1 Laboratoire de Physiologie-Pharmacie Clinique, UPRES 2706, Faculté de Pharmacie, Châtenay-Malabry and 2 Laboratoire du Métabolisme Minéral des Mammifères, EPHE-Physiologie, Faculté de Pharmacie, Châtenay-Malabry, France
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Abstract |
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Methods. Chronic renal failure was surgically induced in rats by partial (7/8) nephrectomy. After 5 weeks, intestinal transport of rhodamine 123, a P-glycoprotein substrate, was carried out using an in vitro model of everted gut sacs. P-glycoprotein protein content was quantified by enzyme-linked immunosorbent assay and P-glycoprotein mRNA expression was evaluated by semi-quantitative reverse transcriptase polymerase chain reaction.
Results. A decrease of intestinal rhodamine 123 transport was observed in chronic renal failure rats, pointing to an inhibition of P-glycoprotein activity. Transport was inhibited in both sham-operated rats and rats with chronic renal failure by verapamil and cyclosporin A, but relative inhibition vs baseline was less marked in chronic renal failure than in sham-operated rats. In contrast, no significant differences in levels of P-glycoprotein protein or mRNA were observed between the two groups.
Conclusions. Intestinal secretion of rhodamine 123 is mainly mediated by P-glycoprotein. It was reduced in rats with chronic renal failure, reflecting reduced intestinal drug elimination via a decrease in P-glycoprotein transport activity rather than via protein underexpression.
Keywords: chronic renal failure; intestine; P-glycoprotein; rat; rhodamine 123; semi-quantitative RT-PCR
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Introduction |
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The pharmacokinetics of various drugs are largely modified in chronic renal failure (CRF). The renal excretion of some drugs is dramatically reduced, due to decreases in glomerular filtration and/or in tubular secretion. This causes increasing plasma levels leading to toxic effects of these drugs.
Furthermore, functional and anatomical changes in the intestine associated with CRF can affect somewhat the absorption of drugs given orally [4,5]. Thus, intestinal absorption of pyridoxine [6] or L-ascorbic acid [7] is reduced in renal failure. Furthermore, intestinal permeability, using polyethylene glycols of several molecular weights, is impaired in uraemia, suggesting possible alterations in paracellular pathways [8].
However, little is known about the possible effects of CRF on the expression of drug elimination transporters in intestine, and only two studies that examined Pgp in renal failure. The first study reported a reduction in renal and biliary excretion of rhodamine 123, a Pgp substrate, in rats with acute renal failure, suggesting an impairment of Pgp function [9]. The second study reported an upregulation of MRP2 expression with an unchanged Pgp protein expression in kidney and liver of rats with CRF [10].
The aim of this study was to investigate the effects of CRF in rats on the expression and activity of Pgp in intestine, both at the mRNA and protein levels. In rodents, Pgp is encoded by three genes, mdr 1a, mdr 1b, and mdr 2. However, only mdr 1a and mdr 1b appear to have a role in xenobiotic or metabolite transport [11]. To quantify mdr 1a protein levels specifically, an original approach using an ELISA method has been developed. In rat small intestine, mdr 1a is the major RNA transcript expressed [12] and its expression was quantified by reverse transcriptase polymerase chain reaction (RT-PCR) ELISA. To estimate Pgp activity, we used an in vitro model of everted gut sacs using rhodamine 123 as substrate for the transporter.
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Subjects and methods |
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CRF induction
Rats weighing between 140 and 160 g were operated 5 weeks before the intestinal studies. In one group, CRF was induced by surgical partial nephrectomy (7/8) [13]. Rats of the other group were sham-operated. The degree of CRF was evaluated by determination of plasma creatinine concentration at the day of the intestinal studies. In the anaesthetized animals, blood was collected from the aorta into preheparinized vials just after the sampling of the intestine.
In vitro transport of rhodamine 123 across rat everted gut sacs
These studies were carried out in animals without surgery (control rats), in sham-operated rats, and in CRF rats. Fasted rats were anaesthetized with urethane (1 g/kg bodyweight, intraperitoneal injection).
