The liver plays a major role in biotransformation and elimination of various therapeutic agents and xenobiotics, many of which are organic cations and substrates of the organic cation transporter 1 (Oct1, Slc22a1). Oct1 is expressed at the basolateral membranes of hepatocytes and proximal renal tubules. Although Oct1 is the major uptake mechanism in hepatocytes for many pharmaceutical compounds, little is known about the effects of liver injury on this process. Our aim was to investigate the effects of obstructive cholestasis on Oct1 expression and function in liver and kidney. The effects of bile duct ligation (BDL) on Oct1 protein, messenger RNA (mRNA) expression, and tissue localization were determined in rat liver and kidney with Western analysis, real-time reverse transcriptase-mediated polymerase chain reaction (RT-PCR), and immunofluorescence. To assess Oct1 function, the model substrate tetraethylammonium ([14C]TEA) was administered intravenously to BDL and control rats and distribution of radioactivity was determined. Oct1 protein significantly decreased in cholestatic livers to 42.1 ± 17.7% (P < .001), 15.5 ± 4.7% (P < .05), and 8.6 ± 2.7% (P < .05) of controls after 3, 7, and 14 days, respectively, but not in kidneys. Hepatic Oct1 mRNA decreased to 77.2 ± 12.7%, 40.7 ± 8.1% (P < .05), and 50.3 ± 7.5% (P < .05) 3, 7, and 14 days after BDL, respectively. Tissue immunofluorescence corroborated these data. Hepatic accumulation of [14C]TEA in 14-day BDL rats was reduced to 29.6 ± 10.9% of controls (P < .0005). In conclusion, obstructive cholestasis down-regulates Oct1 and impairs Oct1-mediated uptake in rat liver, suggesting that hepatic uptake of small cationic drugs may be impaired in cholestatic liver injury. (HEPATOLOGY 2004;39:1382–1389.)
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The liver plays a major role in the biotransformation and elimination of various therapeutic agents and xenobiotics, many of which are at least partly of cationic character.1 In 1994, Grundemann et al.2 isolated the first organic cation transporter (Oct1, Slc22a1) from rat kidney through expression cloning. Functional studies in Xenopus laevis oocytes showed electrogenic, pH-independent, and Na+-independent uptake of the model organic cation tetraethylammonium (TEA).2 Further studies in different expression systems identified a wide range of xenobiotics as substrates of Oct1, including tributylmethylammonium (TBuMA),3 1-methyl-4-phenylpyridinium,4–6 azidothymidine,7 cytosine arabinoside,7 and physiological substances such as choline4, 5 thiamine,5 tyramine,6 N-1-methylnicotinamide,4, 5 dopamine,5, 6, 8 serotonin,6, 8 adrenaline,6 noradrenaline,5, 6, 8 acetylcholine,8 and histamine.5, 8 Studies in Xenopus laevis oocytes demonstrated that rat Oct1 was functionally similar to the hepatic uptake system type I for small organic cations3 described some years ago.9 In contrast, Oct1 did not transport larger organic cations type II whose hepatic uptake9 seems to be primarily a function of the organic anion-transporting polypeptide 2 (Oatp2, Slc21a5).3
In rodents, Oct1 is expressed at the basolateral membranes of hepatocytes,10 proximal renal tubules,11, 12 and enterocytes,5 whereas in humans it is expressed mainly in the liver.13, 14 While Oct1 messenger RNA (mRNA) is distributed throughout the liver lobule, the expression of Oct1 protein is only observed in hepatocytes surrounding the central veins.10 Several additional members of the Oct family have now been identified, including Oct2 (Slc22a2),12, 15 Oct3 (Slc22a3),16 and the more distantly related novel proton/organic cation transporters (Octn) Octn1 (Slc22a4)17 and Octn2 (Slc22a5).18 However, Oct2 is almost exclusively expressed in the basolateral membranes of proximal renal tubules and has some overlap in substrate specificity with Oct1,12, 15 whereas Oct3 seems to be mainly expressed in the placenta and shows less overlap in substrate specificity with Oct1.16 Recent studies in Oct1 knockout mice further suggest that Oct1 is the major if not the only physiologically significant hepatic uptake system for small organic cations.19
Cholestatic liver injury results in the down-regulation of several basolateral membrane transport proteins, including sodium-dependent taurocholate cotransporting polypeptide (Ntcp, Slc10a1) and Oatp1 (Slc21a1).20–22 These alterations are regarded as adaptive responses to cholestasis that may serve to diminish the hepatic accumulation of toxic bile salts and other substances.20 However, the effects of cholestasis on hepatic cation homeostasis and transport are not known. Therefore, the aim of the present study was to utilize bile duct ligation (BDL) as a well-established model of cholestasis to determine its effect on the expression of Oct1 in rat liver and kidneys and on the distribution of the model organic cation TEA in vivo. The results demonstrate that obstructive cholestasis results in significant down-regulation of the organic cation transporter Oct1 in rat liver and impairment of Oct1-mediated hepatic TEA-uptake.
