapolated at the protein level. For example, Brundel et al have found a discrepancy in Ka v 1.5 gene and protein expression in the hearts of patients with atrial fibrillation. While K v 1.5 mRNA did not change, its protein is downregulated [64]. Inflammation did not affect the mRNA expression of adrenergic receptors. However, it has been found that potency of propranolol (a β1 adrenoceptor blocker) is reduced in inflammation [17,18] and this has been attributed to reduced expression of β1 receptor proteins [65]. The reason for this discrepancy can be explained at the level of protein rather than the geneexpression; in other words, the effect is posttranscriptional as in the case of L-type calcium channels.
Previous reports are suggestive of the involvement of inflammatory conditions (20, [117][118][119][120] and the effect of various drugs with antiinflammatory effects (9,17,121) in the outcome of pharmacotherapy at least for cardiovascular drugs. The exact mechanisms behind these involvements remain to be explored. Nevertheless, the overall influence of inflammation on the target receptors must be kept in mind.
In conclusion, inflammation, the host defense mechanism against exogenous pathogens, alters the gene expression of some mediators and drug transporters that can influence the behavior of drugs in the body and contribute to therapeutic failure. The observed expression changes needs to be confirmed at the protein and functional level. Although preliminary, this is an exploratory investigation into the general pattern of the effect of inflammatory conditions on the status of some important proteins. 20. Mayo, P.R., Skeith, K., Russell, A.S., and Jamali, F.