After laparotomy, the ileum was removed above the caecum, washed in ice-cold 0.9% saline and cut into 6 cm long segments. They were everted, ligated at each extremity, and filled with 0.8 ml of a rhodamine 123 solution. Subsequently, this everted sac was placed in 50 ml of buffer A (117.6 mmol/l NaCl, 25 mmol/l NaHCO3, 1.2 mmol/l MgCl2, 1.25 mmol/l CaCl2, 11 mmol/l glucose and 4.7 mmol/l KCl, pH 7.4), then given an O2CO2 mixture (95/5%), and maintained at 37°C throughout the experiment. Excretion of rhodamine 123 from the serosal to the mucosal side was measured by sampling 1 ml of the external medium every 20 min up to 100 min. Rhodamine 123 was dissolved in buffer A, at concentrations indicated in the Figure legends.
The rate of rhodamine 123 transport was expressed in µmoles or in percentage excreted per minute in the mucosal compartment.
In the inhibition studies, inhibitors were added to both mucosal and serosal sides at the same concentration. Several inhibitors of Pgp and other intestinal drug transporters were used to test the specificity of rhodamine 123 transport in this model. Verapamil and cyclosporin A were used as Pgp blockers, at concentrations of 300 µmol/l and 10 µmol/l respectively. Cyclosporin A was dissolved in a mixture of ethanolcremophor EL in buffer A (0.015 and 0.27% w/v), and the same vehicle was used as control.
Tetra ethyl ammonium (TEA), para-aminohippurate (PAH) and probenecid were used as non-Pgp blockers, at concentrations of 300 µmol/l. TEA was used as an OCT1 blocker, PAH as an organic anion transporter (OAT) blocker and probenecid as an MRP blocker. Genistein was used to block both Pgp and MRP, at a concentration of 200 µmol/l.
In some experiments, mannitol was used as a marker of paracellular pathways. Mannitol 2 mmol/l (+0.5 µCi [3H]mannitol) was added in the serosal side with the rhodamine 123. The rate of mannitol transport was expressed as percentage excreted per minute in the mucosal compartment.
Rhodamine 123 was measured by spectrofluorimetry (excitation and emission wavelengths, 500 and 525 nm respectively), and the radioactivity of mannitol was counted in a ß scintillation counter.
ELISA for determination of Pgp protein
Two Pgp antibodies were used in the present study, (i) the commercial monoclonal C219 antibody (Valbiotech), and (ii) a specific anti-mdr 1a antiserum directed against two peptides of the rat protein sequence within a unique region of mdr 1a, TKNDTPEIQR and KDGIDNVDMSSKD (Covalab, France). Peptides were coupled to Keyhole Limpet haemocyanin protein. It was verified using the BLAST program that these sequences were not conserved in the sequence from other rodent Pgps. Furthermore, these sequences were not contained in sequences of other rat mdr.
One gram of jejunal mucosa of each rat from both sham-operated and CRF groups was scraped on ice and homogenized in a glass Teflon potter (20 strokes) in a lysis buffer containing 250 mmol/l sucrose, 50 mmol/l TrisHCl, pH 7.4, 1 mmol/l PMSF, 50 mmol/l iodoacetamide, and 0.1 U/ml aprotinin. The homogenates were centrifuged 10 min at 3000 g, and the supernatant was centrifuged again for 30 min at 15 000 g. The pellets containing the crude membranes were resuspended in 0.5 ml of 50 mmol/l mannitol, 50 mmol/l Tris pH 7.4, 50 mmol/l iodoacetamide, 1 mmol/l PMSF, 0.1 U/ml aprotinin, and 1 mg/ml trypsin inhibitor and stored at -20°C.