Male Sprague-Dawley rats (200–230 g) were obtained from Charles River (Wilmington, MA) and underwent BDL or sham operation as previously described.21, 23 Animals were sacrificed at 3, 7, and 14 days and tissues were harvested for Western analysis, real-time reverse transcriptase-mediated polymerase chain reaction (RT-PCR), and immunofluorescence studies. Tissues for immunofluorescence were snap frozen in freon that had been cooled in liquid nitrogen. Tissues for real-time RT-PCR and Western-blotting were snap frozen directly in liquid nitrogen. Functional studies were carried out in a separate group of animals 14 days after BDL or sham surgery. After pentobarbital anesthesia, TEA (0.2 mg [14C]TEA/kg body weight [bw] [55 Ci/mol; American Radiolabeled Chemicals, St. Louis, MO] plus 1.1 mg TEA/kg bw [Sigma, St. Louis, MO] dissolved in 1 mL saline) was injected intravenously (iv) in BDL and sham-operated rats to achieve an initial TEA blood concentration close to the Michaelis constant (Km) of rat Oct1. Mannitol was infused at a slow rate (40 mg/mL in 0.9% saline, 2 mL/h) via the external jugular vein to assure a steady flow of urine24 and blood samples were taken 15, 30, 45, and 60 minutes after TEA injection. After 1 hour, the livers were harvested and urine was collected from the urinary bladder. The liver samples were homogenized in a 4% (wt/vol) bovine serum albumin solution and radioactivity levels in the homogenates, urine, and plasma samples were determined by liquid scintillation counting (Wallac 1414 WinSpectral® Liquid Scintillation Counter, Perkin-Elmer, Turku, Finland) in 5 mL Opti-Fluor® (Packard Bioscience, Meriden, CT) liquid scintillation fluid. All protocols were approved by the Yale Animal Care and Use Committee, and animals received humane care as outlined in the “Guide for the Care and Use of Laboratory Animals” (NIH publication 86-23, revised 1985).
Livers and kidneys were minced with scissors in ice-cold Tris-sucrose buffer (livers; 100 mmol/L Tris, 250 mmol/L sucrose, pH 7.6) or ice-cold HEPES-mannitol buffer (kidneys; 80 mmol/L HEPES, 200 mmol/L mannitol, 41 mmol/L KOH, pH 7.5) in the presence of protease inhibitors (Complete® protease inhibitor cocktail, Roche Diagnostics, Mannheim, Germany) and then homogenized with a motor driven Teflon glass homogenizer (Thomas Scientific, Philadelphia, PA) operating at 3000 rpm. A membrane-enriched microsomal pellet was obtained from the postnuclear supernatant after a 100,000g ultracentrifugation for 1 hour at 4°C. The pellet was resuspended in HEPES-sucrose buffer (livers; 10 mmol/L HEPES, 300 mmol/L sucrose, pH 7.5, protease inhibitors) or HEPES-mannitol buffer (kidneys).23 The protein concentration was determined according to Bradford25 with the protein assay from Bio-Rad (Hercules, CA) and samples were stored at –80°C. Membrane fractions from either total liver or kidney were resuspended in sample buffer and adequate amounts of protein (50 μg liver total membrane, 25 μg kidney total membrane) were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).26 After electrotransfer to nitrocellulose membranes (Blot® Transfer Medium, Bio-Rad) equal protein loading and sufficient transfer were confirmed by Ponceau staining. Then the blots were blocked with Tris-buffered saline containing 0.1% Tween and 5% dry milk for 1 hour at room temperature. The blots were then incubated overnight at 4°C with affinity-purified polyclonal rabbit anti-Oct1 antiserum raised against amino acids 524 to 542 of rat Oct110, 12 at a dilution of 1:3,000 for liver samples and 1:5,000 for kidney samples. Immune complexes were detected using horseradish-conjugated goat anti-rabbit immunoglobulin G (Sigma) and the enhanced chemiluminescence (ECL) Western blotting kit (Amersham Biosciences, Little Chalfont, UK). Immunoreactive bands were quantified by densitometry with Multi-Analyst (Bio-Rad). Because Oct1 consistently showed two broad bands in Western analysis, deglycosylation studies were performed to confirm the assumed glycosylation. Deglycosylation of Oct1 protein in liver and kidney total membrane fractions was done with N-glycosidase F (PNGase F; New England Biolabs, Beverly, MA) according to the manufacturer's protocol.