The crude membrane suspensions were solubilized in a minimum of 0.1N NaOH solution and the pH was adjusted to 8.5 by addition of 2.2 mol/l TrisHCl. Solubilized membranes were coated onto Immulon 4 HBX extra bind plates (Dynex, France) for 3 h at room temperature. Primary antibodies, C219 or specific anti mdr 1a antiserum, diluted 500- and 1000-fold in PBS3% BSA, respectively were incubated overnight at 4°C. Secondary antibodies, mouse anti-IgG peroxidase conjugate (Biosys) and anti rabbit IgG peroxidase conjugate (Sigma) were diluted 800- and 5000-fold respectively, and incubated at room temperature for 3 h.
The Pgp content of each individual crude membrane preparation, expressed in arbitrary units and reported to the protein content of the sample, was compared to the Pgp content of a pool of jejunal crude membrane defined as standard. C219 antibodies detected all mdr while anti-mdr 1a detected only the mdr 1a form.
Semi-quantitative determination of mdr 1a mRNA in sham-operated rats and in rats with CRF
Total RNA was extracted from jejunal mucosa using Trizol® (Sigma). Reverse transcription of 5 µg of total RNA using 2 pmol of antisense rat specific primers for mdr 1a and rat beta-actin (used as housekeeping gene) was performed for 50 min at 42°C using Superscript II Rnase H-reverse transcriptase (Gibco BRL). The single-strand cDNA generated by each sample was then subjected to semi-quantitative PCR amplification using primers for mdr 1a and beta-actin as previously described [14,15].
The technique of Murphy [16] was applied to quantify the expression of specific mRNAs in each sample. The PCR was found to be both sensitive and quantitative during the exponential range of the reaction. To reach these experimental conditions, serial dilutions of the RNA reverse transcription products (from 0.8 to 12.5 ng) were amplified by PCR during 30 cycles.
Following an initial denaturation at 94°C for 5 min, each cycle consisted of 30 s at 94°C, 30 s at 55°C and 60 s at 72°C.
PCR products were either non-labelled or digoxigenin labelled with a PCR DIG (digoxigenin) mix containing 0.2 mmol/l dATP, dCTP, dGTP each, 1.9 mmol/l dTTP, 0.1 mmol/l digoxigenin-11-dUTP (DIG UTP) during the PCR.
Non-labelled PCR products were subjected to electrophoresis in 1.2% agarose gel and visualized by staining the gel with ethidium bromide. DIG-labelled PCR products were detected with a DIG-detection kit (PCR-ELISA, Boehringer) on microtitration plates. The sequence of the biotinyled capture probe for mdr 1a and for beta-actin directed against an internal sequence of PCR product and necessary to its fixation on the microtitration plates was biotin-TGACGACGTACCTCCAGCTTC and biotin-CACGGCATTGTAACCAACT respectively. The optical density was read with an ELISA reader at 405 nm (ABTS was used as a peroxidase substrate).
In all experiments, after the sampling of the intestine, rats were killed by a lethal injection of urethane.
Expression of results and statistical analysis
The results are expressed as means±SEM. Analysis of variance (ANOVA) was used to compare the results between the different groups.
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Results |
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Transport of rhodamine 123 in everted gut sacs with increasing initial serosal load in sham-operated and CRF rats
A dose-response curve of transport of rhodamine 123 vs increasing initial load of rhodamine 123 is shown in Figure 1. Rhodamine 123 efflux revealed a linear process up to 500 µmol/l in both groups. The slope of linear regression was significantly reduced by 41% in the CRF group as compared to the sham-operated group (0.094, r2=0.92 vs 0.16, r2=0.99). The concentration of 270 µmol/l, chosen in the linear range of the transport, was selected for the following experiments.
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Effect of non-Pgp blockers and genistein on rhodamine 123 efflux in everted gut sacs of control rats
Probenecid, TEA, and PAH did not significantly modify the rate of rhodamine 123 transport. A significant inhibition of about only 30% was observed in the presence of genistein (Figure 2).
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Effect of CRF on rhodamine 123 efflux in everted gut sacs in the presence or absence of Pgp blockers
The basal rate of rhodamine 123 transport (without inhibitor) was significantly decreased by 33%, in the CRF rats as compared with sham-operated rats. The addition of verapamil (300 µmol/l) or cyclosporin A (10 µmol/l) led to a significant inhibition of rhodamine 123 transport, which was smaller in rats of the CRF group (56 and 58% respectively) than in rats of the sham-operated group (73 and 70% respectively) (Figure 3). The cyclosporin solvent was without effect on basal rhodamine 123 transport in either group (data not shown).
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Effects of CRF and of Pgp blockers on mannitol efflux in everted gut sacs
Mannitol efflux rate was measured at the same time as rhodamine 123 efflux in some experiments. It was very weak, representing only 0.006% per min of the mannitol initially present at the serosal side, compared with 0.40 or 0.27% per min for rhodamine 123 efflux in sham-operated and CRF rats respectively. Mannitol efflux was not affected by uraemic state nor by Pgp inhibitors (data not shown).
Effect of CRF on Pgp protein content in jejunal mucosa
The samples were assayed for mdr 1a protein and for total mdr proteins. Initially, we compared Pgp content in the ileum and the jejunum of control rats. No difference was observed between the two segments whether the Pgp was evaluated with C219 antibodies (7.2±0.48 vs 6.44±0.72 arbitrary units/mg of crude membranes respectively, n=8 for both groups) or with specific anti mdr 1a antibodies (5.47±0.38 vs 5.98±0.66 arbitrary units/mg of crude membranes respectively, n=8 for both groups).
Pgp content (mdr 1a and total mdr proteins) was similar in jejunal crude membranes of sham-operated rats and of rats with CRF (Table 1). However, a more marked inter-individual variability was observed in CRF rats, as shown by greater SEM values in this group. In sham-operated rats, a positive correlation was found between the Pgp level determined using C219 antibodies and basal rhodamine 123 transport (R2=0.64).
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Effect of CRF on mdr 1a mRNA expression in jejunal mucosa
Figure 4A shows PCR amplification products of 324 bp and of 513 bp which correspond to the bp predicted from the cDNA sequence of rat mdr 1a and of rat beta-actin. In both groups, a weak inter-individual variation was observed in the ethidium bromide-stained PCR products amplified during the linear range of PCR. To obtain a more accurate quantification than is possible using densitometric analysis of stained PCR products, three dilutions of reverse transcription products of each sample were also analysed by PCR-ELISA in the linear range of PCR. No significant difference in mdr 1a expression after normalization for beta-actin was found between the two groups of rats (Figure 4B
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Discussion |
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Rhodamine 123, a substrate largely used for Pgp activity measurement, is able to detect the activity of other drug efflux proteins such as MRP. Only a few inhibitors are entirely specific for one transporter system. To evaluate the contribution of non-Pgp-related mechanisms to rhodamine 123 transport, we attempted to modulate rhodamine 123 efflux using inhibitors of non-Pgp drug transport systems. Ileal rhodamine 123 transport was not altered by the addition of 300 µmol/l probenecid, but was inhibited in the presence of 200 µmol/l genistein (30%). Among these two MRP inhibitors, genistein has been reported to inhibit rhodamine efflux when used at a high concentration (200 µmol/l) in a Pgp-expressing cell line [17]. Thus, in our in vitro model, the portion of rhodamine 123 transport mediated by MRP was not predominant, as suggested by the lack of inhibition by probenecid and the small inhibition by genistein.
Ileal rhodamine 123 transport was not modified in our study by PAH, a substrate for OAT nor by TEA, a substrate for OCT1, suggesting that the rhodamine 123 secretion was essentially independent of these two transporters.
Cyclosporin A is a potent inhibitor of transport process mediated by Pgp. At least two independent laboratories have suggested that rhodamine 123 efflux in the presence or absence of cyclosporin A could be used to estimate Pgp activity [18,19]. Verapamil is also a substrate for Pgp and has been reported to exert a competitive inhibition with rhodamine 123. We observed a strong inhibition of the basal excretion of rhodamine 123 in sham-operated rats, in the presence of verapamil and cyclosporin A by 73 and 70% respectively.