Real-time RT-PCR Analysis.
Total RNA was extracted from the different tissues using TRIzol® (Invitrogen Life Technologies, Carlsbad, CA). Concentration and purity were confirmed by spectrophotometry (Ultrospec® 3000, Pharmacia Biotech, Cambridge, UK). RNA was stored at –80°C. Reverse transcription was performed on 5 μg of isolated rat total RNA per sample and 5 μg of commercial pooled normal rat liver and kidney total RNA (Stratagene, La Jolla, CA) using established protocols and the ProSTAR® RT-PCR kit (Stratagene). TaqMan real-time quantitative PCR assay was performed on an ABI Prism 7700 Sequence Detection System, according to the manufacturer's protocol (Applied Biosystems, Foster City, CA). The following primers and probes were used for the TaqMan PCR assay: Oct1: forward primer: TGGTGTTCAGGCTGATGGAA; reverse primer: GCCCAAAACCCCAAACAAA; probe: TTTGGCAAGCCCTGCCCCTCA (all designed by ABI Primer Express software and obtained from Applied Biosystems); rodent glyceraldehyde-3-phosphate dehydrogenase (GAPDH): proprietary (Applied Biosystems). Amplification of GAPDH was performed to standardize the quantification of target complementary DNA (cDNA), allowing relative quantitation using the ABI Prism 7700 SDS software. Briefly 2.0 μL, 1.0 μL, 0.5 μL, and 0.25 μL of synthesized normal rat liver and kidney cDNA, respectively, were amplified in triplicate for both Oct1 and GAPDH to create standard curves. Likewise, 2.0 μL of cDNA was amplified in triplicate for Oct1 and GAPDH for all isolated rat liver and kidney samples, respectively. Each sample was supplemented with both forward and reverse primer and fluorescent probe and made up to 50 μL using TaqMan Master-Mix (Applied Biosystems). The amplification was done in a 96-well plate. All samples were incubated at 50°C for 2 minutes and at 95°C for 10 minutes, and then cycled at 95°C for 15 seconds and 60°C for 1 minute for 40 cycles. After calculating the standard curves, the input amounts of cDNA of the unknown samples were calculated for Oct1 and GAPDH. After normalizing the input amount of the Oct1 sample to the GAPDH expression, the relative amount of the expressed RNA species in each unknown sample was calculated.
Indirect immunofluorescence was conducted on liver specimens from sham-operated and BDL rats.27 Small cubes of snap frozen tissue were embedded in O.C.T. embedding medium (Miles, Elkhart, IN) and 5- to 7-μm frozen sections were cut and placed on poly-L-lysine-coated glass slides. Before antibody application, the sections were treated with acetone at –20°C for 10 minutes, and non-specific sites were blocked with 1% bovine serum albumin in phosphate-buffered saline containing 0.05% Triton X100. The polyclonal antibody against Oct1 was used at a dilution of 1:50. The secondary antibody was Alexa 594 anti-rabbit immunoglobulin G (Molecular Probes, Eugene, OR). All fluorescent imaging was performed on a Zeiss LSM510 confocal scanning microscope (Zeiss, Thornwood, NJ) and digital images were recorded on an Iomega® Zip disc and processed with Adobe Photoshop (Adobe Systems, San Jose, CA).
Data are expressed as means ± SD. Differences between experimental groups were assessed for significance by using the two-tailed unpaired Student's t test. A P value less than .05 was considered to be statistically significant.
To determine whether the expression of Oct1 is altered at the protein level in obstructive cholestasis in livers and kidneys, Western analysis was performed in total membrane fractions of these tissues obtained from non-operated and sham-operated controls and BDL rats. The protein blots, probed with the polyclonal anti-Oct1 antibody,10, 12 identified bands at 50 and 70 kilodaltons (Figs. 1A, 1B, 2A, 3A). After deglycosylation with N-glycosidase F, both bands made a shift to one single band at 45 kilodaltons indicating significant posttranscriptional glycosylation of the Oct1 protein (Fig. 1A and B). As the glycosylated form represents the mature protein, densitometrical analysis of the protein bands was performed on the upper band.