Finally, the effects of these various inhibitors suggest that rhodamine 123 excretion in the ileal everted sacs occurs essentially through Pgp. In the test, the residual activity (verapamil or cyclosporin A independent) could correspond to MRP activity or other unknown efflux transporters.
Rhodamine 123 was exported linearly as function of time from intestine in both groups. This transport was decreased in rats with CRF compared to sham-operated rats as shown by the significant reduction of the slope.
The goal of the present work was to study the influence of CRF on the function and expression of intestinal Pgp. Basal rhodamine 123 efflux in the absence of inhibitors was significantly decreased in rats with CRF. The effects of cyclosporin A and verapamil were more pronounced in sham-operated rats than in rats with CRF. We therefore conclude that the part of rhodamine 123 transport dependent on verapamil and cyclosporin was decreased by CRF, and represented the portion of the transport dependent on Pgp activity. In contrast, the residual part that could not be inhibited by verapamil and cyclosporin A was similar in sham-operated rats and in rats with CRF, suggesting a similar activity of MRP and/or other transporters in the intestine of both groups of rats.
Pgp protein levels were measured using two different Pgp antibodies and showed that, as with mdr 1a mRNA levels, Pgp protein was not different in both groups of rats. We have described here for the first time an ELISA method which can be used to analyse Pgp protein levels in crude membranes of rat intestine, thereby offering an alternative method to Western blots for the quantification of Pgp protein. Unfortunately, the immune serum of the rabbit injected with the two specific peptides cross-reacted only against one of the two peptides, which prohibited the development of a sandwich ELISA method. However, the intestine is sufficiently rich in Pgp protein to allow for a detection method based on direct antigen binding by ELISA.
The discrepancy between the normal Pgp protein expression in enterocytes and the decreased rhodamine 123 transport in everted gut sacs in CRF rats suggests a possible modification of the transport capacity of Pgp in relation to PKC associated with uraemia. Recently, an inverse relationship between Pgp-mediated transport and PKC activity has been reported in fish renal proximal tubule, reflecting the possible involvement of one or more intermediate steps between the kinase and the transporter [20], which excludes a direct phosphorylation of Pgp by protein kinase C [21]. According to this hypothesis, endothelin, via endothelin B receptors (ETB), has been reported to decrease Pgp-mediated transport in renal proximal tubules [22] by a mechanism that implicated a negative regulation between PKC and Pgp. Jejunum also expresses ETB receptors [23] and an increase of endothelin 1 excretion has been reported in rats with CRF [24]. It is possible that a similar link between endothelins, protein kinase C, one or more regulatory proteins, and Pgp could explain the decrease of rhodamine 123 transport observed in this work.
During renal failure, intestinal compensatory mechanisms for elimination of some drugs are often suggested and attributed to increases in passive pathways, because of the modification in intestinal permeability [8]. The decrease in intestinal Pgp activity, as found in this work, indicates that these hypotheses are not transposable to drugs with active excretion.
Total clearance of rhodamine 123 was reported to be reduced during renal failure in the rat, and attributed to alterations in renal and hepatic pathways [9]. The reduction in active intestinal secretion of rhodamine 123 during renal failure, displayed in this work, could contribute to the diminution in its total elimination.
In addition to the defect of intestinal secretion of xenobiotics in CRF rats observed in the present study by the decrease of Pgp activity, these rats also show an increase of intestinal absorption of some drugs such as ciprofloxacin, which are substrates of intestinal transporters [25]. Added to the effect of reduced renal elimination, these actions could increase the bioavailability of some xenobiotics, leading to the need for a careful adjustment of the dosage of several drugs in patients with chronic renal failure.
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Notes |
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Correspondence and offprint requests to: Bernard Lacour, Laboratoire de Physiologie-Pharmacie Clinique, Faculté de Pharmacie, F-92296 Châtenay-Malabry Cedex, France.
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References |
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