As seen in Figs. 2A and B, three days after BDL, there was a significant decrease of the hepatic Oct1 protein level to 42.1 ± 17.7% of sham-operated controls (P < .001). More prolonged periods of obstructive cholestasis, to 7 and 14 days, resulted in further progressive loss of Oct1 protein in the liver to 15.5 ± 4.7% (P < .05) and 8.6 ± 2.7% (P < .05) of sham-operated controls, respectively (Fig. 2A and B). In contrast, there were no significant changes of Oct1 protein levels in kidneys after BDL (Fig. 3A and B).
To determine whether the changes of Oct1 on the protein level were also associated with changes of Oct1 mRNA, a quantitative analysis of Oct1 mRNA in liver and kidney was performed with real-time RT-PCR. As illustrated in Fig. 4, 3, 7, and 14 days after BDL hepatic Oct1 mRNA levels decreased to 77.2 ± 12.7%, 40.7 ± 8.1%, and 50.3 ± 7.5% of sham-operated controls, respectively (P < .05 after 7 days). In contrast (Fig. 5), 3 days after BDL there was a significant 79.4% increase of the Oct1 mRNA level in kidneys of the BDL animals (P < .005) compared to sham-operated controls. Thereafter, kidney Oct1 mRNA levels returned to normal, suggesting a transient up-regulation during acute obstructive cholestasis.
Tissue Immunofluorescence of Oct1 in Liver.
Indirect immunofluorescence studies were performed to analyze the qualitative distribution of Oct1 protein in the livers of sham-operated control and BDL rats. Figure 6A and B illustrate that the findings were consistent with the immunoblotting results and confirm that obstructive cholestasis leads to a strong decrease of Oct1 protein in the liver in contrast to sham-operated control liver which showed the previously observed10 basolateral distribution pattern of Oct1 in the hepatocytes of the pericentral zone of the liver lobule. This typical staining pattern for Oct1 was only weakly seen 14 days after BDL. There was no clear Oct1 staining in cholangiocytes from normal liver or following bile duct ligation.
Distribution and Excretion of [14C]TEA in Sham-operated and BDL Rats.
To investigate the functional effects of BDL induced Oct1 down-regulation on the hepatic uptake of small organic cations, the distribution and excretion of [14C]TEA, a model Oct1 cationic substrate, was determined in sham-operated and 14-day BDL rats. [14C]TEA is not substantially metabolized in rodents28 and is an established substrate for Oct1.2, 5 When TEA (0.2 mg [14C]TEA/kg bw plus 1.1 mg TEA/kg bw) was administered iv to achieve an initial TEA blood concentration close to the Km of rat Oct1 (95 μmol/L),2, 5 the hepatic accumulation of [14C]TEA in BDL rats was only 30% of sham-operated controls (96.0 ± 35.2 vs. 323.8 ± 69.8 nanogram-equivalent (ng-eq) [14C]TEA/g liver, P < .0005) 1 hour after iv injection (Fig. 7). In contrast, there was no significant difference in respect to urinary [14C]TEA recovery (52.4 ± 15.9% vs. 44.9 ± 21.3% of the administered dose in urine of BDL and sham-operated rats, respectively, collected over 1 hour after [14C]TEA iv injection). Although both experimental groups received the same dose of [14C]TEA, [14C]TEA levels in plasma were significantly lower in BDL than in sham-operated rats 30 minutes after iv injection and later (Fig. 8).
This study demonstrated that BDL in the rat, an established model of obstructive cholestasis,21, 23 resulted in profound down-regulation of the organic cation transporter Oct1 on mRNA and protein level and resulted in the impairment of the hepatic uptake of a prototype Oct1 substrate, [14C]TEA. A 36% reduction in Oct1 liver mRNA has been previously reported after 3 days of BDL in the rat29 and intraperitoneal injection of lipopolysaccharide, another cholestatic experimental model, has resulted in impaired hepatic uptake of the Oct1 substrate TBuMA,30 consistent with our findings.
Prior studies have also found that certain other transport proteins located at the basolateral membrane of the hepatocyte are down-regulated at the mRNA and protein level during cholestatic liver injury, resulting in impairment of hepatic uptake of their respective substrates.20 Particular examples are the sodium-taurocholate cotransporter Ntcp21 and the organic-anion-transporting polypeptide Oatp122 that transport bile salts and a variety of organic anionic substrates into the liver, respectively. Detailed studies have shown that Ntcp is down-regulated by transcriptional mechanisms, involving the effects of cytokines that lead to the loss of the nuclear receptors retinoid X receptor α/retinoic acid receptor α (RXRα/RARα), an Ntcp promoter agonist.31 Also, inhibitory effects on Ntcp expression occur and are mediated by the nuclear receptor short heterodimeric protein (Shp) that is induced by the binding of bile acids to the nuclear receptor farnesoid X activated receptor (FXR) that then binds to the Shp promoter.32 Whether similar mechanisms account for the down-regulation of Oct1 is not known.
Oct1 protein expression in the liver was more profoundly diminished than mRNA levels following BDL. However, it is highly likely that Oct1 is at least in part post-transcriptionally regulated because Oct1 in situ hybridization studies of liver sections indicate that Oct1 mRNA is homogeneously expressed throughout the liver lobule, whereas Oct1 protein is normally observed only in hepatocytes surrounding the central veins.10
In contrast to the effects of obstructive cholestasis on hepatic Oct1 expression, Oct1 gene expression in the kidneys of BDL rats was not impaired and even appeared to be up-regulated transiently, three days after BDL, when kidney Oct1 mRNA levels increased by 79% compared to sham-operated controls. Thereafter, the elevated mRNA levels returned to normal. However, this increase in Oct1 mRNA expression was not associated with significant changes of the Oct1 protein level in kidneys of cholestatic rats at any of the measured time points during the 14 days of bile duct obstruction.
In accordance with the Western analysis and immunofluorescence findings that reflected significant loss of Oct1 protein expression in liver 14 days after BDL, the hepatic accumulation of the Oct1 substrate [14C]TEA was decreased to 30% of sham-operated controls 1 hour after iv injection indicating an impairment of Oct1-mediated uptake. These results are consistent with findings in Oct1 knockout mice where the hepatic accumulation of [14C]TEA was only 23% of that in wild-type mice.19 Because Oct1 is an electrogenic transporter,2 changes in the membrane potential could theoretically also affect [14C]TEA accumulation in the liver.
The long-term effects of impaired hepatic uptake of Oct1 substrates that largely consist of small organic cationic drugs and important physiological substrates like choline,4, 5 thiamine,5 tyramine,6 N-1-methylnicotinamide,4, 5 dopamine,5, 6, 8 serotonin,6, 8 adrenaline,6 noradrenaline,5, 6, 8 acetylcholine,8 and histamine5, 8 are not known but could be important in clinical cholestatic disorders, should the hepatic uptake of these and other cationic solutes and drugs be impaired. It is attractive to speculate whether reductions in the hepatic uptake of neurotransmitters such as serotonin might account for the chronic fatigue seen in many patients with chronic cholestasis.33
Although BDL and sham-operated rats received the same dose of [14C]TEA, plasma [14C]TEA levels were lower in BDL than in sham-operated rats, which was significant 30 minutes and later after iv injection. The lower [14C]TEA plasma levels in BDL rats could reflect altered pharmacokinetics due to the reduced hepatic uptake of TEA after BDL. Thus, shortly after iv administration [14C]TEA plasma levels, i.e., plasma drug availability, may be higher in BDL than in sham-operated rats resulting in an initially more rapid clearance of TEA from plasma via the kidney, which is the main excretory route for small organic cations in contrast to biliary excretion,33 which contributes only minimally to the overall clearance of TEA.19 However, because we did not analyze [14C]TEA levels in plasma or urine at earlier time points, this possibility remains speculative.
In summary, the findings in the present study have potential implications for the pharmacokinetics of drug distribution in human cholestatic liver disease. Hepatic uptake of small organic cations includes essential substrates for biosynthetic pathways as well as xenobiotics and is of major importance because the liver represents the main site for biotransformation in the body. For example, hepatic uptake of metformin in Oct1 knockout mice is reduced more than 30 times below levels of uptake in wild-type mice,35 and results in lower blood concentrations of lactate.36 Transport of metformin into the liver is essential for its antidiabetic effect that is mediated by inhibition of complex 1 of the mitochondrial respiratory chain in the hepatocyte.37 Thus, the primary function of Oct1 in liver appears to be to facilitate hepatic biotransformation of small organic cations, rather than to mediate their excretion, which is a likely function of Oct1 in kidney. However, in humans, OCT1 is expressed almost exclusively in the liver13, 14 so that other Oct family members may be of greater importance in the elimination of cationic substrates in human kidney.
The authors thank Kathy Harry for excellent technical assistance.